Eukaryotic cells have solved the genome-packaging problem combining chromatin, the nucleoprotein fiber consisting of DNA and histones, with factors that modify it and regulate its dynamics. Among them are the ATP- dependent chromatin remodeling complexes or "remodelers", large and conserved multi-subunit assemblies that use ATP hydrolysis to non-covalently alter nucleosome structure. Remodelers can be classified into four families and differ both in composition and the products they generate both in vivo and in vitro. What is the mechanism of chromatin remodeling? How are different products generated? These questions remain unanswered due, to a large extent, to a combination of the remodelers'complexity and a paucity of structural information to provide a context for the accumulated biochemical and genetic data. Current mechanistic models are difficult, if not impossible, to test with biochemical approaches alone. Cryo-electron microscopy (Cryo-EM) is ideally suited for the analysis of remodelers and of what appears to be their intrinsic conformational flexibility. While the high resolution required to answer many mechanistic questions is a challenging long-term goal, more easily achievable lower resolution structures can provide important constraints to our modeling as well as insights into remodeler specialization. We will apply our expertise in electron microscopy of large, asymmetric and heterogeneous macromolecular assemblies to begin a comparative structural study of two remodelers with very different activities: histone octamer sliding and histone dimer exchange. Our model systems, both ~1MDa, are the S. cerevisiae remodelers RSC, which slides octamers, and SWR1, which exchanges H2A.Z/H2B dimers for the native H2A/H2B.
In Aim 1 we will obtain reconstructions of the SWR1 complex both by itself and bound to a nucleosome.
In Aim 2 we will obtain the structure of a remodeling-competent 4-subunit subcomplex of RSC (RSCsub) bound to a nucleosome and will refine our current RSC structure to higher resolution with frozen-hydrated samples.
In Aim 3 we will extract biological information from the structures obtained in Aims 1 and 2 by mapping the location of a few key subunits in SWR1 and RSC/RSCsub. We have selected targets that will maximize the mechanistic insight we can gain even at the medium-resolution we expect to obtain within this funding period. The targets for RSC are also designed to allow us to dock the RSCsub-nucleosome structure into full RSC. An important component of Aim 3 is the development of novel methods for labeling subunits in frozen-hydrated samples.

Public Health Relevance

ATP-dependent chromatin remodeling complexes are large macromolecular assemblies capable of altering the structure of nucleosomes, the packaging units of DNA, to regulate DNA accessibility.
We aim to understand, at the molecular level, how these complexes interact with and remodel nucleosomes. Our research could lead to new insights in the fields of epigenetics and cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM092895-03
Application #
8449304
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Carter, Anthony D
Project Start
2011-04-01
Project End
2016-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
3
Fiscal Year
2013
Total Cost
$309,047
Indirect Cost
$126,179
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138