The packaging of DNA as chromatin regulates many important cellular processes that require access to the cell's genetic material. One major class of enzymes responsible for regulating the structure of chromatin is the ATP-dependent chromatin remodelers. These enzymes play essential roles in a variety of biological processes ranging from DNA replication, repair, recombination and transcription to the regulation of cell fate decisions. Dysfunction of chromatin remodeling enzymes can cause a variety of cancers, such as breast, lung, pancreatic, prostate, and rhabdoid cancers, as well as a number of multisystem developmental disorders. Dissecting the functional roles of chromatin remodelers and developing novel therapies to combat diseases related to remodeler dysfunction require a mechanistic understanding of the biochemical and biophysical principles underlying chromatin remodeling. Our long-term goal is to develop a detailed mechanistic understanding of how chromatin remodeling enzymes use the free energy of ATP hydrolysis to disrupt histone- DNA contacts and alter the position, structure, and composition of nucleosomes. In this project, we will focus on investigating the remodeling mechanisms of the ISWI and SWI/SNF family remodelers. Determining the mechanisms of chromatin remodeling requires quantitative characterizations of the dynamics of the remodeling reaction. Single-molecule techniques are well suited for this purpose as they allow us to monitor complex molecular processes in real time, directly observe intermediate states, and dissect reaction pathways. In this project, we will use single-molecule fluorescence resonance energy transfer, in conjunction with complementary biochemical assays, to study the mechanisms of chromatin remodeling and its regulation. We will address three specific aims.
Aim 1 : We will investigate the nucleosome remodeling dynamics catalyzed by ISWI family remodelers. In particular, we will determine the structural dynamics of the nucleosome during remodeling and probe how remodeling actions at different nucleosomal sites are coordinated.
We aim to test different mechanistic models and advance our understanding of how ISWI family enzymes translocate nucleosomes along DNA.
Aim 2 : The activity of ATP-dependent chromatin remodelers is under intricate regulation by a variety of factors. In this aim, we plan to study how several biologically relevant nucleosomal features, including DNA linker length and histone modifications/variants, regulate ISWI remodeling activity.
Aim 3 : While remodeling enzymes from different families share a homologous ATPase domain, they display different remodeling activities and regulate different biological processes.
In Aim 3, we will extend our studies to SWI/SNF family remodelers and compare them with the ISWI family, aiming to identify key commonalities and differences in the nucleosome remodeling mechanisms used by these two major families of chromatin remodelers.

Public Health Relevance

In this project, we will study the nucleosome remodeling mechanisms of ATP-dependent chromatin remodeling enzymes. The significance of this project lies in the development of powerful, single-molecule methodologies for investigating chromatin remodeling and in the derivation of critical mechanistic insights that are difficult to obtain previously. The improved mechanistic understanding of chromatin remodeling may shed light on the roles of chromatin remodelers in development and physiology as well as the links between remodeler dysfunction and diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM105637-01
Application #
8478864
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Lewis, Catherine D
Project Start
2013-08-01
Project End
2017-03-31
Budget Start
2013-08-01
Budget End
2014-03-31
Support Year
1
Fiscal Year
2013
Total Cost
$314,194
Indirect Cost
$124,194
Name
Harvard University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Hwang, William L; Deindl, Sebastian; Harada, Bryan T et al. (2014) Histone H4 tail mediates allosteric regulation of nucleosome remodelling by linker DNA. Nature 512:213-7