The goal of this research is to understand chromatin mechanisms that maintain gene expression states during development. Molecular, genetic and biochemical methods will be used to study the Polycomb group (PcG) transcriptional repressors of Drosophila, which provide one of the premier models for deciphering chromatin mechanisms in development. PcG proteins control selective expression of homeotic (Hox) genes along the anterior-posterior (A-P) axis which, in turn, program differentiation of body structures. For normal body patterning to occur, Hox genes must be kept silent in A-P positions where they are not normally expressed. Hox expression patterns are set by segmentation gene products in 2-hour-old fly embryos, but these initial regulators decay by about 4 hours. The Polycomb group proteins then assume control to maintain Hox gene silencing during the rest of development. Thus, PcG proteins provide molecular memory of spatial cues present in early embryos. Current models suggest that PcG silencing is maintained through covalent histone modification and stable association of PcG protein complexes in the local chromatin. The PcG proteins, and the chromatin complexes they form, are highly conserved from flies to humans. Human PcG proteins play critical roles to maintain pluripotency of embryonic stem cells. Overabundance of PcG proteins is also implicated in disease progression in 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. This project will determine PcG mechanisms using Drosophila, which provides one of the best-characterized systems for in vivo investigation of PcG silencing. This research will investigate molecular roles of PcG complexes and their subunits. Much of the work focuses on a PcG complex called PRC2 (Polycomb repressive complex 2). PRC2 has four core subunits and an enzyme activity that methylates histone H3 on lysine-27.
One Aim i s to determine how the noncatalytic subunits make key inputs to PRC2 function in vitro and in vivo.
A second Aim addresses in vivo consequences of histone methylation and whether PRC2 has any function besides enzyme activity.
A third Aim i s to define the molecular role of another critical PcG repressor, called Sex comb on midleg (SCM), which appears to work independently of PRC2. The methods will include loss-of-function and over-expression studies, site-directed mutagenesis, transgene manipulation, chromatin immuneprecipitation, enzyme assays, protein purification and chromosome immunostaining. Fulfillment of these Aims should advance knowledge of basic PcG mechanisms 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, keep genes turned off during animal development. In humans, PcG proteins are critical for embryonic stem cell maintenance and they are implicated in breast cancer, prostate cancer, 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 #
5R01GM049850-16
Application #
8303402
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
1996-08-01
Project End
2013-12-31
Budget Start
2012-08-01
Budget End
2013-12-31
Support Year
16
Fiscal Year
2012
Total Cost
$315,428
Indirect Cost
$99,806
Name
University of Minnesota Twin Cities
Department
Genetics
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Herzog, Veronika A; Lempradl, Adelheid; Trupke, Johanna et al. (2014) A strand-specific switch in noncoding transcription switches the function of a Polycomb/Trithorax response element. Nat Genet 46:973-81
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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