The retinoblastoma tumor suppressor (pRB) and two related proteins (P107 and P130;collectively referred to as pocket proteins) playa fundamental role in regulating proliferation, cell cycle progression, and cell cycle exit. pRB has also been shown to play an essential role in differentiation of several tissues, including muscle. Nevertheless, our understanding of the regulatory controls involving the transition from a proliferative to a quiescent and, finally, terminally differentiated state is incomplete. Our work during the previous funding period has focused on the unique roles for each of these pocket proteins in responding to growth arrest cues and in both promoting and maintaining differentiation of muscle. In addition, we have identified transcriptional and epigenetic regulatory mechanisms associated with cell cycle exit and terminal differentiation. In particular, 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 myotube-specific 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 (H3K27IDe3) 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 Ezhl, 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.

Public Health Relevance

Cancer results in some cases when cells fail to properly differentiate. Differentiation is coupled to exit from the cell cycle, and the retinoblastoma (pRB) tumor suppressor and E2F transcription factor play pivotal roles in controlling growth arrest and differentiation. pRB and associated proteins regulate these processes in part by directing chromatin modifications. Using muscle differentiation as a model, this proposal seeks 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 #
2R01GM067132-09A1
Application #
8238898
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Carter, Anthony D
Project Start
2003-02-01
Project End
2016-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
9
Fiscal Year
2012
Total Cost
$369,265
Indirect Cost
$150,765
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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Strikoudis, Alexandros; Lazaris, Charalampos; Trimarchi, Thomas et al. (2016) Regulation of transcriptional elongation in pluripotency and cell differentiation by the PHD-finger protein Phf5a. Nat Cell Biol 18:1127-1138
Yang, Yan; Li, Wencheng; Hoque, Mainul et al. (2016) PAF Complex Plays Novel Subunit-Specific Roles in Alternative Cleavage and Polyadenylation. PLoS Genet 12:e1005794
Blum, Roy (2015) Stepping inside the realm of epigenetic modifiers. Biomol Concepts 6:119-36
Cheng, Jemmie; Blum, Roy; Bowman, Christopher et al. (2014) A role for H3K4 monomethylation in gene repression and partitioning of chromatin readers. Mol Cell 53:979-92
Blum, Roy (2014) Activation of muscle enhancers by MyoD and epigenetic modifiers. J Cell Biochem 115:1855-67
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
Blum, Roy; Vethantham, Vasupradha; Bowman, Christopher et al. (2012) Genome-wide identification of enhancers in skeletal muscle: the role of MyoD1. Genes Dev 26:2763-79

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