The goal of this research is to define chromatin mechanisms that control gene expression during development. Molecular, genetic, genomic, and biochemical methods will be used to study the Polycomb group (PcG) transcriptional repressors of Drosophila, which provide a premier model for revealing chromatin mechanisms in a developing organism. PcG silencing is performed by multiprotein complexes that selectively occupy genomic sites. The constituents and activities of two PcG complexes, called Polycomb repressive complex 1 (PRC1) and PRC2, are the best-defined. The focus of this work is on PRC2, which is a chromatin- modifying enzyme that methylates histone H3 on lysine 27 (K27). The trimethylated product, H3-K27me3, is a hallmark of transcriptionally silenced chromatin in genomes of higher eukaryotes. The PcG proteins, and the chromatin complexes they form, are highly conserved from flies to humans. Human PcG proteins play key roles in the transcriptional circuitry that controls pluripotency and differentiation of embryonic stem cells. They are also central regulators in adult tissue-specific stem cells, such as in skin, muscle and blood. Overabundance or hyperactivity of PcG proteins is implicated in leukemias and cancers of the breast, prostate, and other tissues. Their expanding importance in stem cell biology and cancer epigenetics underscores the need to understand basic PcG chromatin mechanisms. The main goals of this work are to determine mechanisms of PRC2 function and molecular roles of H3-K27 methylation in gene silencing. PRC2 has four core subunits, three of which are required for histone methyltransferase activity. The subunits contain key regulatory modules, including binding sites that detect chromatin features, that profoundly influence PRC2 activity.
One Aim will determine how critical subunit elements, located outside the catalytic center, control PRC2 function in vitro and in vivo.
A second Aim defines in vivo consequences of histone methylation at normal sites of PcG silencing and at naive sites not normally impacted by PcG machinery. The methods include loss-of-function and over-expression studies, site-directed mutagenesis, transgene manipulation, chromatin immuneprecipitation, protein purification, enzyme assays, chromosome immunostaining, and targeted genomic modifications. Fulfillment of these Aims should advance knowledge of basic PcG mechanisms in gene silencing and also of epigenetic processes that control human stem cell fates and that underlie certain human cancers.

Public Health Relevance

This research is to determine how a set of highly conserved regulatory proteins, called Polycomb group (PcG) proteins, turn genes off during animal development. In humans, PcG proteins are critical regulators in embryonic stem cells and adult tissue stem cells and they are implicated in breast cancer, prostate cancer, leukemias, and cancers of other tissues. This research will advance basic understanding of gene regulatory mechanisms and provide knowledge that could impact stem cell applications in medicine and development of anti-cancer strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM049850-17
Application #
8650589
Study Section
Special Emphasis Panel (ZRG1-GGG-L (02))
Program Officer
Carter, Anthony D
Project Start
1996-08-01
Project End
2017-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
17
Fiscal Year
2014
Total Cost
$339,217
Indirect Cost
$116,048
Name
University of Minnesota Twin Cities
Department
Genetics
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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Simon, Jeffrey A; Kingston, Robert E (2013) Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put. Mol Cell 49:808-24
O'Meara, M Maggie; Simon, Jeffrey A (2012) Inner workings and regulatory inputs that control Polycomb repressive complex 2. Chromosoma 121:221-34
Smith, Matthew; Mallin, Daniel R; Simon, Jeffrey A et al. (2011) Small ubiquitin-like modifier (SUMO) conjugation impedes transcriptional silencing by the polycomb group repressor Sex Comb on Midleg. J Biol Chem 286:11391-400
Wang, Liangjun; Jahren, Neal; Miller, Ellen L et al. (2010) Comparative analysis of chromatin binding by Sex Comb on Midleg (SCM) and other polycomb group repressors at a Drosophila Hox gene. Mol Cell Biol 30:2584-93
Chen, Shuai; Bohrer, Laura R; Rai, Aswathy N et al. (2010) Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2. Nat Cell Biol 12:1108-14
Zhu, Changqi C; Bornemann, Douglas J; Zhitomirsky, David et al. (2008) Drosophila histone deacetylase-3 controls imaginal disc size through suppression of apoptosis. PLoS Genet 4:e1000009
Wang, Liangjun; Jahren, Neal; Vargas, Marcus L et al. (2006) Alternative ESC and ESC-like subunits of a polycomb group histone methyltransferase complex are differentially deployed during Drosophila development. Mol Cell Biol 26:2637-47
Ketel, Carrie S; Andersen, Erica F; Vargas, Marcus L et al. (2005) Subunit contributions to histone methyltransferase activities of fly and worm polycomb group complexes. Mol Cell Biol 25:6857-68
Fang, Jia; Feng, Qin; Ketel, Carrie S et al. (2002) Purification and functional characterization of SET8, a nucleosomal histone H4-lysine 20-specific methyltransferase. Curr Biol 12:1086-99

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