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Work Breakdown Structure (WBS): The Complete Guide

Written by samantha ferguson.

Last updated on 11th April 2024

Work Breakdown Structure (or WBS, as it’s sometimes known) is about dividing a project into smaller, more digestible chunks, making it easier to plan, execute, and monitor. 

In this guide, we’ll cover everything you need to know about work breakdown structures: what they are, how to create them, and how to use them effectively in your project planning. 

We’ll also provide some templates and examples to get you started. So let’s dive in!

What is a work breakdown structure?

A work breakdown structure is a planning tool used by project managers to break down the work of a project into smaller, more manageable ‘pieces’ in order to make it easier to track progress – as well as identify potential issues. 

As an organisational tool, WBS helps to assign roles for each task and subtask and define who’s responsible for what. 

Typically created from the project scope , a WBS lets teams map out all tasks that need to be completed from beginning to end, starting with the larger activities and breaking them down into more granular detail until every element of the project has been accounted for. 

With its flexibility and scalability, this popular planning tool can easily be modified along the way to adjust for changes or environmental factors that arise during the lifetime of a project.

The benefits of using a WBS

So, we’ve established that a work breakdown structure is an incredibly useful tool for project managers. What are the actual benefits of working in this way?

1. Improved project planning

A WBS breaks down complex projects into smaller, more manageable tasks, making it easier to plan and schedule the work that needs to be completed. 

By identifying all the stuff that needs to be done, you can create a more accurate project plan, including timelines, milestones, and deliverables – keeping your entire project running smoothly and optimising chances of success.

2. Better resource allocation

With a detailed WBS, you can identify the specific resources needed for each task, including people, equipment, time and materials. 

This lets you allocate resources more efficiently and effectively – ensuring that everyone and everything is being used to their fullest potential.

3. Greater project control

A work breakdown structure provides a clear and comprehensive overview of the project, allowing you to monitor progress, identify potential issues, and make necessary adjustments as issues arise.

By breaking the project down into smaller pieces, you can track progress more easily and keep everyone on the same page.

4. Enhanced communication

A WBS can serve as a valuable communication tool, since it lets you share project information with team members, stakeholders, and other relevant parties. 

By presenting the project in a clear, structured format, you can facilitate communication and ensure that everyone understands what needs to be done and when. 

People can grasp not only their own role in the bigger picture of your project, but also understand what others are working on.

5. Improved risk management

With a work breakdown structure, you can identify potential risks and develop strategies to mitigate them. 

By breaking the project down into smaller pieces, you can identify areas where risks are more likely to occur and take steps to address them before they become major issues.

The different types of work breakdown structure

Further underlining the flexibility of this way of working, there are a variety of different types of WBS to be used and adapted depending on the needs of your project.

1. Deliverable-oriented WBS

This type of WBS focuses on the end deliverables of the project and breaks them down into smaller, more manageable tasks. Each task is assigned to a specific team or individual responsible for completing it. 

WORKS BEST FOR: Projects with clearly defined outcomes.

2. Phase-oriented WBS

This type of WBS breaks down the project into phases, with each phase representing a major milestone or objective. Each phase is further broken down into smaller tasks, allowing for better project management and monitoring.

WORKS BEST FOR: Projects with distinct stages. 

Each phase would then be given its own set of tasks and assigned to specific people.

3. Organisational-oriented WBS

This type of WBS is based on the organisational structure of the project team. Tasks are grouped according to the team or department responsible for completing them, making it easier to allocate resources and track progress.

WORKS BEST FOR: Projects with multiple departments or stakeholders involved.

4. Activity-oriented WBS

This type of WBS breaks down the project into specific activities or tasks that need to be completed, regardless of the end deliverable. Each activity is assigned to a specific team or individual responsible for completing it.

WORKS BEST FOR: Projects with many interdependent tasks. 

5. Hybrid WBS

This type of WBS combines two or more of the above types, depending on the needs of the project. For example, a hybrid WBS may include a phase-oriented WBS for overall project management, with an activity-oriented WBS for specific tasks or deliverables.

WORKS BEST FOR: Projects where you have a ‘mix’ of any or all of the other types of WBS and need to use a highly customised WBS to suit the needs of the project team.

A hybrid WBS would be necessary in this project because it would allow for both deliverable-based and phase-based management, as well as departmental management.

This would provide a more comprehensive and flexible structure for managing the project, ensuring that all deliverables are completed on time and within budget while also allowing for more efficient departmental coordination and resource allocation.

In essence a hybrid WBS offers the best of both worlds – a high-level overview of the project’s phases and deliverables, as well as a more detailed breakdown of the tasks and responsibilities within each department. 

How to Create a WBS

Developing a WBS isn’t difficult – all it takes is understanding the basics of project management and following a few steps. 

1. Identify the major deliverables

The first step is to identify the major deliverables or outcomes that the project aims to achieve. These are usually the key objectives or milestones of the project.

2. Break down deliverables into sub-deliverables

Once the major deliverables have been identified, break them down into smaller, more manageable sub-deliverables. This step involves breaking down the major objectives into smaller, more specific tasks that need to be completed to achieve them.

3. Continue breaking down until you reach manageable tasks

Continue breaking down the sub-deliverables into smaller and more manageable tasks until you have reached a level of detail that is sufficient for project planning and management. This level of detail will depend on the complexity and size of the project.

4. Organise the tasks

Organise the tasks into a hierarchical structure that shows the relationship between the different tasks. This structure will help in project planning and tracking progress.

5. Assign resources and estimate time

Assign resources and estimate the time required to complete each task. This will help in determining the project schedule and budget.

6. Review and refine

Review and refine the WBS to ensure that it accurately reflects the scope of the project and that all necessary tasks have been included.

7. Use the WBS as a reference

Once the WBS has been created, use it as a reference tool throughout the project to ensure that all tasks are completed as planned and that the project stays on track.

Work Breakdown Structure: A Summary

To recap, creating a work breakdown structure (WBS) is a critical step in planning and managing a project. 

By breaking down a project into smaller, manageable deliverables, the project team can organise their work, allocate resources effectively, and track progress more easily. 

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Developing a Work Breakdown Structure

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Cicala, G. (2020). Developing a Work Breakdown Structure. In: The Project Managers Guide to Microsoft Project 2019 . Apress, Berkeley, CA. https://doi.org/10.1007/978-1-4842-5635-0_6

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Work Breakdown Structure (WBS)

This guide to wbs in project management is presented by projectmanager, project and work management software loved by 35,000+ users. make a wbs in minutes.

What Is a Work Breakdown Structure (WBS)?

Why use a wbs in project management, work breakdown structure example, types of wbs, wbs elements, how to create a work breakdown structure in six steps, wbs software, must-have features of wbs software, how to create a wbs in projectmanager.

• Work Breakdown Structure Template

When to Use a WBS?

Work breakdown structure best practices.

A work breakdown structure (WBS) is a visual, hierarchical and deliverable-oriented deconstruction of a project. It is a helpful diagram for project managers because it allows them to break down their project scope and visualize all the tasks required to complete their projects.

All the steps of project work are outlined in the work breakdown structure chart, which makes it an essential project planning tool. The final project deliverable, as well as the tasks and work packages associated with it rest on top of the WBS diagram, and the WBS levels below subdivide the project scope to indicate the tasks, deliverables and work packages that are needed to complete the project from start to finish.

Project managers make use of project management software to lay out and execute a work breakdown structure. When used in combination with a Gantt chart that incorporates WBS levels and task hierarchies, project management software can be especially effective for planning, scheduling and executing projects.

ProjectManager is an online work management software with industry-leading project management tools like Gantt charts, kanban boards, sheets and more. Plan using WBS levels in our tool, then execute with your team via easy-to-use kanban boards and task lists. Try it for free today.

ProjectManager’s online Gantt charts feature a column for the WBS code— learn more

Making a WBS is the first step in developing a project schedule . It defines all the work that needs to be completed (and in what order) to achieve the project goals and objectives. By visualizing your project in this manner, you can understand your project scope, and allocate resources for all your project tasks.

A well-constructed work breakdown structure helps with important project management process groups and knowledge areas such as:

• Project Planning, Project Scheduling and Project Budgeting
• Risk Management, Resource Management, Task Management and Team Management

In addition, a WBS helps avoid common project management issues such as missed deadlines, scope creep and cost overrun, among others.

In other words, a work breakdown structure serves as your map through complicated projects. Your project scope may include several phases or smaller sub-projects—and even those sub-projects can be broken down into tasks, deliverables, and work packages! Your WBS can help you manage those items, and gain clarity into the details needed to accomplish every aspect of your project scope.

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WBS Template

Use this free WBS Template for Excel to manage your projects better.

Now that we’ve gone through the definition of a WBS and learned why they are a great project management tool, let’s take a look at a work breakdown structure example.

For our WBS example, we’ll be creating a work breakdown structure to lay down the work plan for a commercial building construction project. This is potentially a complex project, but a WBS chart will take that complexity and boil the project scope down to simpler tasks to make the project manageable.

Study the phase-based work breakdown structure example of a construction project below:

At the top of the work breakdown structure is your final deliverable (in this instance, the construction project). Immediately beneath that is the next WBS level, which are the main project phases required to complete the project. The third and lowest level shows work packages . Most WBS charts have 3 levels, but you can add more depending on the complexity of your projects.

Each of those five project phases—initiation, planning, execution, control and closeout, also act as control accounts and branch off the main deliverable at the top. Once decided, they are then broken down into a series of deliverables. For example, the initiation phase includes site evaluation work and creating the project charter.

You’ll also need a work package to go with each of those project deliverables. In the execution phase of our construction example, we can look at the interior work deliverable. That deliverable is divided into two work packages, which are installing the plumbing and setting up the electricity.

The WBS, when created as thoroughly as possible, is the roadmap to guide you to the completion of what would seem to be a very complicated project scope. However, when broken down with a WBS, project planning, scheduling and resource planning suddenly become much more manageable.

There are two main types of WBS: deliverable-based, and phase-based. They depend on whether you want to divide your project in terms of time or scope.

Deliverable-Based Work Breakdown Structure

A deliverable-based WBS first breaks down the project into all the major areas of the project scope as control accounts and then divides those into project deliverables and work packages.

Here’s an example of a deliverable-based WBS that’s taken from our free work breakdown structure template. Download the template today to practice building your own work breakdown structure in Excel.

A deliverable-based WBS example showing control accounts, work packages and tasks.

Phase-Based Work Breakdown Structure

The phase-based WBS displays the final deliverable on top, with the WBS levels below showing the five phases of a project (initiation, planning, execution, control and closeout). Just as in the deliverable-based WBS, the project phases are divided into project deliverables and work packages. Our previous graphic in the “Work Breakdown Structure Example” section contained a phase-based WBS example.

Types of WBS Charts

Once you’ve chosen a deliverable-based or phase-based WBS, you can also choose between different types of WBS diagrams. Let’s take a look at the main types of work breakdown structure charts.

Work Breakdown Structure List: Also known as an outline view, this is a list of work packages, tasks and deliverables. It’s probably the simplest method to make a WBS, which is sometimes all you need.

Work Breakdown Structure Tree Diagram: The most commonly seen version, the tree structure depiction of a WBS is an organizational chart that has all the same WBS elements of the list (phases, deliverables, tasks and work packages) but represents the workflow or progress as defined by a diagrammatic representation.

Work Breakdown Structure Gantt Chart: A Gantt chart is both a spreadsheet and a timeline. The Gantt chart is a WBS that can do more than a static task list or tree diagram. With a dynamic Gantt chart, you can link dependencies, set milestones, even set a baseline. This is the most common version in project management software.

Build a work breakdown structure Gantt chart diagram in ProjectManager in just a matter of minutes. Get started for free today.

A Gantt chart with WBS codes in ProjectManager. Learn more

A typical project work breakdown structure is made up of several key components. We’ll use our WBS example above to identify each of the main WBS elements.

• WBS Dictionary: A WBS dictionary is a document that defines the various WBS elements. It’s an important component of a WBS because it allows the project participants and stakeholders to understand the work breakdown structure terminology with more clarity.
• WBS Levels: The WBS levels are what determines the hierarchy of a WBS element. Most work breakdown structures have 3 levels that represent the project’s main deliverable, control accounts, project deliverables and work packages.
• Control Accounts: Control accounts are used to group work packages and measure their status. They’re used to control areas of your project scope. In our example, the execution project phase could be a control account because it has several deliverables and work packages associated with it.
• Project Deliverables: Project deliverables are the desired outcome of project tasks and work packages. In our WBS example, we can observe some examples of project deliverables such as the project budget or interior work. Both of them are the result of smaller tasks and work packages.
• Work Packages: As defined by the project management institute (PMI) in its project management body of knowledge book (PMBOK) a work package is the “lowest level of the WBS”. That’s because a work package is a group of related tasks that are small enough to be assigned to a team member or department. As a project manager, you can estimate costs and duration of these work packages, which makes them an essential WBS element.
• Tasks: Your tasks make up your work packages and therefore, your project scope. A WBS will help you define each task requirements, status, description, task owner, dependencies, and duration.

If you prefer a visual and verbal explanation of this information on work breakdown structures, watch this video.

To create a WBS for your project, you’ll need information from other project management documents. Here are six simple steps to create a work breakdown structure.

1. Define the Project Scope, Goals and Objectives

Your project goals and objectives set the rules for defining your project scope. Your project scope, team members, goals and objectives should be documented on your project charter .

2. Identify Project Phases & Control Accounts

The next level down is the project phases: break the larger project scope statement into a series of phases that will take it from conception to completion. You can also create control accounts, which are task categories for different work areas you want to keep track of.

3. List Your Project Deliverables

What are your project deliverables ? List them all and note the work needed for those project deliverables to be deemed successfully delivered (sub-deliverables, work packages, resources, participants, etc.)

4. Set WBS Levels

The WBS levels are what make a work breakdown structure a “hierarchical deconstruction of your project scope”, as defined by the project management institute in its project management body of knowledge book (PMBOK). You’ll need to start at the final project deliverable and think about all the deliverables and work packages needed to get there from the start.

5. Create Work Packages

Take your deliverables from above and break them down into every single task and subtask that is necessary to deliver them. Group those into work packages.

6. Choose Task Owners

With the tasks now laid out, assign them to your project team. Give each team member the work management tools , resources and authority they need to get the job done.

Work breakdown structure software is used to outline a project’s final deliverable and define the phases that are necessary to achieve it.

Software facilitates the process in several different ways. Some use a network diagram and others use a Gantt chart. All of them, however, are a visual representation of the project, literally breaking down the various stages and substages needed to assemble the final project deliverable.

There are many types of work breakdown structure software available, so when you’re looking for one to help you plan your project, be sure it offers these features:

Break Tasks Down

Deliverables are important to define, as are the tasks that get you there—but most tasks require being broken down further in order to complete them. That’s where subtasks come in. They’re part of a more complex task, and you want that feature in your WBS software.

Link Dependent Tasks

Not all tasks are the same. Some can’t start or stop until another has started or stopped. These dependent tasks can create a bottleneck later in the project’s execution phase, unless you identify them early. Having a task dependency feature is essential.

Set Task’s Priority and Duration

The point of WBS software is to build a feasible schedule. Therefore, you need features that feed into this process by defining the priority of the task, so you know which phase it goes with; as well as describing the task and estimating how long it will take to complete.

Keep Your Team Working

The WBS sets up your tasks and deliverables, but once the project is in the execution stage, it’s key that you have a way to allocate resources to your team to keep the tasks moving as planned. That includes a feature to make sure their workload is balanced.

Get a High-Level View

Being able to monitor your progress is what keeps your project on schedule. A WBS software sets up the plan and you must have features to maintain it throughout all the phases of the project. Dashboards can give you a view of the landscape across several metrics.

Make Better Decisions

As you move from the planning to the execution stage, you’ll need a reporting feature that can deliver critical project data on progress and performance. This information will feed your decision-making and help you steer the project to a successful conclusion.

The purpose of work breakdown structure software in project management is to organize and define the scope of your project. Using ProjectManager’s online Gantt charts to build your WBS is not only more efficient, it dovetails into every other aspect of your project, because of our robust suite of project management features.

Here’s a quick summary of how to create a WBS using a Gantt chart. Sign up for a free trial of our software and follow along !

1. Identify Project Deliverables

There are 5 stages in the project life cycle, initiation, planning, execution, monitoring and closure. Each of them produces deliverables that are required to produce the final deliverable, which is the completion of your project.

Identify the phases in your project to create more than a mere task list. Set them apart with our milestone feature on the Gantt chart tool. They can also be color coded to better differentiate the phases.

2. List Subtasks, Describe Tasks & Set Task Owner

Subtasks are part of a larger, more complex task. In this case, your WBS work packages are perfect for this feature. Add summary tasks or work packages above the related tasks, which can be your project phases or project deliverables, depending on your WBS type preference and indent them. The image below shows our WBS example represented on a Gantt chart, showing the project phases and work packages associated with them.

3. Link Dependencies

Task dependencies are tasks that cannot start until another is finished or started. Link tasks that are dependent on one another by dragging one to the other. We link all four types of task dependencies. By identifying these tasks at this stage, you’ll avoid bottlenecks during execution.

4. Set Resources & Costs

Resources are anything that you need to complete the project phases, deliverables and work packages. They range from the people on your team to materials, supplies and equipment. Your WBS allows you to break down your project scope into work packages so that you can estimate resources and costs.

5. Add Start & End Dates & Estimated Completion

Every task has a start and an end date. Add the date when the task needs to start in the planned start date column and when it should be completed in the planned finished date. There’s also an estimated completion column for the amount of time you plan for the task to take.

6. Track Status of Control Accounts & Work Packages

Tracking is how to know if a project is performing as planned. That’s why a WBS has control accounts and work packages. When speaking of tasks, tracking tells you multiple things: logged hours, costs, priority, new communications, the percentage complete and how its actual progress compares to your planned progress.

7. Write Notes

Having a section in which to jot down notes is always advisable. While the WBS is thorough, there might be something you need to address that doesn’t fit into its rigid structure.

8. Generate Reports

Project reports pull data from the project to illuminate its progress, overall health, costs and more. Generate a report on your WBS by using our reporting tool. Our reports summarize your project data and allow you to filter the results to show just want you want. Reports can also be shared with stakeholders.

If you’re not ready to take the plunge and use ProjectManager’s work breakdown structure software, but you’re still interested in seeing how using this tool can help you construct a sturdier plan for your next project, don’t worry. We have an intermediate step you can take.

We also have a library of free project management templates , including a free WBS template, to get you started off right.

If you decide to try out our project management software, we offer a free 30-day trial. You can upload the project work breakdown structure template into ProjectManager, and it automatically creates a new project in our software. Now you can use that template to plan, schedule, monitor and report on your project.

Because our software is cloud-based, all your data is collected and displayed in real time. This makes us different from on-premises project management software like Microsoft Project . We take your WBS and make it more dynamic with our online planning tools .

There are many ways in which you can use a work breakdown structure to help you manage work. Here are three common examples of how to use a WBS for different purposes.

Scope of Work

A scope of work is a comprehensive document that explains your project scope, which is all the work to be performed. A WBS is the perfect tool to break down the scope of a project into work packages that are easier to control. On top of that, a work breakdown structure allows you to easily identify milestones, deliverables and phases.

Statement of Work

A statement of work is a legally binding document between a client and the organization who’s responsible for executing a project. It details project management aspects such as the timeline, deliverables, requirements of the project.

A work order is similar to a statement of work, but it’s main purpose is to show the costs associated with each task. A WBS is essential for an accurate cost estimation.

As you’re working on your WBS it is helpful to maintain some best practices. Here are some things to keep in mind.

• 100% Rule: This is the most important work management principle to construct a WBS. It consists in including 100% of the work defined by the project scope, which is divided into WBS levels that contain control accounts, project deliverables, work packages and tasks. This rule applies to all the levels of the WBS, so the sum of the work at a lower WBS level must equal the 100% of the work represented by the WBS level above without exception.
• Use Nouns: WBS is about deliverables and the tasks that will lead to your final deliverable. Therefore, you’re dealing more on the what than the how. Verbs are great for action, and should be used in your descriptions, but for clarity, stick to nouns for each of the steps in your WBS.
• Be Thorough: For a WBS to do its job, there must be no holes. Everything is important if it’s part of the course that leads to your final deliverable. To manage that schedule, you need a complete listing of every task, big and small, that takes you there.
• Keep Tasks Mutually Exclusive: This simply means that there’s no reason to break out individual tasks for work that is already part of another task. If the work is covered in a task because it goes together with that task, then you don’t need to make it a separate task.
• Go Just Deep Enough: You can get crazy with subtasks on your WBS. The WBS has to be detailed, but not so deep that it becomes confusing. Ideally, think maybe three or five at most levels.

All our tools are geared to making your project more efficient and effective. See for yourself by starting your free 30-day trial of our software.

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Work Breakdown Structure Resources

• Scope of Work Template
• Gantt Chart Template
• Work Plan Template
• Work Schedule Template
• Statement of Work Template
• Work Order Template
• Project Management Trends (2022)
• How to Make a Resource Breakdown Structure
• 5 Project Management Techniques Every PM Should Know
• Cost Estimation for Projects: How to Estimate Accurately
• Sample Project Management Flow Chart
• Sample Project Plan For Your Next Project

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Surgery and Research: A Practical Approach to Managing the Research Process

Peter r. swiatek.

1 Medical Student, University of Michigan Medical School, Ann Arbor, MI, USA

Kevin C. Chung

2 Professor of Surgery, Section of Plastic Surgery, Assistant Dean for Faculty Affairs, University of Michigan Medical School, Ann Arbor, MI, USA

Elham Mahmoudi

3 Assistant Research Professor, Department of Surgery, Section of Plastic Surgery, University of Michigan Medical School, Ann Arbor, MI, USA

Following a practical project management method is essential in completing a research project on time and within budget. Although this concept is well developed in the business world, it has yet to be explored in academic surgical research. Defining and adhering to a suitable workflow would increase portability, reusability, and therefore, efficiency of the research process. In this article, we briefly review project management techniques. We specifically underline four main steps of project management: (1) definition and organization, (2) planning, (3) execution, and (4) evaluation, using practical examples from our own multidisciplinary plastic surgery research team.

Introduction

The healthcare landscape in the United States is rapidly changing. Today, with an increasing emphasis on improving access to quality care, reducing healthcare costs, and preventing surgical complications, there is an urgent need for medical professionals and surgeons to intensify their research efforts. From clinical trial studies [ 1 ] to economic analyses of different treatments, [ 2 ] new topics in surgical research range the gamut. Despite opportunities for surgeons to engage in impactful research, time constraints facing surgeons on a day-to-day basis hamper progress. Research requires a commitment to drafting literature reviews, writing grants, working with Internal Review Boards (IRB), managing study logistics, analyzing data, and writing manuscripts. Devoting bandwidth to such time-intensive processes is difficult for surgeons who spend more than 50 hours per week on surgery, patient visits, paperwork, and other administrative obligations. [ 3 ]

To effectively pursue their research goals, many surgeons choose to work with teams of clinical investigators, health economists, statisticians, and other health professionals. Although teams can augment the capability of any one surgeon in the research process, the additional responsibility of managing team dynamics and group workflow can pose significant challenges. For surgeons interested in collaborating with other researchers, there exists a set of project management principles and practices to maximize their effectiveness and efficiency in the research process. Borrowing from the realm of business and industry, project management is the combination of people, systems, and techniques required to coordinate the activities and resources needed to complete such projects within the defined parameters.[ 4 ] In addition to providing structure to workflow, project management enables principal investigators (PIs) to sense potential problems and course-correct before the problems become major setbacks or failures. [ 4 ]

Our aim in this paper is to discuss the specific systems and techniques surgeons can leverage to effectively manage the research process. Specifically, we discuss best practices related to defining and organizing the research effort, planning the project, and managing progress so that surgeons and their teams can deliver impactful results on time and within budget. Many of the management techniques discussed have been pressure-tested within our own multidisciplinary surgical research group. To ensure the generalizability of our study to surgical groups of all types and sizes, we dovetail practical examples from our group with general principles and paradigms of project management.

Projects and Teams

A project is defined as the unique set of activities intended to produce an outcome within the parameters of cost, time, and quality. [ 4 , 5 ] In surgery, projects can range from case reports, which might involve a half-day of work and minimal financial cost, to larger, more resource-intensive prospective cohort studies which can necessitate months to plan, approve, execute, and publish. [ 1 ] Every research project requires a different level of resource commitment from a PI and his or her team. It is important to highlight that different project types require research teams of different sizes, functions, and expertise. These teams, then, require various levels of management and engagement. For example, our surgical research group is unique in that we pursue a wide range of research interests within the realm of hand and plastic surgery. Thus, the composition of our research projects can vary from a team member working independently with minimal support from the research group to a team of three to six researchers from different fields of study who communicate on a regular basis. By employing some best practices in project management, we ensure that each project is completed on time and receive the needed level of oversight and attention for quality and effectiveness.

Engaging the Management Process Model

Regardless of project size and complexity, our group follows one management process model that provides the necessary structure and directionality to the project workflow. The four primary stages of the process model include (1) defining and organizing the project, (2) planning the tactical aspects of the project, (3) tracking and managing the project, and (4) evaluating outcomes ( Figure 1 ). [ 5 ]

Management Process Model

1. Define and Organize

First, a PI spearheads the establishment of the research protocol, frames the team’s organizational structure, defines team norms for communicating and collaborating, and identifies a funding source for a project. [ 5 ] In our lab, for example, a PI takes a primary role in staffing the project team based upon availability within the resource group and the specific skills and expertise needed to tackle the research project. For larger teams, the PI may designate one team member as a project lead. The project lead plays a surrogate role for the PI in managing day-today responsibilities. After recruiting a team, the PI assigns roles and responsibilities to each team member and establishes a reporting structure within the team. For larger teams, knowing “who reports to whom” is imperative for ensuring accountability and minimizing unproductive communications.

To kick-off the project, the PI communicates high-level project goals to the team, sets time, quality, and cost parameters for the project, and charges the team to devise a comprehensive research plan. For us, this communication typically occurs in person. Face-to-face interaction allows team members to better communicate their concerns and receive immediate feedback. Moreover, engaging in informal and small-group meetings helps the team members to know one another better. This is particularly helpful in meetings between surgeons and researchers from different fields. For example, just recently we were working on a project relating to studying national variation of care in thumb carpometacarpophalangeal joint (CMCJ) arthritis. By taking advantage of a one-on-one meeting and a simple drawing board, one of our hand surgeons clearly described various CMC treatment options for our team’s health economist. Open, informal, and regular communications among team members who are engaged in the same project ensures that they all understand the aims and constraints of the project from the beginning. When face-to-face meeting is not possible we find that “video conferencing” and “teleconferencing” are effective alternatives.

After onboarding the team, our PI designates one team member to manage research plan development. The research plan, which consists of objectives, research questions, hypotheses, conceptual frameworks, and methods, serves to highlight the scientific value and feasibility of carrying out research on a particular topic. In addition to providing a framework for the general research project, the research plan serves as the basis for grant and IRB applications. Although one team member can take on the entire research plan, we find that allocating workload to match the strengths, bandwidths, and developmental goals of each team member to be most effective. For example, we engage a number of medical students and research assistants to sift through published literature and data, prepare literature reviews, and write the content in many of our research plans. These students and research assistants work closely with health economists, statisticians, and surgeons, who are often engaged in several other research efforts. Leveraging the abilities of each team member early in the research process ensures timely completion of the research plan.

In Stage 2, the PI or project lead works with the team to devise a Work Breakdown Structure (WBS) of all activities and tasks necessary for executing on the project objectives ( Figure 2 ). [ 5 ] Benefits of leveraging the WBS are three-fold: (1) the WBS provides a framework for organizing scope of work; (2) it ensures that all tactical activities necessary for project completion are identified; and (3) it provides a framework for allocating resources. [ 6 ]

Work Breakdown Structure

For a surgical research project, the WBS usually consists of at least four major activities: (1) managing the funding and ethics approval process, (2) organizing study logistics and managing the execution process, (3) analyzing data, and (4) publishing results. Each major activity is disaggregated into smaller, sub-activities or tasks to which the PI can assign an “owner” who remains accountable for completion of the task. [ 7 ] In Figure 2 , for example, “Activity A: Managing Funding and Ethics Approval,” includes all tasks involved with organizing project finances and managing communications with IRB. For example, our PI has designated one team member to manage all financial transactions within our institution across all research projects. Because this process requires unique expertise, our group leverages the skills of this one team member to enhance the efficiency of the entire group. Our labs take a similar approach to managing IRB requirements. Rather than asking each team member to process his or her IRB applications individually, our lab leverages the IRB-expertise of one or two team members to effectively manage the IRB approval process for all projects.

In addition to accounting for all activities and ensuring ownership, the WBS allows the PI to pinpoint resource-costs for each activity, identify potential risks, and devise a plan from mitigating those risks. [ 7 ] With respect to the “Activity A” in Figure 2 , the PI must assess the likelihood of receiving institutional funding. If the likelihood is uncertain, the PI must determine an alternative source. In seeking ethics approval from the IRB, the PI must anticipate how delayed IRB approval could affect the timeline for the project. From our experience working with the IRB, some projects may require months of iteration with the IRB before achieving approval. Knowing this up front allows our PI to plan more downstream activities and tasks.

After identifying all activities and tasks required to complete the project, the PI can then fit the WBS into a timeline, represented by a Gantt chart ( Figure 3 ). In the chart, each activity and task is plotted on a linear calendar. The process of building the chart requires the PI to consider all interdependencies between tasks. For example, in Figure 2 , IRB approval is linked to study subject recruitment. Since the IRB approves methods for subject recruitment, the PI must wait for IRB approval before recruiting subjects. In addition to serving as a planning tool, the Gantt chart provides us with an effective way to communicate timelines to teams and relevant stakeholders. Importantly, Gantt charts can be easily created in Excel® [ 8 ] or other third-party programs like Smartsheet®, [ 9 ] transferred to an email or Microsoft PowerPoint®, and disseminated. The chart is an easy and efficient way to keep track of progress of the project, and it can be easily created and updated.

Gantt Chart

With a WBS and Gant chart in hand, the PI can begin developing activity milestones for using in-progress tracking during the execution phase. Milestones are achievement dates that stand as “guideposts” for monitoring critical completion dates within a given activity. [ 4 ] A milestone chart graphically displays all milestones for a given activity, along with a description of the milestone, scheduled deadline, actual date completed, and delay ( Figure 4 ). In the execution phase of the project, team members should regularly update columns for “Actual Date Completed” and “Delay (days)” to provide a realistic timetable for completion of the project. Additionally, team members can add specific comments each week in the “Notes” column to provide further granularity on progress made since the last update and any anticipated challenges or roadblocks ahead. Knowing what projects are delayed or are at risk of delay is imperative to PIs who may need to intervene or reallocate resources accordingly.

Milestone Chart

During Stage 3, or the Execution phase, the PI actively tracks the progress of the project. Best practices in reporting strive to minimize time and efforts required of each team member and maximize the quality of information reported. [ 10 ] Regular updating of the aforementioned milestone charts allows team members to quickly and effectively update the PI on their process ( Figure 4 ). At a gross level, the PI sees the milestones that are on track for completion along with milestones that are delayed. For additional information, the PI may refer to the notes provided by the activity owner. In our group, for example, updates are provided weekly, not only to the PI but also to our extended research group. This internal and regular socialization allows our group to leverage its diversity in expertise to the fullest capacity and stay engaged and interested in other projects being carried out by our team. Although milestone charts are generally useful tools, PIs must determine what method of progress tracking is most effective for their teams. In a small team, a simple email thread for weekly updates may suffice. For larger projects with more complex interdependencies, use of a formal reporting system (e.g., Microsoft Project®) may be necessary. [ 11 ]

In addition to establishing a method for the tracking progress, the PI must determine how all study data is gathered, stored, and shared. One of the main challenges facing data-driven research is the lack of effective data and file management. This includes inadvertently deleting, losing track of changes, duplicating, and mislabeling primary data files ( Figure 5 ). Challenges in effective data management extend to the analysis of the primary source data. Failure to track the code used in generating a given analysis or failure to update the version number for the analysis may create significant obstacles for research teams, especially if the analysis is revisited weeks or months later.

Data Management

To ensure effective data management within our research team, we all follow a practical and easy-to-use data and file management protocol, which addresses the following: (1) naming files, (2) tracking updates within files, (3) sharing files, (4) backing-up files and (5) ensuring file security ( Figure 5 ). Establishing a common file-naming convention ensures that the most up-to-date version is used by all team members. Communicating best practices in tracking updates within a file enables transferability of that file to other team members and ensures that, if the file is revisited at a later point, the user understands any modifications made to the file. For example, we name our files based on “project name” + “YYYYMMDD of the last edit” + “initials of the last team member to edit the file.” Therefore, when a folder containing multiple iterations of an individual documents or analysis is opened, the most recent version appears at either the top or bottom of the list when sorted by date.

Additionally, our workflow structure has substantially helped us avoid losing important files. We maintain copies of all files related to educational purposes (e.g., seminars, workshops, etc.), manuscripts submitted for publication, and grant proposals submitted to different agencies for funding. For each category, we have project-specific folders containing the latest versions of all files (e.g., manuscript, tables, figures, cover letter, etc.) and earlier revisions. This structure facilitates all our team members to access files as needed for different projects. For example, recently, our lab needed to develop a questionnaire for a cost-effectiveness analysis. Knowing that our research group had developed a similar questionnaire in the past, we were able to quickly search though our document database to obtain desired files and to modify them accordingly. Documenting workflow of all databases and all final files reduces redundancy, protects against data loss and makes the team more efficient, ultimately saving time and money. Having a workflow in place and following it is an investment that pays substantial dividends over time.

Furthermore, following a practical file management protocol increases reusability and therefore efficiency within a group. In our group, for example, we occasionally used to lose or misplace source codes of our data-driven projects. Instead of doing research, we wasted precious time looking for files. Then, we decided to collect all the source codes and keep them in one central location under the project name. This has helped us to (1) reuse the code for similar projects, (2) teach our new data analysts how to code, and (3) over time, expand the aims for the same project without losing time redoing initial data management. Additionally, we check that everything, including source codes, is fully annotated and can be followed by other programmer analysts. This ensures reusability and portability of source codes within our research group.

Sharing files via a “common folder” on a network server or in a secure third party “cloud” can effectively reduce risk of data and file management mishaps. Moreover, all files should be backed-up via an alternative storage device. An external hard-drive provides an easy safeguard and can be locked in a cabinet for security assurance. Diligent tracking and effective data and file management empowers the PI to successfully progress the project from execution to data analysis to publication and dissemination of findings.

4. Evaluate Outcomes and “Kaizen”

W. Edward Deming, a business consultant credited with inspiring Japan’s rise as an economic superpower after World War II, believed that success in management is dependent upon continuous improvement of systems and process. [ 12 ] Deming’s philosophy of continuous improvement, known as “Kaizen” in the Japanese workplace, champions activities that continually improve functions throughout an organization. [ 13 ] The Toyota Production System, a socio-technical system developed by the automotive giant Toyota, is one example of Kaizen put into practice. [ 14 ] The system allows Toyota employees to provide regular feedback to management regarding their work and productivity. By inviting feedback from those closest to inherent inefficiencies in a process, as might be seen on an assembly line, Toyota’s management is able to best assess potential problems and take corrective action. [ 14 ]

For busy surgeons interested in conducting medical research, the principles of project management are a powerful tool for maximizing efficacy and efficiency in the research setting. Whether working independently or teaming with a large multidisciplinary research team, PIs can leverage the management process model and management practices, such as progress tracking and data management, to drive the successful completion of their research projects. Although overt practice of project management in the realm of medical research has been limited, [ 15 ] we hope that this brief introduction to the principles and practices of project management will both encourage and enable surgeons as they strive to advance medical knowledge.

Acknowledgments

Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number 2 K24-AR053120-06 (to Dr. Kevin C. Chung). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Research Project Work Breakdown Structure Template

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Embarking on a research project can be both thrilling and overwhelming. With so many moving parts and tasks to tackle, it's crucial to stay organized and focused. That's where ClickUp's Research Project Work Breakdown Structure Template comes in handy!

The Research Project Work Breakdown Structure Template helps you break down your research project into manageable tasks, ensuring that you:

• Plan and structure your project effectively, from data collection to analysis
• Assign and track tasks, so everyone knows their responsibilities
• Stay on top of deadlines and milestones, keeping your project on track

Whether you're conducting scientific research or working on a market study, this template will help you stay organized and achieve research success—all in one place! So why wait? Dive into your next research project today with ClickUp!

Benefits of Research Project Work Breakdown Structure Template

When it comes to managing a research project, having a clear and organized plan is essential. The Research Project Work Breakdown Structure Template can help you:

• Break down complex research projects into smaller, manageable tasks
• Assign responsibilities to team members and track progress
• Ensure that all necessary research components are accounted for
• Identify potential bottlenecks or areas of concern
• Streamline communication and collaboration among team members
• Stay on track and meet project deadlines
• Improve overall project efficiency and success.

Main Elements of Research Project Work Breakdown Structure Template

ClickUp's Research Project Work Breakdown Structure template is designed to help you efficiently manage and track your research projects. Here are the main elements of this template:

• Custom Statuses: Keep track of the progress of your research tasks with 6 different statuses, including Open, Cancelled, Complete, Delayed, In Progress, and Needs Input.
• Custom Fields: Utilize 9 custom fields such as Project Phase, Allocated Budget, Consulted, Progress, Remaining Effort Hours, Responsible, Accountable, Cost Type, and Informed to capture important information about each task and ensure accurate project tracking.
• Custom Views: Access 5 different views to visualize and manage your research project, including Activities List View, Status List View, Gantt Chart View, Getting Started Guide, and Timeline View.
• Project Management: Leverage ClickUp's powerful project management features, including task dependencies, time tracking, collaboration tools, and integrations, to streamline your research project workflow and improve productivity.

How to Use Work Breakdown Structure for Research Project

Completing a research project can be a complex task, but with the help of ClickUp's Research Project Work Breakdown Structure (WBS) template, you can break it down into manageable steps. Follow these five steps to effectively use the template and stay organized throughout your research project:

1. Define your project scope

Start by clearly defining the scope of your research project. Determine the objectives, deliverables, and expected outcomes. This will help you understand the specific tasks and activities that need to be included in your WBS.

Use the Goals feature in ClickUp to outline the scope and objectives of your research project.

2. Break down the project into phases

Divide your research project into logical phases. Each phase should represent a major milestone or stage of your project. Examples of phases could include literature review, data collection, data analysis, and report writing.

Use the Board view in ClickUp to create columns for each phase and organize your tasks accordingly.

3. Identify the key tasks and activities

Within each phase, identify the key tasks and activities that need to be completed. These tasks should be specific, measurable, achievable, relevant, and time-bound (SMART). Consider the resources and time required for each task.

Create tasks in ClickUp and assign them to team members responsible for completing each task.

4. Create dependencies and set deadlines

Determine the dependencies between tasks. Some tasks may need to be completed before others can begin. Establishing these dependencies will help you plan your project timeline effectively.

Use the Gantt chart feature in ClickUp to visualize task dependencies and set realistic deadlines.

5. Monitor progress and make adjustments

Regularly monitor the progress of your research project and make adjustments as needed. Update task statuses, track time spent on each task, and communicate with your team to ensure everyone is on track.

Use the Dashboards feature in ClickUp to track the progress of your research project at a glance and identify any bottlenecks or areas that require attention.

By following these five steps and utilizing ClickUp's Research Project Work Breakdown Structure template, you can effectively plan, execute, and complete your research project with ease.

Get Started with ClickUp's Research Project Work Breakdown Structure Template

Researchers and project managers can use this Research Project Work Breakdown Structure Template to help everyone stay organized and on track when conducting research projects.

First, hit “Get Free Solution” to sign up for ClickUp and add the template to your Workspace. Make sure you designate which Space or location in your Workspace you’d like this template applied.

Next, invite relevant members or guests to your Workspace to start collaborating.

Now you can take advantage of the full potential of this template to manage your research project effectively:

• Use the Activities View to break down your project into smaller tasks and assign them to team members
• The Status View will help you track the progress of each task and keep everyone updated
• The Gantt View will provide you with a visual representation of your project timeline and dependencies
• Reference the Getting Started Guide View for an overview of the template and how to use it effectively
• Utilize the Timeline View to see a chronological view of your project and make adjustments as needed
• Organize tasks into six different statuses: Open, Cancelled, Complete, Delayed, In Progress, Needs Input, to track project milestones and progress
• Update statuses as tasks progress to keep stakeholders informed and ensure project success
• Monitor and analyze tasks to ensure maximum productivity and meet research project goals.

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How to create a work breakdown structure and why you should

Reading time: about 9 min

Rules to create a work breakdown structure

• Include 100% of the work necessary to complete the goal.
• Don't account for any amount of work twice.
• Focus on outcomes, not actions.
• A work package should take no less than 8 hours and no more than 80 hours of effort.
• Include about three levels of detail.
• Assign each work package to a specific team or individual.

It’s your first dive into the world of project management and everyone around you seems to be speaking a different language. Worse than anything, your co-workers hurl acronyms left and right, and there’s not enough time for you to look one up before a new one is casually tossed onto the field—QCD, PMBOK, ACWP, QFD, RBS, SOW, SWOT, FPIF, and WBS.

Mercifully, the deluge stops. But now you are left to ponder over your list. You start at the bottom and think to yourself, “What does WBS stand for?" 

In project management, WBS stands for work breakdown structure. This is a foundational tool that will help you to plan, manage, and evaluate large projects, so let’s learn a little bit more.

What is a work breakdown structure?

A work breakdown structure is a diagram that shows the connections between the objectives, measurable milestones, and deliverables (also referred to as work packages or tasks). The main reason for a work breakdown structure is to make a project more manageable and quantifiable by breaking up the work into smaller tasks.

Why use a WBS in project management?

There are a number of reasons why breaking down a large project is beneficial. It helps you to:

• Estimate the cost of a project.
• Establish dependencies.
• Determine a project timeline and develop a schedule.
• Write a statement of work (or SOW, one of your other acronyms).
• Assign responsibilities and clarify roles (use our roles and responsibilities template to outline duties).
• Track the progress of a project.
• Identify risk.

All of these benefits essentially arise from working with chunks of a project that you can accurately visualize rather than trying to digest and interpret a mysterious and overwhelming task in one fell swoop.

How to create a work breakdown structure

• Define the scope and objectives.  Record the overarching objective you are trying to accomplish. This objective could be anything from developing a new software feature to building a missile. Document these details in your project charter. This will be your guiding reference.
• Break it down into key phases and deliverables. Depending on the nature of your project, start dividing by project phases, specific large deliverables, or sub-tasks. Divide the overarching project into smaller and smaller pieces, but stop before you get to the point of listing out every action that must be taken. Remember to focus on concrete deliverables rather than actions.
• Organize deliverables into work packages. Break down each deliverable into all the tasks and sub-tasks required to complete them. Organize the tasks into work packages. Work packages are the lowest level of the breakdown and should define the work, duration, and costs for each task, as well task owners. Each work package should provide assignments that can be completed within a reporting period. 

Tips for making a work breakdown structure

As you make a work breakdown structure, use the following rules for best results:

• The 100% rule. The work represented by your WBS must include 100% of the work necessary to complete the overarching goal without including any extraneous or unrelated work. Also,  child tasks on any level must account for all of the work necessary to complete the parent task.
• Mutually exclusive. Do not include a sub-task twice or account for any amount of work twice. Doing so would violate the 100% rule and will result in miscalculations as you try to determine the resources necessary to complete a project.
• Outcomes, not actions. Remember to focus on deliverables and outcomes rather than actions. For example, if you were building a bike, a deliverable might be “the braking system” while actions would include “calibrate the brake pads.”
• The 8/80 rule. There are several ways to decide when a work package is small enough without being too small. This rule is one of the most common suggestions—a work package should take no less than eight hours of effort, but no more than 80. Other rules suggest no more than ten days (which is the same as 80 hours if you work full time) or no more than a standard reporting period. In other words, if you report on your work every month, a work package should take no more than a month to complete. When in doubt, apply the “if it makes sense” rule and use your best judgment.
• Three levels. Generally speaking, a WBS should include about three levels of detail. Some branches of the WBS will be more subdivided than others, but if most branches have about three levels, the scope of your project and the level of detail in your WBS are about right.
• Make assignments. Every work package should be assigned to a specific team or individual. If you have made your WBS well, there will be no work overlap so responsibilities will be clear.

Work breakdown structure example

As you are thinking about how to make a work breakdown structure, let’s look at an example. This is a work breakdown structure for building a house.

Notice how the rules of building a WBS are applied in this example. First, the house building project is subdivided into three large sections: foundation, exterior, and interior. Those sections are further subdivided into one or two more levels for a maximum of three levels. The effort needed to build a house has been allocated across all of the work packages for a total of 100% effort. There is no duplication of work represented in this diagram. To further enhance this diagram, you could add the budget for each work package and assign a team.

Work breakdown structure formats

When creating a work breakdown structure, you can choose from several different format options, such as a hierarchical table, an outline or numbered list, a tabular view, or a tree diagram. The example above uses a tree format, which is the most visual option. It structures the WBS like an org chart and shows the hierarchy of tasks, providing space for additional information about each work package.

Outline structure

A text outline is the simplest WBS format. It is easy to put together and shows the hierarchy of tasks. However, it is difficult to add additional information about budget, duration, and assignment using this format.

                    Build a House

                         1 Foundation

                              1.1 Excavate

                                   1.1.1 Dig

                                   1.1.2 Level

                              1.2 Frame

                              1.3 Concrete

                                   1.3.1 Pour

                                   1.3.2 Cure

                         2 Exterior

                         3 Interior

Hierarchical structure

This format is less visually intuitive but shows the hierarchy of tasks. Because it is a table, this format fits easily onto a page.

Tabular view

A tabular view is a more visually intuitive way to show hierarchy using a table.

WBS dictionary

What is a WBS dictionary? A WBS dictionary is formatted like the hierarchical structure, but it includes a brief description of each work package. When documenting a project, a WBS dictionary is often included in addition to a visualization of the WBS. It helps to clarify the scope of each task so that all team members understand their responsibilities.

Work breakdown structure template

To get you started, here are a number of work breakdown structure templates you can use. Simply click to open the template, and then customize the information, layout, and design.

Work breakdown structure chart

How to make a work breakdown structure in Microsoft Office

Work breakdown structures and WBS dictionaries are often included as part of a larger set of documentation or data analysis made using Microsoft Office. The best way to put a WBS into Word or Excel is to use Lucidchart and its free integrations with MS Office. Build your diagram in a software optimized for diagrams, and leave the words and data crunching to Microsoft. Just follow the steps below:

1.  Register for a free account with Lucidchart.

2. To make a work breakdown structure in Excel, install the free Lucidchart add-in for Excel .

3. Install the free Lucidchart add-in for Word to create a WBS in Word.

Get started with our work breakdown structure software.

About Lucidchart

Lucidchart, a cloud-based intelligent diagramming application, is a core component of Lucid Software's Visual Collaboration Suite. This intuitive, cloud-based solution empowers teams to collaborate in real-time to build flowcharts, mockups, UML diagrams, customer journey maps, and more. Lucidchart propels teams forward to build the future faster. Lucid is proud to serve top businesses around the world, including customers such as Google, GE, and NBC Universal, and 99% of the Fortune 500. Lucid partners with industry leaders, including Google, Atlassian, and Microsoft. Since its founding, Lucid has received numerous awards for its products, business, and workplace culture. For more information, visit lucidchart.com.

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The jet-like chromatin structure defines active secondary metabolism in fungi

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Wenyong Shao, Jingrui Wang, Yueqi Zhang, Chaofan Zhang, Jie Chen, Yun Chen, Zhangjun Fei, Zhonghua Ma, Xuepeng Sun, Chen Jiao, The jet-like chromatin structure defines active secondary metabolism in fungi, Nucleic Acids Research , Volume 52, Issue 9, 22 May 2024, Pages 4906–4921, https://doi.org/10.1093/nar/gkae131

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Eukaryotic genomes are spatially organized within the nucleus in a nonrandom manner. However, fungal genome arrangement and its function in development and adaptation remain largely unexplored. Here, we show that the high-order chromosome structure of Fusarium graminearum is sculpted by both H3K27me3 modification and ancient genome rearrangements. Active secondary metabolic gene clusters form a structure resembling chromatin jets. We demonstrate that these jet-like domains, which can propagate symmetrically for 54 kb, are prevalent in the genome and correlate with active gene transcription and histone acetylation. Deletion of GCN5 , which encodes a core and functionally conserved histone acetyltransferase, blocks the formation of the domains. Insertion of an exogenous gene within the jet-like domain significantly augments its transcription. These findings uncover an interesting link between alterations in chromatin structure and the activation of fungal secondary metabolism, which could be a general mechanism for fungi to rapidly respond to environmental cues, and highlight the utility of leveraging three-dimensional genome organization in improving gene transcription in eukaryotes.

The eukaryotic genome is packaged hierarchically into multiscale structural units ( 1 ). Chromosomes often occupy distinct subnuclear territories ( 2 ), with transcriptionally active regions located at their surface ( 3 ). Compartments, topologically associating domains (TADs) and loops are typic chromatin architectures commonly found in plants and animals ( 1 , 4 , 5 ). TADs define the boundaries of regulatory domains ( 6 ), within which promoters interact with enhancers to form loops ( 7 ) or stripes when enhancers are superactive ( 8 ). Compared with TADs, which are relatively conserved across tissues and species ( 9 , 10 ), chromatin structures are more extensively reorganized locally ( 11 ). The fungal genomes often show a Rabl-like chromosome configuration, in which centromeres cluster and chromosome arms are organized in parallel ( 12–16 ). Self-associating domains in fungi are notably shorter, with sizes scaled by gene number rather than by genomic distance ( 17 ). Unlike in animals and plants, chromatin structures and their functions in fungi remain largely unexplored.

Fungi are known to produce a vast array of secondary metabolites (SMs) that are critical for their survival and adaptation ( 18 ). Biosynthesis of SMs is regulated by a complex interplay of environmental cues such as temperature, light, humidity, pH and nutrient availability ( 19–21 ). Genes involved in secondary metabolism are rapidly induced or repressed in response to these environmental signals, leading to significant changes in SM production within a short time ( 22 ). The rapid response is likely facilitated by the presence of clustered biosynthetic genes, which are often located in proximity to regulatory elements ( 23 ). However, transcriptional co-regulation of genes does not necessarily require physical proximity on chromosomes ( 24 ), indicating that the evolution of biosynthetic gene clusters (BGCs) may provide additional advantages. It has been proposed that gene clustering can establish local chromatin environments that are more permissive for transcriptional activation or repression, thus enhancing the efficiency of gene expression controlled by transcriptional and epigenetic machinery ( 25 , 26 ). Epigenetic mechanisms, such as histone modifications, allow the fine-tuning and coordination of temporal gene expression in fungi ( 27 , 28 ). Among them, the trimethylation of histone H3 lysine 27 (H3K27me3), a conserved epigenetic mark of facultative heterochromatin and gene silencing, has been found to be enriched in BGCs in several fungal species ( 29–32 ). Deletion of histone methyltransferases responsible for H3K27me3 leads to the upregulation of genes involved in secondary metabolism and an increase in SM production ( 29 , 30 ). Interestingly, epigenetic modifications, including H3K27me3, acetylation of histone H3 lysine 27 (H3K27ac) and trimethylation of histone H3 lysine 9, have been shown to play a role in genome compartmentalization in mammals ( 33 ), and contribute to a ‘layer cake’ model of chromosome 3D organization in plants and metazoans ( 34 ). However, the impact of epigenetic modifications on the spatial organization of fungal genomes and secondary metabolism remains unclear.

To understand whether and how fungal chromosome architectures affect SM production, we focused on Fusarium graminearum , a devastating fungal pathogen causing Fusarium head blight in wheat. Fusarium   graminearum produces a number of SMs, among which the deoxynivalenol (DON) toxin poses a great threat to public health ( 35 ). Substantial studies have unveiled key biosynthetic genes ( Tri genes) and various regulators involved in DON production ( 20 ). In this study, we report for the first time that the active DON BGC forms a jet-like domain, which likely facilitates Tri gene transcription. Moreover, the jet-like domains are prevalent in the genome and correlate with active gene transcription and histone acetylation. Deletion of GCN5 , which encodes a core and functionally conserved histone acetyltransferase (HAT), results in the absence of the domains. Notably, insertion of an exogenous gene within the jet-like domain significantly enhances its transcription. Collectively, our results establish a conceptual framework for understanding the regulation of chromatin structures and secondary metabolism, and highlight the utility of genome organization in improving target gene transcription in fungi. In addition, we elucidated the regulatory role of a previously uncharacterized acetyltransferase in DON biosynthesis, providing critical information for future management of this fungal pathogen.

Strain and medium

The F . graminearum strain PH-1 was used in this study. The PH-1 was cultured in PDA (200 g potato, 20 g glucose and 18 g agar per 1 l ddH 2 O) or PDB (200 g potato and 20 g glucose per 1 l ddH 2 O) medium for morphological examination. The putrescine medium (30 g sucrose, 1 g KH 2 PO 4 , MgSO 4 ·7H 2 O, 0.5 g KCl, 0.01 g FeSO 4 ·7H 2 O and 1.5 g putrescine per 1 l ddH 2 O) and NaNO 3 medium (30 g sucrose, 1 g KH 2 PO 4 , MgSO 4 · 7H 2 O, 0.5 g KCl, 0.01 g FeSO 4 · 7H 2 O and 2 g NaNO 3 per 1 l ddH 2 O) were used to culture the fungus for DON measurement and sequencing library preparation.

qRT-PCR assay

Ten mycelial plugs, taken from the margin of PH-1 colony grown on PDA for 3 days at 25°C, were added into a 2-ml centrifuge tube containing one steel ball and 1 ml PDB. The mixture was ground using a tissue lyser and then transferred to a 250-ml triangular flask containing 200 ml of putrescine or NaNO 3 medium. The flask was incubated in a shaker at 28°C and 180 rpm for 18, 24, 30, 32, 34, 36, 38 and 40 h. After incubation, the mycelia were collected and ground into powder in liquid nitrogen. Total RNA was isolated using the RNA isolation kit (Tiangen, China). Approximately 0.8 μg of total RNA from each sample was used for complementary DNA (cDNA) synthesis with the HiScript II 1st strand cDNA synthesis kit (Vazyme, China). Gene expression was quantified using HiScipt II Q RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme) with the primer sequences listed in Supplementary Table S1 . The actin gene was used as the reference. Each experiment was repeated three times.

DON production assay

PH-1 was cultured under the condition mentioned in the quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay section. For each sample, the mycelia and nutrient solution were collected using gauze filtration. In the time-series experiments, 50 μl of the nutrient solution was taken and used to measure the concentration of DON using the DON assay kit (Weisai, China). To measure DON concentration more accurately in the gene deletion mutant, 1 ml of cell-free supernatant was subjected to filtration and subsequently purified by passing through SampliQ Amino (NH 2 ) solid-phase extraction columns (Agilent Technologies). The purified extract (4 ml) was evaporated to dryness under a nitrogen stream. The resulting residue was dissolved in 1 ml mixture of methanol and water (40:60, v/v) and subjected to centrifugation at 10 000 rpm. The final solution was then analyzed by using liquid chromatography–tandem mass spectrometry, and the concentration of DON was estimated based on the curve of standard DON solutions (Sigma–Aldrich, USA). The DON production in vitro was expressed as a ratio of DON concentration weight to mycelia dry weight (μg/g) ( 36 ). The entire experiment was repeated three times.

Transcriptome sequencing and analysis

Total RNA was extracted using the RNA isolation kit (Tiangen, China). The quantity and integrity of the RNA samples were assessed using the RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). RNA sequencing (RNA-seq) library was prepared from the high-quality RNA samples (RNA integrity number ≥8) with the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB) following the manufacturer’s instructions. Subsequently, the quality of the libraries was assessed using Qubit 2.0 and Bioanalyzer 2100, and the libraries were sequenced on an Illumina NovaSeq platform under the 2 × 150-bp mode.

The raw RNA-seq reads were processed to remove adapters and low-quality bases with Trimmomatic (v0.39) ( 37 ). The cleaned reads were mapped to the PH-1 reference genome (FungiDB release 58, https://fungidb.org/fungidb/app ) using HISAT2 (v.2.2.1) ( 38 ). Finally, the unique alignments were counted using HTSeq-count (v.0.11.3) ( 39 ) and the differentially expressed genes were identified with DESeq2 (v.1.36.0) ( 40 ) under the cutoff of adjusted P -value ≤0.05 and |log 2 (fold change)| ≥ 1. Coverage tracks of gene expression of 1-kb genomic bin were calculated using bamCoverage from the deepTools package (v3.5.0) ( 41 ) with the read coverage normalized in FPKM (fragments per kilobase of transcript per million mapped fragments).

Nuclei isolation

A total of 0.25 g of mycelia were ground into powder using a mortar and pestle in liquid nitrogen. The powder was then incubated with 4 ml lysis buffer (20 mM Tris–HCl, pH 7.5, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl 2 , 25% glycerol and 250 mM sucrose) for 10 min on ice to release the nuclei. The mixture was subsequently filtered through Miracloth to remove any cell debris, and the filtrate was centrifuged at 1500 ×  g and 4°C for 10 min. The resulting supernatant was carefully transferred to a new 1.5-ml tube and centrifuged again at 4°C and 13 000 rpm for 15 min. The sediment containing the cell nuclei was then resuspended in 4 ml of the NRBT buffer (20 mM Tris–HCl, pH 7.5, 2.5 mM MgCl 2 , 25% glycerol and 0.2% Triton X-100). The suspension was centrifuged at 1500 ×  g and 4°C for 10 min. This step was repeated four times to ensure thorough washing of the nuclei. Consequently, the NRBT buffer was removed, and the sediment was resuspended in lysis buffer for downstream experiments.

ATAC-seq library preparation and sequencing

Intact nuclei were isolated from 0.25 g of mycelia in each experiment. The nuclei were pelleted by centrifugation and resuspended in 20 μl of 1× TTBL buffer (VAHTS, TD501). The integrity and quantity of the isolated nuclei were carefully assessed prior to further experiments. The ATAC-seq libraries were prepared using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, China). In each run, ∼10 000 nuclei were subjected to Tn5 transposase treatment. The transposition reaction was performed at 37°C for 30 min. After that, DNA fragments were conjugated with adapters, followed by PCR amplification for 10–13 cycles. The DNA fragments were then size selected with VAHTS DNA Clean Beads (Vazyme, China), and the final libraries were quality assessed with Bioanalyzer 2100 and sequenced on an Illumina NovaSeq platform under the 2 × 150-bp mode.

CUT&Tag library preparation and sequencing

CUT&Tag libraries were constructed using the Hyperactive Universal CUT&Tag Assay Kit for Illumina (TD903, Vazyme, China). Briefly, the intact nuclei were isolated from 0.25 g of mycelia of each treatment and resuspended in 10 μl binding buffer with ConA beads. After incubating for 10 min at 25°C, the ConA bead–nucleus complex was pelleted by hopper magnet and resuspended in 50 μl of cold antibody buffer. Then, 2.5 μl of primary antibody of H3K4me1, H3K4me3, H3K9ac, H3K27ac (A2355, A2357, A7255 and A7253, Abclonal) and H3K27me3 (ab195477, Abcam) was added, respectively, and the reaction solution was incubated at 4°C overnight. The ConA bead–nucleus complex was pelleted again by hopper magnet and resuspended in 50 μl Dig-wash buffer containing secondary antibody (1:100 dilution, Vazyme). After incubation at 25°C for 60 min, the ConA bead–nucleus complex was pelleted and washed with 200 μl of Dig-wash buffer three times. Subsequently, the complex was resuspended in 100 μl of Dig-300 buffer containing 2 μl of pA/G-Tn5 and kept for reaction at 25°C for 60 min. After that, the beads were pelleted and washed with 200 μl of Dig-300 buffer three times. The beads were resuspended in 50 μl of Dig-300 buffer containing 10 μl of 5× TTBL and incubated at 37°C for 60 min. Then, 5 μl of proteinase K, 100 μl of buffer L/B and 20 μl of DNA extract beads were added and incubated at 55°C for 10 min. The beads were pelleted and washed with 200 μl of WA buffer once and WB buffer twice. After the beads were air dried, the DNA fragments binding on beads were eluted with 22 μl of pure water, 20 μl of which was used for library preparation. The libraries were sequenced on an Illumina NovaSeq platform under the 2 × 150-bp mode.

Data analysis of ATAC-seq and CUT&Tag sequencing

Raw reads of ATAC-seq and CUT&Tag sequencing were processed to remove adapters using Cutadapt (v.4.1) ( 42 ). The cleaned reads (≥20 bp) were aligned to the PH-1 genome using Bowtie2 (v.2.4.5) ( 43 ), and the unique alignments of ATAC-seq data were positionally shifted using alignmentSieve from the deepTools package with the parameter ‘--ATACshift’. Peaks were called using MACS2 (v2.2.7) ( 44 ) with ‘--keep-dup all’ parameter for ATAC-seq, H3K4me3, H3K9ac and H3K27ac, and ‘--keep-dup all --broad’ parameter for H3K4me1 and H3K27me3. Coverage tracks with a bin size of 50 bp were calculated using the bamCoverage and FPKM normalized data. The generated datasets were visualized by pyGenomeTracks ( 45 ).

Hi-C library preparation and data analysis

The mycelia of PH-1 were fixed in a 1% (v/v) formaldehyde solution for 30 min at room temperature. The cross-linking reaction was terminated by the addition of a glycine solution. The fixed mycelia were resuspended in 1 ml of lysis buffer and incubated on ice for 20 min. Nuclei were pelleted through centrifugation at 600 ×  g for 5 min at 4°C, followed by thorough washing with 1 ml of lysis buffer ( 46 ). Once pelleted, the nuclei were resuspended in 400 μl of a restriction enzyme buffer and transferred to a safe lock tube. The chromatin was solubilized by the addition of dilute sodium dodecyl sulfate (SDS) and incubated at 65°C for 10 min. After quenching the SDS with Triton X-100, overnight digestion was carried out using the restriction endonuclease MboI at 37°C on a rocking platform. The resulting cohesive ends were filled with a biotin marker to generate blunt ends, followed by ligation using T4 DNA ligase (New England Biolabs Inc.). DNA cross-linking was then disrupted using proteinase K (Thermo Fisher, Waltham, MA), and DNA was purified through phenol–chloroform extraction. Nonligated fragment ends carrying biotin labels were removed using T4 DNA polymerase. Subsequently, the DNA fragments were sheared by sonication to achieve a size range of 200–600 bp. The fragments were end-repaired by a mixture of T4 DNA polymerase, T4 polynucleotide kinase and Klenow DNA polymerase. Biotin-labeled DNA fragments were purified through pulldown using streptavidin magnetic beads. The DNA fragment ends were then subjected to A-tailing using Klenow DNA polymerase, followed by the addition of Illumina paired-end sequencing adapters using a ligation mix ( 47 ). Finally, the Hi-C libraries were amplified by PCR with 12–14 cycles and sequenced on an Illumina NovaSeq platform under the 2 × 150-bp mode.

Raw Hi-C reads were processed to remove adapters and low-quality sequences. The cleaned Hi-C reads were aligned to the PH-1 genome using bwa (v.0.7.17) ( 48 ) with the parameters set to ‘-A1 -B4 -E50 -L0’. The paired alignments were then processed using HiCExplorer (v.3.7.2) ( 49 ) to generate the contact matrices at the resolution of 1 kb. Hi-C matrices with different bin resolutions were generated using the HicMergeMatrixBins tool in HiCExplorer. All interaction matrices were normalized using HicNormalize, followed by Knight–Ruiz matrix balancing using hicCorrectMatrix. Principal component analysis (PCA) was performed on matrices with a bin size of 25 kb using hicPCA with the Lieberman method. Average Hi-C contacts around selected genes or peaks were computed using HicAverageRegions. Hi-C data from biological replicates were combined before downstream analysis.

Identification and analysis of the jet-like domain

The Hi-C interaction matrix was transformed into a dense format using the sparseToDense.py script from HiC-Pro ( 50 ). This matrix was used to calculate two metrics, namely the insulation score and the delta value ( 51 ), using the script matrix2insulation.pl ( https://github.com/dekkerlab/cworld-dekker ) with parameters set to ‘-is 10000 -ids 6000’. Specifically, to calculate the insulation score of each 1-kb bin, we slid a 10 kb × 10 kb (10 × 10 bins) square along the matrix diagonal and aggregated all signals within the square. The mean signal within the square was then assigned to the 1-kb diagonal bin and this procedure was then repeated for all 1-kb diagonal bins ( Supplementary Figure S1A ). The insulation score was then normalized relative to all of the insulation scores across each chromosome by calculating the log 2 ratio of each bin’s insulation score and the mean of all insulation scores. Meanwhile, the delta value, which was defined as the difference between the mean insulation scores 3 kb to the left of the central bin and 3 kb to the right of the central bin, was calculated for each 1-kb diagonal bin ( Supplementary Figure S1A ). Bins at the peak of insulation score profile with a delta value equal to zero were extracted. For these bins, we further calculated the strength of insulation by measuring the difference of delta values between the local maximum (Δ max ) and local minimum (Δ min ) of the bin and removed those with the strength value <0.3. The preserved bins represent the center of candidate jet-like domains. Typical jet-like domains contain two stripes at ∼45° (left stripe) and ∼135° (right stripe) angles off the diagonal, which represent low chromatin interactions between the focal area and the center of the domain ( Supplementary Figure S1B ). To verify the presence of the stripes for each candidate domain, we aggregated the signals in the sliding squares based on the observed/expected matrix. For the left stripe, we performed two rounds of sliding, with the first starting at the center of the domain (higher interaction region) and the second starting immediately upstream of the domain center (lower interaction region). For each round, interaction signals from 10 squares (step size = 1 kb) were calculated. We used t -test to compare the interaction intensities between two sets of sliding squares, and a P -value <0.05 was used to define the presence of the stripe in the candidate domain. Similarly, the presence of the right stripe for each domain was verified by comparing sliding squares starting at or immediately downstream of the domain center. Consequently, domains with both stripes were preserved and plotted for a final manual examination ( Supplementary Figure S1B ).

To measure the length of the jet-like domain, we used the observed/expected matrix to aggregate signals within a 10 kb × 10 kb (10 × 10 bins) square, which moves perpendicularly away from the matrix diagonal starting at the center of each domain ( 52 ). The sliding square quantifies the genomic contacts between two flanking regions at varying distances. We applied the same technique to randomly selected bins to serve as a control. It has been observed that the neighboring regions of a jet-like domain center generally have more contacts compared to the control. The length of the domain is determined by the point at which the contact intensity of the two flanking regions from the domain area decreases to the level of the contacts in the control regions.

Creation of gene deletion mutants

For gene deletion, the flanking sequences of the target gene were amplified from the genomic DNA with primers listed in Supplementary Table S1 . The two amplified fragments were fused together with a hygromycin resistance gene cassette (HPH) through a double-joint PCR reaction ( 53 ). The construct was transferred to the protoplast of PH-1 following a modified protocol ( 54 ). In brief, 1 g of mycelia were suspended in 20 ml of 0.7 M NaCl buffer containing 0.1 g driselase, 0.2 g lysing enzymes and 0.2 g snailase (Sigma, St Louis, MO, USA) and incubated for 2 h at 30°C and 85 rpm. Protoplasts were separated from cell debris with filtration through four layers of lens tissue and washed twice with NaCl buffer. Then, the protoplasts were suspended sequentially in 10 ml of STC buffer and SPTC buffer and incubated with the gene deletion construct on ice for 30 min. Transformed protoplasts were added into 20 ml of the RM medium (1 g yeast extract, 1 g casein hydrolysate and 274 g sucrose per 1 l ddH 2 O). After incubation for 12 h at 25°C and 100 rpm, all culture was mixed with 200 ml of the melt PDA medium containing 100 μg/ml hygromycin B and poured into Petri plates for incubation at 25°C under dark condition. Antibiotic-resistant colonies were transferred to the PDA plate containing hygromycin B (100 μg/ml) and used in subsequent experiments.

Virulence assay

The fungal strain was cultured in the MBL medium (a broth of 30 g mung beans boiled in 1 l of sterile water) at 25°C and 180 rpm for 5 days to induce the conidia ( 55 ). For inoculation, a 10 μl suspension of conidia (10 5 conidia/ml) was injected into a floret located in the central section of the spikelet in single flowering wheat heads of the susceptible cultivar ‘Jimai22’. In contrast, for the control the heads were inoculated with 10 μl of sterile water. The experiment consisted of 20 replicates for each strain. Following inoculation, the plants were maintained under conditions of 100% humidity at a temperature of 22 ± 2°C for 2 days, after which they were transferred to a glasshouse environment ( 56 ). Fifteen days after inoculation, the number of infected spikelets in each inoculated wheat head was recorded. The experiment was repeated twice.

Western blotting

Approximately 0.2 g of finely ground mycelia were resuspended in 1.5 ml of the isolation buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% protease inhibitor cocktail). After homogenization with a vortex shaker, the lysate was centrifuged at 10 000 × g for 20 min at 4°C. The resulting proteins were separated on a 10% SDS–PAGE (polyacrylamide gel electrophoresis) gel and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA) with a Bio-Rad electroblotting apparatus. The PVDF membrane was incubated with the TBS-T buffer (8 g NaCl, 3 g Tris, 0.2 g KCl and 1 ml Tween 20 per 1 l pure water) containing 5% non-fat milk for 1 h at 25°C, and the PVDF membrane was then washed with the TBS-T buffer three times. Next, the membrane was incubated with the primary antibody of H3K9ac, H3K18ac, H3K27ac and H3 for 1 h at 25°C, respectively. After the PVDF membrane was washed with the TBS-T buffer three times, it was incubated with secondary antibodies for 1 h at 25°C. The PVDF membrane was washed again, and the chemical signal was detected using the western blotting detection system (GE, USA). The histone H3 protein was used as the reference ( 57 ). The antibodies of H3 (M1306-4, Huaan Biotechnology), H3K9ac, H3K18ac and H3K27ac (A7255, A7257 and A7253, Abclonal) were used at a 1:500 to 1:10 000 dilution for immunoblot analyses. The experiment was repeated twice.

Other bioinformatics analyses

Gene Ontology term enrichment analysis was performed using GOATOOLS (v2.0) ( 58 ). Enrichment analysis of epigenetic modifications in A/B compartment was conducted with ChromHMM (v.1.23) ( 59 ). BGCs were predicted with antiSMASH (v.6.1.1) ( 60 ) with default parameters. CHESS (v.0.3.7) ( 61 ) was used to compare the two chromatin contact matrices at the 5 kb resolution using a window span of 100 kb and a step size of 20 kb. The 3D folding of chromosomes was reconstructed using LorDG (v.1.0) ( 62 ) with parameters ‘CONVERT_FACTOR = 0.6; LEARNING_RATE = 1.0; MAX_ITERATION = 2000’. The nucleotide diversity and recombination rate of F . graminearum were calculated based on the previously published datasets ( 63 , 64 ).

Chromosome architecture of F. graminearum is shaped by epigenetic status and ancient genome rearrangements

To investigate the 3D organization of chromosomes during DON production, we cultured F . graminearum strain PH-1 in a toxin-inducing liquid medium supplemented with putrescine, a plant defense compound capable of stimulating DON production ( 65 ). The same medium with putrescine replaced by NaNO 3 was used as a control. We set out to identify the optimal time point for sampling by monitoring biosynthetic gene expression as well as DON production in a time-series experiment ( Supplementary Figure S2 ). DON production remained at a low level in the NaNO 3 medium but elevated significantly from 34 h after incubation in the putrescine medium. Expression of DON biosynthetic genes, including Tri1 , Tri4 , Tri5 , Tri6  and Tri10 , reached the peak at 36 h, at which DON content was increased by 7.5-fold ( Supplementary Figure S2 ). Thus, mycelia at 36 h of cultivation were collected to profile transcriptomes, Hi-C-based chromosome interactions, chromatin accessibility (ATAC-seq) and histone modifications (i.e. H3K4me1, H3K4me3, H3K27me3, H3K9ac and H3K27ac). All the libraries were successfully constructed and yielded abundant high-quality and highly reproducible data for the 3D multi-omics analyses ( Supplementary Table S2 ).

The high-order structure of F . graminearum genome showed a Rabl-like configuration ( Supplementary Figure S3 ), consistent with previous observation in other fungi ( 15 ). Genome compartments were inferred based on the PCA using Hi-C contact metrics, which showed that genomic regions occupied by Polycomb proteins, as manifested by the enrichment of H3K27me3 modifications, were frequently interacted (Figure 1A ), leading to a compartmentalized genome with distinctive transcriptional activities (Figure 1B and C, and Supplementary Figure S4 ). The F . graminearum genome has evolved from an ancestral karyotype consisting of 11 or 12 chromosomes ( 66 ). We asked whether ancestral genome fissions and fusions had also influenced chromosome 3D organization in F . graminearum . To address this, we performed comparative genomic and epigenomic analyses with the early divergent relative F . oxysporum ( 67 , 68 ). Our data revealed that genomic regions exhibiting a high degree of collinearity between the two fungi were enriched with active histone modification H3K4me1/2 (Figure 1D ). Conversely, the repressive H3K27me3 marked less conserved genomic regions and subtelomeres. These regions, which were enriched with BGCs, displayed frequent recombination and showed a high level of nucleotide diversity in the population (Figure 1D ). Furthermore, we found that H3K27me3 was enriched at the breakpoints of genome rearrangements in F . graminearum . Notably, certain translocations, such as those on chromosome 4, were labeled with H3K27me3 despite being marked with active H3K4me2 in F. oxysporum (Figure 1D ). In addition, we observed a breakdown of chromosome interactions at the regions where genomic synteny was disrupted by rearrangements (Figure 1A and D), and the intensity of chromosome interactions was much stronger in the continuous syntenic regions (e.g. chromosome 4) than in the frequently rearranged regions (e.g. chromosome 3; Supplementary Figures S4 and S5 ). These results indicate that genome rearrangements likely have affected both epigenetic modifications and genome 3D organization during the evolution of F . graminearum and other Fusarium species.

Epigenetic modifications and three-dimensional structure of the F . graminearum genome. ( A ) Epigenetic modifications and compartmentalization of chromosome 1 (Chr1). The heatmap shows Pearson correlation coefficient ( r ) of Hi-C interactions on Chr1 at 25 kb resolution. Colored bar on the left represents the genome synteny between F . graminearum and F. oxysporum , and each color corresponds to a single chromosome of F. oxysporum shown in Supplementary Figure S5 , while the blank region indicates no synteny. The left arrow points to the centromere position. PCA-based compartmentalization as well as the status of gene expression and epigenetic modifications of each 25-kb window is displayed on the right. Biological replicates were combined to generate the plot. ( B ) Distribution of gene expression in A/B compartments defined by the first principal component (PC1). Expression of each gene was calculated as the averaged log 2 (FPKM + 1) of three biological replicates. ( C ) Enrichment analysis of epigenetic modifications in A/B compartments. Values indicate the fold of overrepresentation or underrepresentation of each mark in corresponding compartments. ( D ) Synteny and histone modifications of the two Fusarium genomes. Dot plot shows the synteny of the two genomes, with the syntenic fragments colored by their average K s values. The recombination rate of F . graminearum genome was calculated as the genetic distance (cM) within the 10-kb window. The nucleotide diversity ( π ) was calculated based on a window size of 10 kb. Black arrows in panels (A) and (D) indicate genomic region where recombination has occurred.

DON production is associated with local chromatin reorganization

Transcriptome analysis of F . graminearum cultured in the putrescine and NaNO 3 media revealed 1349 and 2873 genes that were preferentially expressed in the two conditions, respectively ( Supplementary Table S3 ). Upregulated genes in the putrescine medium were significantly enriched with those involved in the secondary metabolism ( Supplementary Figure S6 ), and genes in the biosynthetic clusters of DON, gramillin and butenolide displayed particularly significant expression differences (Figure 2A ). Comparison of chromatin accessibility identified by ATAC-seq showed that promoter regions of BGCs exhibited much higher levels of open accessibility in putrescine treatment, but such differences were not obvious for genes involved in other biological activities associated with SM production ( Supplementary Figure S7 ). The BGCs showed enrichment of active histone modifications of H3K9ac and H3K27ac, while being depleted of the repressive modification of H3K27me3 in the putrescine treatment ( Supplementary Figure S7 ). These findings were consistent with the expression patterns observed in the two conditions, indicating a concordance between chromatin accessibility, histone modifications and gene expression within the BGCs.

Genetic and epigenetic regulation of BGCs under DON-stimulating condition. ( A ) Differential gene expression of F . graminearum under the two culture conditions. ( B ) Physical interactions of F . graminearum genome revealed by Hi-C sequencing. Colored bars indicate the positions of centromeres. The resolution for the Hi-C map is 10 kb. ( C ) Structure changes of Chr1 and Chr2 under the two culture conditions. Similarity ( z -normalized similarity score, Z -ssim) of Hi-C data generated from the two samples was assessed by CHESS using a sliding window of 100 kb. Highly dissimilar regions ( Z -ssim ≤ −1) are indicated with the horizontal dash line. ( D ) Hi-C interaction maps of selected BGCs in putrescine (P) or NaNO 3 (N) medium. Arrows of the same color indicate the jet-like domain overlapping the BGCs. Different colors of arrows indicate the presence of more than one jet-like domain. The status of gene expression, chromatin accessibility and histone modifications was calculated from all biological replicates. The resolution for the Hi-C map is 1 kb. ( E ) Graphic illustration showing alternation of local chromatin structure of BGC and its flanking regions (150–200 kb) under the two culture conditions. The panel shows the enhanced interaction within BGC (the middle bar) and reduced interaction between BGC and its flanking regions in the putrescine condition.

The overall chromatin structure of F . graminearum in the two conditions appeared to be very similar (Figure 2B ). However, upon examination of the chromatin structure at a higher resolution, we identified several regions that exhibited significant local structural alternations (Figure 2C and Supplementary Figure S8 ). Notably, among the most prominent regions were those encoding the DON, gramillin and butenolide clusters (Figure 2C ). To further investigate the nature of these structural changes in the clusters, we zoomed in on these regions and discovered that all the three clusters were wrapped by one or more ‘V-shape’ domains in the putrescine treatment, with the most prominent case being the butenolide cluster (Figure 2D ). This ‘V-shape’ domain resembled a previously defined chromatin jet (hereafter named as jet-like domain) ( 52 ) and was not observed in the same regions when the fungus was cultured with NaNO 3 (Figure 2D ). Thus, the jet-like domain, which has not been reported previously in fungi, marks the BGCs with enhanced local chromatin interaction, elevated gene transcription and altered active/repressive epigenetic modifications (Figure 2D and E).

The jet-like domain is associated with transcription activation and histone acetylation

We identified a total of 511 and 297 jet-like domains in the genome of F . graminearum cultured in putrescine and NaNO 3 media, respectively (Figure 3A ). Chromatin interactions within the jet-like domain displayed a bubble-like structure (Figure 3A ), suggesting an enhancement of local physical interactions similar to those observed in the butenolide and other clusters (Figure 2D ). The length of bases of the jet-like domains was measured to be ∼8 kb; however, once initiated, the domain could propagate symmetrically for 54 kb in the genome (Figure 3B ). Genomic regions encompassed by these domains were characterized by low gene density but exhibited high transcriptional activity. This aligned with the increased chromatin accessibility and the enrichment of histone acetylation marks, H3K9ac and H3K27ac, as well as the promoter-specific H3K4me3 mark (Figure 3A ).

The jet-like domain is associated with active gene transcription and histone acetylation. ( A ) Characterization of the jet-like domains identified under putrescine ( n  = 511) and NaNO 3 ( n  = 297) conditions. All jet-like domains and their 40-kb upstream and downstream sequences were aggregated to show the Hi-C interactions, insulation score, gene expression, open accessibility and histone modifications. An equal number of randomly selected 80-kb genomic regions lacking jet-like domains were used as a control. ( B ) The strength of Hi-C interactions within a 10 kb × 10 kb square sliding perpendicularly away from the diagonal starting at the center of each jet-like domain or at the center of the randomly selected control regions. Arrows indicate the distance at which interaction strengths are indistinguishable between the jet-like domain and the control. This distance represents the maximum length of the jet-like domain that can propagate in certain culture conditions. ( C ) The top 10% of highly expressed genes or significant peaks of chromosome accessibility and histone modifications were selected, and Hi-C interaction maps (putrescine condition) of the 80-kb window centered at the selected genes or peaks were plotted. Equal numbers of lowly expressed genes or random non-peak regions were selected as the control. A pair of arrows on the sides and a central arrow indicate typical features of the jet-like domain. The resolution for the Hi-C maps (A and C) is 1 kb.

To further associate the domain with different genomic features, the top 10% ( n  = 1415) of genes ranked by expression level in the putrescine treatment were selected ( Supplementary Table S3 ), and chromatin interactions within an 80-kb window centered at the translation start site of each gene were plotted. We found that the jet-like domain was observed in genomic regions harboring these highly expressed genes, while other regions containing lowly expressed genes did not show this structure (Figure 3C ). Similarly, the jet-like domains were found to be present in the top 10% ( n  = 1529) open accessible regions ranked by fold changes ( Supplementary Table S4 ), while they were absent in the 1529 nonaccessible genomic regions selected at random (Figure 3C ). Following the same approach, we found that the jet-like domain was also present in the genomic regions encompassing the top 10% of significant peaks of H3K9ac ( n  = 519; Supplementary Table S5 ) and H3K27ac ( n  = 485; Supplementary Table S6 ); however, it showed no or weak correlation with the status of both active and repressive histone methylation marks, H3K4me1 (10%, n  = 347), H3K4me3 (10%, n  = 338) and H3K27me3 (10%, n  = 185) (Figure 3C and Supplementary Tables S7 – S9 ). Importantly, these patterns persisted across both the putrescine and NaNO 3 treatments ( Supplementary Figure S9 ). In summary, our findings provide compelling evidence supporting the prevalence of the jet-like domain throughout the genome of F . graminearum and establish its association with elevated level of histone acetylation and enhanced genome open accessibility, both of which are known to facilitate gene transcription.

Deletion of the HAT gene blocks the formation of the jet-like domain

Because histone acetylation influences chromatin open accessibility, we hypothesized that it plays a critical role in the formation of the jet-like domain. We focused on the HATs, which are enzymes responsible for histone acetylation. Through domain search and phylogenetic analysis, we identified 73 putative HATs in the PH-1 genome ( Supplementary Figure S10 and Supplementary Table S10 ), among which 28 genes showed differential expression between the two conditions (Figure 4A ). Consequently, our study focused on two specific HATs, namely GCN5 and 01G09073. GCN5 is a central enzyme for histone acetylation and interacts with a variety of regulatory proteins to control gene expression and nutrient adaptation ( 20 , 69 ), whereas the gene 01G09073 , located within the butenolide cluster and encoding a functionally uncharacterized homolog of yeast HPA2/3 ( Supplementary Figure S10 ), was drastically upregulated by 169-fold in the putrescine treatment (Figure 4A ). We created gene deletion mutants for both HATs, and performed RNA-seq, Hi-C sequencing and CUT&Tag sequencing of H3K9ac and H3K27ac under both putrescine and NaNO 3 conditions (Figure 4B and C, Supplementary Figure S11 and Supplementary Table S2 ). Genomic regions encoding the gramillin, butenolide and trichothecene clusters were the most significantly reorganized in the wild-type (WT) strain in the putrescine treatment (Figure 2C ). These regions also showed pronounced local reorganization in the Δ 01G09073 mutant under the two conditions, although the extent of reorganization for butenolide and trichothecene clusters seemed to be slightly attenuated compared to the WT strain (Figure 4B ). In contrast, none of the local reorganizations in the three clusters had occurred in the Δ GCN5 mutant, suggesting that GCN5 mediated local chromosome reorganization of F . graminearum in response to putrescine.

Deletion of GCN5 blocks the formation of the jet-like domain. ( A ) Genes encoding HATs that were differentially expressed between the two conditions. ( B ) Structure changes of Chr1 and Chr2 in the Δ 01G09073 and Δ GCN5 mutants under the two culture conditions. Similarity ( z -normalized similarity score, Z -ssim) of Hi-C data generated from the two samples was assessed by CHESS using a sliding window of 100 kb. Highly dissimilar regions ( Z -ssim ≤ –1) were indicated with the horizontal dash line. ( C ) Hi-C interaction maps of the butenolide cluster in fungi grown in putrescine (P) or NaNO 3 (N) medium. Arrows indicate the jet-like domain encompassing the BGC. The status of gene expression and histone modifications was calculated from all biological replicates. The resolution for the Hi-C maps is 1 kb.

Next, we revisited the genomic regions harboring the butenolide cluster, where the presence of the jet-like domain was initially observed in the WT strain under the putrescine condition (Figure 2D ). We found that the jet-like domain was absent in the WT and both mutants under the NaNO 3 culture condition; however, it became very clear when the WT and Δ 01G09073 mutant were cultured in the putrescine treatment (Figure 4C ). In contrast, the jet-like domain was completely absent in the Δ GCN5 mutant under the putrescine condition, corroborating previous observations regarding chromosomal reorganization patterns (Figure 4B ). Furthermore, gene transcription and the deposition of H3K9ac and H3K27ac within the butenolide cluster remained inducible in the Δ 01G09073 mutant in response to putrescine, whereas such inducibility was entirely abolished in the Δ GCN5 mutant (Figure 4C ). Similar patterns were also observed for the gramillin and trichothecene clusters ( Supplementary Figure S11 ). Overall, our data substantiated the involvement of GCN5 in the establishment of the jet-like domain in F . graminearum .

The jet-like domain enhances transcription of exogenous gene

The presence of the jet-like domain has sparked inquiries into its potential utility for enhancing expression of exogenous genes in future genome engineering of this fungus. The Δ 01G09073 mutant strain was considered a good system to study this, as this gene was housed within the butenolide cluster, and its replacement with hygromycin resistance gene cassette ( hph ) in the Δ 01G09073 mutant did not block the formation of the jet-like domain surrounding the cluster (Figure 4C ). We calculated the expression of hph in the Δ 01G09073 mutant under the two culture conditions, representing the presence or absence of the jet-like domain surrounding itself. We found that the expression of hph was increased by 2.5-fold ( P = 1.5E−17) in the presence of the jet-like domain ( Supplementary Table S11 ). Moreover, the growth inhibition rate for the Δ 01G09073 mutant treated with 100 μg/ml hygromycin B was decreased by 27.7% ( P < 0.01) when cultured in the putrescine medium with the jet-like domain ( Supplementary Figure S12 ). These data suggest that forced chromatin looping as exampled by the formation of the jet-like structure can reprogram the expression of exogenous genes in F . graminearum .

The HAT 01G09073 is a new regulator of DON biosynthesis

Due to the pronounced upregulation of 01G09073 in the DON-inducing medium, we embarked on further investigations to elucidate its underlying biological functions. The Δ 01G09073 mutant showed no obvious growth defect compared to the WT (Figure 5A ). However, DON production in the Δ 01G09073 mutant was decreased by ∼37% (Figure 5B ). Consistently, the mutant showed attenuated virulence on wheat in comparison to the WT (Figure 5C ). Phylogenetic analysis indicated the close relationship between 01G09073 encoding acetyltransferase and GCN5 ( Supplementary Figure S10 ). GCN5 acts as the catalytic subunit responsible for acetylating various lysine residues on different histone subunits, including H2BK11, H2BK16, H3K9, H3K14, H3K18, H3K23 and H3K27 ( 70 , 71 ). We examined the acetylation status of H3K9, H3K18 and H3K27 in the Δ 01G09073 mutant through western blotting. Our results showed that the levels of H3K9ac and H3K27ac in the mutant slightly decreased compared to the WT under the NaNO 3 treatment. In the putrescine treatment, a significant enhancement of H3K9ac and H3K27ac was observed in the WT, whereas the Δ01G09073 mutant failed to show such increase (Figure 5D ). In contrast, the level of H3K18ac remained largely unaltered in both strains and under all conditions examined.

The HAT 01G09073 is a new regulator of DON biosynthesis. ( A ) Colonies of the Δ 01G09073 and WT strains grown on the PDA medium for 3 days. ( B ) DON production in the mutant and WT strains after cultivation in the putrescine medium for 5 days. ( C ) Virulence assay of the two strains. Photos were taken 15 days after inoculation. ( D ) Western blots showing the levels of H3K9ac, H3K18ac and H3K27ac in the mutant and WT strains. Values below each gel figure are the intensities of detected protein bands relative to that of H3 band after immunoprecipitation. ( E ) Levels of H3K9ac and H3K27ac on genes relevant to different functional categories. ( F ) Levels of H3K9ac and H3K27ac on genomic regions housing gramillin, butenolide and trichothecene clusters in the Δ 01G09073 mutant and the WT strain under the putrescine (P) or NaNO 3 (N) condition. Biological replicates were combined to calculate the values.

We categorized genes based on their putative functions to explore the potential link between gene functions and the induced histone acetylation (H3K9ac and H3K27ac) mediated by 01G09073 . Interestingly, we found that deletion of 01G09073 resulted in a significant reduction in the acetylation levels of H3K9 and H3K27 within the biosynthetic clusters of gramillin, trichothecene and butenolide, as well as their associated regulatory factors (Figure 5E and F). It is worth noting that the substrate specificity and precise mechanism of action for 01G09073 are yet to be fully elucidated. Nevertheless, our findings strongly indicate that 01G09073 exerts regulatory control over the BGCs of F . graminearum through its modulation of histone acetylation, such as H3K9ac and H3K27ac. However, such regulatory effects are dependent on specific environmental conditions.

Regulation of BGCs involves intricate control at multiple levels, including transcriptional and post-transcriptional regulation ( 19 , 20 ). In this study, we demonstrate the potential regulation of 3D chromosomal architecture and BGC activity, particularly in the context of the BGC responsible for the DON toxin production (Figure 2 ). Active BGCs exhibit a jet-like domain (Figure 2 ), and this spatial arrangement likely facilitates gene transcription by enabling close proximity to transcriptional resources within the nuclear milieu. Importantly, our findings establish a direct association between the presence of the jet-like domain and histone acetylation marks (Figure 3 ), and reveal that knockout of GCN5 disrupts the formation of the jet-like domain (Figure 4 ). Intriguingly, we find that incorporation of an exogenous gene within the jet-like domain leads to a significant upregulation of its expression ( Supplementary Figure S12 ). These findings provide evidence for an additional regulatory mechanism underlying the biosynthesis and regulation of secondary metabolism in fungi. These results also underscore the potential utility of leveraging the 3D organization of chromosomes to enhance the efficiency of genome engineering in fungal species.

Euchromatin and heterochromatin typically segregate into distinct A/B nuclear compartments in metazoans. This spatial organization arises from the fact that genomic regions with similar transcriptional activities tend to engage in spatial interactions within the nucleus ( 9 , 72 ). Investigation into A/B compartments in fungi has unveiled intriguing insights into genome compartmentalization. For instance, the arbuscular mycorrhizal fungus, Rhizophagus irregularis , manifests clearly defined A/B compartments that align with transcriptional activity and repeat content ( 73 ). Conversely, other fungi show minimal chromatin compartmentalization ( 74 ). In our study, we find that genome compartmentalization of F . graminearum reflects a complex interplay of heterochromatic regions and ancient genome rearrangements (Figure 1A ). Notably, aggregation of heterochromatic regions being apart from euchromatin has been observed in numerous fungi ( 12 , 75–77 ).

The fungal genomes also have TAD-like structures ( 12 , 13 , 76 , 78 , 79 ), which span several hundreds of kilobases, and their boundaries in yeast are associated with gene regulation and DNA replication ( 78 ). Furthermore, the chromatin loops or its analogous form globules are absent in some fungi ( 74 , 47 ) but present in the fission yeast ( 12 , 80 ) and Neurospora crassa ( 76 ). In our examination of F . graminearum , we identified TAD-like domains; however, their strength was notably weaker in comparison to metazoans. Typic chromatin loops were not observed in F . graminearum . However, we discovered a jet-like domain that is prevalent in the genome of F . graminearum . Chromatin jets and analogous structures, such as plumes, fountains and ‘hinge-like’ domains, have been observed in mammals ( 52 , 81 ), worms ( 82 , 83 ) and zebrafish ( 84 , 85 ). There is evidence suggesting that these structures arise from specific cohesin loading in narrow accessible chromatin regions ( 52 ). While CTCF (CCCTC-binding factor), a blocker of cohesin, is not necessary for jet formation, it can impact cohesin looping and thus shape the domain structure ( 52 , 83 ). Consistently, genomes with or without CTCF proteins exhibit jet-like chromatin structures. The precise mechanisms driving preferential cohesin loading remain unclear. Emerging studies indicate that this targeted loading occurs at enhancers, and the absence of certain pioneer transcription factors, which establish open chromatin regions and facilitate H3K27 acetylation on enhancers, can influence the formation of the jet-like domain ( 83 ). Moreover, H3K27ac-associated BET proteins interact with cohesin motors, and thus can affect the loading of cohesin ( 86 , 87 ). In an effort to determine whether cohesin is also involved in the formation of the jet-like domain in F . graminearum , we attempted to create knockout mutants for the core cohesin subunits, including SMC1, SMC3, SCC1, SCC3, SCC2 and SCC4. However, despite examining dozens to hundreds of transformants, we were unable to obtain true mutants for these genes, implying their essential nature for this fungus. Thus, the mechanisms behind the formation of the jet-like domain in F . graminearum remain to be explored.

Enhanced histone acetylation at BGC loci is frequently correlated with active BGCs, leading to increased expression of genes within the cluster. In many fungal species, including F. graminearum , GCN5 has been implicated in the activation of BGCs through its regulatory influence on chromatin accessibility ( 20 ). However, the precise mechanisms underlying the GCN5-mediated changes in chromatin structure remain poorly characterized. Our data uncovered a pivotal role of GCN5 in the establishment of the jet-like domain in F. graminearum . This newly identified jet-like domain constitutes a paradigmatic framework for comprehending the spatial regulation of BGCs orchestrated by GCN5. In addition, we identified a new HAT that is involved in the regulation of DON biosynthesis. This gene is located within the butenolide cluster and strongly induced under the DON-stimulating conditions. Since the BGCs of butenolide and trichothecene are often co-regulated, this gene might have been co-opted to ensure the proper regulation of DON production in response to internal and external signals.

In conclusion, we provide comprehensive insights into the 3D chromosomal organization, epigenetic modifications, chromatin accessibility and gene expression dynamics in F . graminearum during DON production. The observations of the jet-like domains, their association with specific genomic features and the role of HATs highlight the complex interplay between chromatin structure, epigenetic modifications and gene regulation. These findings contribute to our understanding of the molecular mechanisms underlying fungal secondary metabolism and open up potential avenues for future genome engineering and gene expression manipulation in eukaryotes.

Raw reads generated in this study have been deposited in the National Center for Biotechnology Information BioProject database under accession no. PRJNA995191.

Supplementary Data are available at NAR Online.

We would like to thank Xiao-xiao Feng from the Agricultural Experiment Station of Zhejiang University for her assistance during the experiment.

Author contributions : C.J., Z.M. and X.S. conceived and supervised the project. W.S. performed experiments. J.W., C.Z., C.J. and X.S. analyzed the data. Y.Z., Y.C. and J.C. contributed to sample preparation and processing. C.J., X.S., W.S. and J.W. wrote the manuscript. Z.M. and Z.F. revised the paper.

National Key Research and Development Program of China [2022YFD1400100]; National Natural Science Foundation of China (NSFC) [U21A20219, 31930088 and 32102148]; Earmarked Fund for China Agriculture Research System [CARS-03-29]; National Excellent Young Scientists (overseas) Fund of NSFC (to C.J.). Funding for open access charge: National Key Research and Development Program of China [2022YFD1400100].

Conflict of interest statement . The authors declare no conflict of interest.

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