Normal cellular differentiation and development rely extensively on silencing mechanisms. Disruption of silencing can lead to genetic disorders, chromosome loss and contribute to tumorigenesis. Our long term goal is to determine the normal mechanisms that lead to establishment and maintenance of silenced regions (heterochromatin) of the genome. The goal of this proposal is to define how heterochromatin at centromeres is established. A major impediment to research on the establishment of heterochromatin has been difficulty in separating establishment from maintenance. To circumvent this problem, we utilize the exquisite genetic tractability of the fission yeast, where silenced chromatin shares many features with mammals, including reliance on similar chromatin marking systems and a shared dependence on non-coding RNA and the RNAi pathway for silencing control. Through characterization of a macromolecular complex that is important for the assembly of heterochromatin, we generated a novel mutant strain of yeast that can maintain pre-assembled heterochromatin, but cannot support its de-novo establishment. We will use this mutant to screen genes that are known to play a role in heterochromatin stability to identify those that are required for heterochromatin establishment. We have already found that one "establishment" gene that encodes a histone methyltransferase, Clr4, (the homolog of Suv39 proteins in flies and mammals) which methylates histone H3 on lysine 9 (K9) only in regions of heterochromatin. We anticipate that other "establishment" genes will regulate Clr4's recruitment to chromatin and / or its activity. Second, we will determine the DNA sequences that are required for the establishment of heterochromatin. We will define "hot-spots" for Clr4 activity using high resolution chromatin immunoprecipitation analyses, and will assess whether these sequences suffice to establish de novo heterochromatin. We will determine which establishment genes work through which establishment sequences. Finally, we will determine if K9 methylation of histone H3 suffices to establish heterochromatin, and the role that the proteins that bind this mark play in the establishment of heterochromatin. These studies will yield a basic knowledge of the mechanisms that dictate heterochromatin establishment and will provide a framework for future work directed towards understanding and developing therapies for aberrant heterochromatin assembly and disease-related silencing defects in humans.

Public Health Relevance

The "turning-off" or silencing of specific regions of chromosomes is as important for the normal growth of cells in an organism as is "turning-on" genes. Silencing is important for cell-type identity and for enabling all cells to divide normally, such that improper silencing can contribute to genetic disorders and cancer. The proposed studies are directly relevant as they will yield a basic knowledge of silencing and help direct future work towards understanding and developing therapies for disease- related silencing defects in humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM084045-05
Application #
8258797
Study Section
Molecular Genetics C Study Section (MGC)
Program Officer
Carter, Anthony D
Project Start
2008-05-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2014-04-30
Support Year
5
Fiscal Year
2012
Total Cost
$316,964
Indirect Cost
$128,295
Name
St. Jude Children's Research Hospital
Department
Type
DUNS #
067717892
City
Memphis
State
TN
Country
United States
Zip Code
38105
Creamer, Kevin M; Job, Godwin; Shanker, Sreenath et al. (2014) The Mi-2 homolog Mit1 actively positions nucleosomes within heterochromatin to suppress transcription. Mol Cell Biol 34:2046-61
Alper, Benjamin J; Job, Godwin; Yadav, Rajesh K et al. (2013) Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast. EMBO J 32:2321-35
Schalch, Thomas; Job, Godwin; Shanker, Sreenath et al. (2011) The Chp1-Tas3 core is a multifunctional platform critical for gene silencing by RITS. Nat Struct Mol Biol 18:1351-7
Creamer, Kevin M; Partridge, Janet F (2011) RITS-connecting transcription, RNA interference, and heterochromatin assembly in fission yeast. Wiley Interdiscip Rev RNA 2:632-46
Hansen, Klavs R; Hazan, Idit; Shanker, Sreenath et al. (2011) H3K9me-independent gene silencing in fission yeast heterochromatin by Clr5 and histone deacetylases. PLoS Genet 7:e1001268
Shanker, Sreenath; Job, Godwin; George, Olivia L et al. (2010) Continuous requirement for the Clr4 complex but not RNAi for centromeric heterochromatin assembly in fission yeast harboring a disrupted RITS complex. PLoS Genet 6:e1001174
Schalch, Thomas; Job, Godwin; Noffsinger, Victoria J et al. (2009) High-affinity binding of Chp1 chromodomain to K9 methylated histone H3 is required to establish centromeric heterochromatin. Mol Cell 34:36-46