We have begun a systematic investigation of all chromatin modifications associated with myogenic differentiation using a combination of ChIP-sequencing (ChIP-seq) and expression profiling. Here, we propose the following aims to further dissect the mechanisms underlying pocket protein involvement in cell cycle exit and differentiation. In our first Aim, we will examine gene regulation during myogenic differentiation by specifically focusing on the role of histone H2B ubiquitylation (H2BUb) during differentiation. We will examine the extent of histone cross-talk in myotubes and investigate the impact of ablating RNF20, the enzyme responsible for H2B ubiquitylation, on differentiation. Remarkably, we have found that H2BUb essentially disappears during differentiation, and therefore we will examine the underlying mechanistic basis for myotubespecific loss of H2Bub. In our second Aim, we will examine the genome-wide role of pRB and co-repressors in directing chromatin modifications. We will first perform ChIP-seq on pRB and p130 in differentiated myotubes, enabling us to identify regions bound by these factors. We will also perform expression profiling after removal of pRB or p130 from myotubes. By merging these data, we will determine how chromatin modifications and gene expression are affected by loss of pocket proteins. Using our extensive ChIPseq data as a guide, we will test the hypothesis that pRB directs tri-methylation of H3K27 (H3K27me3) and ask whether Polycomb repressive complex (PRC2) is involved and to what extent. We will examine whether Ezh2 or an alternative histone methyltransferase (HMT), such as Ezh1, may be involved in H3K27me3 deposition. We will determine whether pRB influences the recruitment of either or both HMTs. The acquisition of our extensive ChIP-seq dataset allows us an unprecedented ability to examine on a genome-wide scale the relationship between pRB binding, chromatin modifications, and gene expression in a developmentally relevant setting, thereby enhancing our understanding of regulatory controls that are essential for both reversible and permanent gene silencing, withdrawal from the cell cycle, and muscle differentiation. Progress on both Aims was impacted by Hurricane Sandy. Funds are requested to continue the Aims listed above.

Public Health Relevance

Differentiation is coupled to exit from the cell cycle, and the retinoblastoma (pRB) tumor suppressor plays pivotal roles in controlling growth arrest and differentiation. pRB and associated proteins regulate these processes in part by directing chromatin modifications. We request funds to extend work under the parent proposal, delayed by Hurricane Sandy, in which we seek to understand the underlying pRB-dependent and pRB-independent mechanisms that drive chromatin modifications, regulate gene expression, and thereby control the decision to permanently stop dividing and to terminally differentiate.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM067132-10S1
Application #
8665617
Study Section
Program Officer
Carter, Anthony D
Project Start
2014-01-01
Project End
2015-12-31
Budget Start
2014-01-01
Budget End
2015-12-31
Support Year
10
Fiscal Year
2014
Total Cost
$93,728
Indirect Cost
$38,431
Name
New York University
Department
Pathology
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
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Bowman, Christopher John; Ayer, Donald E; Dynlacht, Brian David (2014) Foxk proteins repress the initiation of starvation-induced atrophy and autophagy programs. Nat Cell Biol 16:1202-14
Blum, Roy; Dynlacht, Brian D (2013) The role of MyoD1 and histone modifications in the activation of muscle enhancers. Epigenetics 8:778-84
Vethantham, Vasupradha; Yang, Yan; Bowman, Christopher et al. (2012) Dynamic loss of H2B ubiquitylation without corresponding changes in H3K4 trimethylation during myogenic differentiation. Mol Cell Biol 32:1044-55
Micsinai, Mariann; Parisi, Fabio; Strino, Francesco et al. (2012) Picking ChIP-seq peak detectors for analyzing chromatin modification experiments. Nucleic Acids Res 40:e70
Asp, Patrik; Blum, Roy; Vethantham, Vasupradha et al. (2011) Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc Natl Acad Sci U S A 108:E149-58
van Oevelen, Chris; Bowman, Christopher; Pellegrino, Jessica et al. (2010) The mammalian Sin3 proteins are required for muscle development and sarcomere specification. Mol Cell Biol 30:5686-97
Tsikitis, Mary; Acosta-Alvear, Diego; Blais, Alexandre et al. (2010) Traf7, a MyoD1 transcriptional target, regulates nuclear factor-?B activity during myogenesis. EMBO Rep 11:969-76
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Blais, Alexandre; van Oevelen, Chris J C; Margueron, Raphael et al. (2007) Retinoblastoma tumor suppressor protein-dependent methylation of histone H3 lysine 27 is associated with irreversible cell cycle exit. J Cell Biol 179:1399-412

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