The intricate programs of mammalian cell differentiation ultimately target chromatin, formulating chromatin environments accessible to or deflective of the transcriptional machinery and thus conducive to distinct gene expression profiles. How these chromatin structures are first established to set the transcription program and how these established structures are then re-instated on newly replicated DNA during cell division is the crux of epigenetics. Based on preliminary findings obtained during the previous funding period, we will comprehensively explore two major modulators of epigenetic information that we have investigated extensively: PRC1 and PRC2, and a third potential epigenetic modulator, CTCF. We will continue our investigation of the molecular basis of their specificity in targeting discrete regions of the genome, the dynamics controlling their activity, and how their activities convey appropriate transcription outputs. Two mammalian complexes that comprise Polycomb Group proteins, PRC1 and PRC2 are recognized epigenetic conveyers of transcriptional repression. PRC1 transmits repression through catalysis of monoubiquitination of histone H2A at lysine 119 and chromatin compaction. Through our extensive biochemical analyses, we demonstrated that PRC1 embodies discrete yet heterogenous complexes.
In aim 1, we expand on our preliminary results showing that the distinguishing proteins for some of the PRC1 subgroups either convert PRC1 into a transcriptional activator, as in the case of the neuronal-specific protein, AUTS2, or exhibit cell-type specificity and a possible novel repressive mechanism as in the case of FBRSL1 in cardiomyocytes, or modulate PRC1 recruitment to chromatin as in the case of motor neuron enriched YAF1. The underlying mechanistic basis and the outcome to specific gene expression will be studied using biochemical and genomic approaches, respectively and with mouse models in the case of AUTS2 and FBRSL1.
In aim 2, we continue our extensive analyses of PRC2 that catalyses methylation of histone H3 at lysine 27, a modification of repressive chromatin. We explore parameters regulating PRC2 activity including post-translational modifications of its catalytic subunit Ezh2, and the negative effects of naturally occurring, dominant negative histone mutants using biochemical analyses and CRISPR technology. With the foundation of our studies of interactions of long noncoding RNA with both Ezh2 and the PRC2 associated protein Jarid2, we expand into the role of these interactions in mediating specificity in PRC2 recruitment to chromatin.
In aim 3, we pursue our preliminary results of RNA-mediated CTCF multimerization and its possible role in CTCF- mediated regulation of chromatin boundaries within the HOX gene cluster using ChIP-seq, sequence capture Hi-C and ChIA-PET technologies.

Public Health Relevance

The establishment, maintenance, and dynamic regulation of distinct gene expression patterns originating from the same genome is central to cellular identity in multicellular organisms and alterations in these regulatory pathways underlie a number of diseases, including cancer. Genetic information is packaged into a structure called `chromatin', which has key roles in regulating gene expression. The aim of this proposal is to understand how particular chromatin domains are established within a cell, propagated during cell division, and altered during development, and how these processes regulate transcription, the most fundamental step in gene expression.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA199652-16
Application #
9555806
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Mietz, Judy
Project Start
2002-01-01
Project End
2020-07-31
Budget Start
2018-08-01
Budget End
2019-07-31
Support Year
16
Fiscal Year
2018
Total Cost
Indirect Cost
Name
New York University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
Stafford, James M; Lee, Chul-Hwan; Voigt, Philipp et al. (2018) Multiple modes of PRC2 inhibition elicit global chromatin alterations in H3K27M pediatric glioma. Sci Adv 4:eaau5935
Lee, Chul-Hwan; Holder, Marlene; Grau, Daniel et al. (2018) Distinct Stimulatory Mechanisms Regulate the Catalytic Activity of Polycomb Repressive Complex 2. Mol Cell 70:435-448.e5
Lee, Chul-Hwan; Yu, Jia-Ray; Kumar, Sunil et al. (2018) Allosteric Activation Dictates PRC2 Activity Independent of Its Recruitment to Chromatin. Mol Cell 70:422-434.e6
Krishnan, Swathi; Smits, Arne H; Vermeulen, Michiel et al. (2017) Phospho-H1 Decorates the Inter-chromatid Axis and Is Evicted along with Shugoshin by SET during Mitosis. Mol Cell 67:579-593.e6
Narendra, Varun; Bulaji?, Milica; Dekker, Job et al. (2016) CTCF-mediated topological boundaries during development foster appropriate gene regulation. Genes Dev 30:2657-2662
Justin, Neil; Zhang, Ying; Tarricone, Cataldo et al. (2016) Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2. Nat Commun 7:11316
Campos, Eric I; Smits, Arne H; Kang, Young-Hoon et al. (2015) Analysis of the Histone H3.1 Interactome: A Suitable Chaperone for the Right Event. Mol Cell 60:697-709