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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics B Study Section (MGB)
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Carter, Anthony D
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Massachusetts General Hospital
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Kundu, Sharmistha; Ji, Fei; Sunwoo, Hongjae et al. (2018) Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation. Mol Cell 71:191
Tchasovnikarova, Iva A; Kingston, Robert E (2018) Beyond the Histone Code: A Physical Map of Chromatin States. Mol Cell 69:5-7
Jaensch, Elizabeth S; Kundu, Sharmistha; Kingston, Robert E (2017) Multitasking by Polycomb response elements. Genes Dev 31:1069-1072
Mueller, Britta; Mieczkowski, Jakub; Kundu, Sharmistha et al. (2017) Widespread changes in nucleosome accessibility without changes in nucleosome occupancy during a rapid transcriptional induction. Genes Dev 31:451-462
Tchasovnikarova, Iva A; Timms, Richard T; Douse, Christopher H et al. (2017) Hyperactivation of HUSH complex function by Charcot-Marie-Tooth disease mutation in MORC2. Nat Genet 49:1035-1044
Lau, Mei Sheng; Schwartz, Matthew G; Kundu, Sharmistha et al. (2017) Mutation of a nucleosome compaction region disrupts Polycomb-mediated axial patterning. Science 355:1081-1084
Kundu, Sharmistha; Ji, Fei; Sunwoo, Hongjae et al. (2017) Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation. Mol Cell 65:432-446.e5
Ardehali, M Behfar; Anselmo, Anthony; Cochrane, Jesse C et al. (2017) Polycomb Repressive Complex 2 Methylates Elongin A to Regulate Transcription. Mol Cell 68:872-884.e6
Ray, Mridula K; Wiskow, Ole; King, Matthew J et al. (2016) CAT7 and cat7l Long Non-coding RNAs Tune Polycomb Repressive Complex 1 Function during Human and Zebrafish Development. J Biol Chem 291:19558-72
Wani, Ajazul H; Boettiger, Alistair N; Schorderet, Patrick et al. (2016) Chromatin topology is coupled to Polycomb group protein subnuclear organization. Nat Commun 7:10291

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