For development of complex organisms to occur properly, the genes that determine cell type must be regulated both spatially and temporally. One key aspect of this regulation is the effective lineage-specific repression of master regulatory genes that must be maintained in an ?off? status. This problem has been studied since the 1940s in Drosophila, which has led to the identification of a group of genes called the Polycomb- Group (PcG) after the founding member, the Polycomb gene. The PcG encodes proteins that form complexes that direct a repressed state. The central repressive complex in this family is called Polycomb Repressive Complex 1 (PRC1), and a second complex that methylates histones and helps target PcG function is called PRC2. PRC1 and PRC2 are targeted to appropriate locations by Polycomb Response Elements (PREs), which are usually about 1 kb in length and nucleosome depleted. This application describes experiments designed to understand function of the PcG system, with a focus on the role of nucleosome compaction, PRE function and the ability of non-coding RNAs to regulate this system. Three areas are addressed in three specific Aims in this application.
Aim 1 examines the ability of a mammalian Polycomb protein called Cbx2 to compact nucleosomes in vitro and in cell culture. It also describes approaches to crystallize proteins involved in creating a compacted state with the goal of defining these mechanisms for Polycomb at this high level of resolution.
Aim 2 explores the location and function of PREs in the human and mouse HOX clusters. Nucleosome depletion is used as one method to identify potential PRE sequences, which are then tested for function using reporter constructs. The hypothesis that the Jarid2 protein is generally involved in mammalian PRE function is tested using these PRE sequences.
Aim 3 describes the development of a new technology to map the binding sites for ncRNAs. This technology will be used to test the hypothesis that specific ncRNAs are involved in targeting PcG function to specific loci.

Public Health Relevance

We describe experiments to characterize the several aspects of the complex gene system, called the Polycomb-Group, that silences genes to maintain proper cell identity in mammals. We will characterize the protein domain and the molecular mechanism that creates a compacted chromatin structure perhaps causal to silencing, will identify DNA elements that target this system to specific genes, and will develop techniques to determine where non-coding RNAs involved in regulation function on the genome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM043901-21A1
Application #
8371445
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
1991-05-01
Project End
2016-06-30
Budget Start
2012-08-01
Budget End
2013-06-30
Support Year
21
Fiscal Year
2012
Total Cost
$660,703
Indirect Cost
$281,385
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
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Mieczkowski, Jakub; Cook, April; Bowman, Sarah K et al. (2016) MNase titration reveals differences between nucleosome occupancy and chromatin accessibility. Nat Commun 7:11485
Deaton, Aimee M; Gómez-Rodríguez, Mariluz; Mieczkowski, Jakub et al. (2016) Enhancer regions show high histone H3.3 turnover that changes during differentiation. Elife 5:

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