The long-term goal of this work is to understand in precise molecular terms how large DNA domains are packaged into an inactive chromatin structure called heterochromatin or silent chromatin. This type of chromatin plays a central role in the regulation of gene expression and maintenance of chromosome stability in organisms ranging from yeast to human. Studies in yeast have identified several gene products that are involved in the assembly of silent chromatin, and the types of complexes that these gene products form have been characterized. We have recently shown that a conserved component of silent chromatin, the Sir2 protein, has a functionally essential ADP-ribosyltransferase activity. This proposal will use purified silencing complexes, together with a chromatin assembly system, to reconstitute silent chromosome domains in vitro. This system will then provide the basis for biochemical experiments to deduce molecular mechanisms and the significance of the enzymatic activity of Sir2 in gene silencing. Silent chromatin is a highly conserved feature of eukaryotic chromosomes. It therefore seems likely that many of the principles developed for the yeast proteins covered in this proposal will apply to other settings. A basic understanding of the molecular events that control chromosome structure and ensure the stable inheritance of cell fate provides not only a frame work for understanding how the process can fail, but also provides substrates and knowledge to design therapeutic strategies based on intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061641-02
Application #
6387198
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Carter, Anthony D
Project Start
2000-07-01
Project End
2005-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
2
Fiscal Year
2001
Total Cost
$249,400
Indirect Cost
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
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Wang, Feng; Li, Geng; Altaf, Mohammed et al. (2013) Heterochromatin protein Sir3 induces contacts between the amino terminus of histone H4 and nucleosomal DNA. Proc Natl Acad Sci U S A 110:8495-500
Tung, Shu-Yun; Hong, Jia-Yang; Walz, Thomas et al. (2012) Chromatin affinity-precipitation using a small metabolic molecule: its application to analysis of O-acetyl-ADP-ribose. Cell Mol Life Sci 69:641-50
Moazed, Danesh (2011) Mechanisms for the inheritance of chromatin states. Cell 146:510-8
Halic, Mario; Moazed, Danesh (2010) Dicer-independent primal RNAs trigger RNAi and heterochromatin formation. Cell 140:504-16
Mekhail, Karim; Moazed, Danesh (2010) The nuclear envelope in genome organization, expression and stability. Nat Rev Mol Cell Biol 11:317-28
Gerace, Erica L; Halic, Mario; Moazed, Danesh (2010) The methyltransferase activity of Clr4Suv39h triggers RNAi independently of histone H3K9 methylation. Mol Cell 39:360-72
Sinha, Manisha; Watanabe, Shinya; Johnson, Aaron et al. (2009) Recombinational repair within heterochromatin requires ATP-dependent chromatin remodeling. Cell 138:1109-21
Johnson, Aaron; Li, Geng; Sikorski, Timothy W et al. (2009) Reconstitution of heterochromatin-dependent transcriptional gene silencing. Mol Cell 35:769-81

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