Post-translational modifications of histone proteins play a fundamental role in the organization of chromatin and in the regulation of gene transcription. Our long-term objective is to elucidate how histone ubiquitinases and histone methyltransferases regulate gene expression and control cell growth. Two such enzymes, which are highly conserved, are the histone H2B ubiquitinase Rad6 and the histone H3 lysine 4 (K4) methyltransferase Set1. We have recently demonstrated that Rad6-mediated histone H2B K123 ubiquitination is essential for histone H3 K4 methylation in yeast, thereby linking these two forms of histone modification to gene activation. Rad6 is an E2 ubiquitin-conjugating enzyme whose functions are evolutionary conserved from yeast to human. Importantly, the human RAD6 homolog (hHR6B) was found to be over-expressed in breast cancer cell lines and in breast carcinomas. Moreover, over-expression of exogenous hHRGB resulted in chromosome instability and transformation of human epithelial cells. Many human homologs of the H3 K4 methyltransferase SET1 (e.g., MLL, Mixed Lineage Leukemia) are also mutated or disrupted in a variety of human cancers. Collectively, these findings establish a strong link between histone ubiquitination and methylation with cancer. Given the highly conserved functions of Rad6 in regulating H2B ubiquitination and H3 methylation, it is therefore of paramount importance to understand the roles of Rad6 in mediating these histone modifications, and ultimately, controlling gene expression and cell growth. Our overall hypothesis is that through H2B ubiquitination and subsequent H3 K4 methylation, Rad6 regulates the transcription of specific genes that are important for controlling cell growth. Therefore, it is imperative to understand the basic molecular mechanisms of action of Rad6-mediated H2B ubiquitination in establishing histone H3 methylation, and thereby, gain insight into the role of Rad6 in controlling gene transcription. Using budding yeast as a model organism, we plan to use a combination of biochemistry and genetics to dissect the function of Rad6 in these processes. To achieve this, three Specific Aims are proposed.
Aim 1. Identify and characterize factors that mediate the functions of Bre1 and Rad6.
Aim 2. Define the roles of phosphorylation and acetylation on Rad6.
Aim 3. Determine the molecular mechanisms of action of Rad6-mediated H2B ubiquitination in establishing histone H3 methylation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA109355-04
Application #
7617019
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Okano, Paul
Project Start
2006-07-13
Project End
2011-05-31
Budget Start
2009-06-01
Budget End
2010-05-31
Support Year
4
Fiscal Year
2009
Total Cost
$238,105
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Stengel, Kristy R; Barnett, Kelly R; Wang, Jing et al. (2017) Deacetylase activity of histone deacetylase 3 is required for productive VDJ recombination and B-cell development. Proc Natl Acad Sci U S A 114:8608-8613
Zhao, Yue; Liu, Qi; Acharya, Pankaj et al. (2016) High-Resolution Mapping of RNA Polymerases Identifies Mechanisms of Sensitivity and Resistance to BET Inhibitors in t(8;21) AML. Cell Rep 16:2003-16
Stengel, Kristy R; Zhao, Yue; Klus, Nicholas J et al. (2015) Histone Deacetylase 3 Is Required for Efficient T Cell Development. Mol Cell Biol 35:3854-65
Wells, Christina E; Bhaskara, Srividya; Stengel, Kristy R et al. (2013) Inhibition of histone deacetylase 3 causes replication stress in cutaneous T cell lymphoma. PLoS One 8:e68915
Summers, Alyssa R; Fischer, Melissa A; Stengel, Kristy R et al. (2013) HDAC3 is essential for DNA replication in hematopoietic progenitor cells. J Clin Invest 123:3112-23
Joo, Heui-Yun; Jones, Amada; Yang, Chunying et al. (2011) Regulation of histone H2A and H2B deubiquitination and Xenopus development by USP12 and USP46. J Biol Chem 286:7190-201
Chandrasekharan, Mahesh B; Huang, Fu; Sun, Zu-Wen (2011) Decoding the trans-histone crosstalk: methods to analyze H2B ubiquitination, H3 methylation and their regulatory factors. Methods 54:304-14
Chandrasekharan, Mahesh B; Huang, Fu; Chen, Yi-Chun et al. (2010) Histone H2B C-terminal helix mediates trans-histone H3K4 methylation independent of H2B ubiquitination. Mol Cell Biol 30:3216-32
Mulugeta Achame, Eskeatnaf; Wassenaar, Evelyne; Hoogerbrugge, Jos W et al. (2010) The ubiquitin-conjugating enzyme HR6B is required for maintenance of X chromosome silencing in mouse spermatocytes and spermatids. BMC Genomics 11:367
Huang, Fu; Chandrasekharan, Mahesh B; Chen, Yi-Chun et al. (2010) The JmjN domain of Jhd2 is important for its protein stability, and the plant homeodomain (PHD) finger mediates its chromatin association independent of H3K4 methylation. J Biol Chem 285:24548-61

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