How chromatin domains such as euchromatin and heterochromatin are established and maintained are poorly understood. At the heart of chromatin organization are the histone proteins, which are deposited and removed by chaperone proteins. One such chaperone is Spt6, which is evolutionarily conserved and promotes transcription elongation through its interaction with the RNA polymerase II (RNAPII) C-terminal domain (CTD). During transcription, Spt6 mediates nucleosome reassembly in the wake of transcribing RNAPII and promotes H3K36 methylation, which recruits histone deacetylase and chromatin remodeling activities that maintain chromatin integrity in the coding region of genes. Our studies have now identified Spt6 as a central component of a novel regulatory circuit that maintains CTD phosphorylation;this circuit is itself controlled through Spt6 protein stability. We have also found that Spt6 is up-regulated during S phase, where it likely contributes to histone gene transcription and/or deposition at this critical stage. Further, we have uncovered an unexpected role for Spt6 in maintaining centromere identity through the potential targeting of the Psh1 E3 ligase that removes ectopically deposited histone H3 variant Cse4. Despite these findings, we know little about how Spt6 performs its chromatin-dependent activities. Thus, this proposal aims to elucidate the fundamental mechanisms by which Spt6 contributes to the maintenance of chromatin structure in the coding and regulatory regions of the genome - thereby contributing to the biology of Spt6 and to the mechanisms by which chromatin domains are established and maintained. To accomplish this goal, we will address the following key questions: 1) How is Spt6 regulated through its interactions with RNAPII and by post-translational modifications that occur on this protein? 2) Why is Spt6 cell cycle regulated, and what role does it play in S phase? 3) Does Spt6 function outside of its canonical role in transcriptional regulation to regulate other aspects of chromatin biology, in particular centromere identity? Given the conservation of Spt6 across evolution, our studies will significantly impact our understanding of the mechanisms that underlie transcriptional elongation and the maintenance of chromatin integrity that govern genome integrity. These studies will also be relevant to our understanding of Spt6 in human development and disease.

Public Health Relevance

Histone chaperones play a pivotal role in the maintenance of chromatin integrity during disruptive processes such as DNA replication, repair and transcription. Our studies will define the roles of the histone chaperone Spt6 in transcription, cell cycle progressio and in surveillance of aberrantly deposited histone variants in chromatin. As defects in transcription elongation, cell cycle progression and chromatin organization are at the heart of many human diseases including cancer, these studies are directly relevant to human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM110058-01
Application #
8671088
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
2014-06-01
Project End
2018-02-28
Budget Start
2014-06-01
Budget End
2015-02-28
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Andrews, Forest H; Shinsky, Stephen A; Shanle, Erin K et al. (2016) The Taf14 YEATS domain is a reader of histone crotonylation. Nat Chem Biol 12:396-8
Andrews, Forest H; Tong, Qiong; Sullivan, Kelly D et al. (2016) Multivalent Chromatin Engagement and Inter-domain Crosstalk Regulate MORC3 ATPase. Cell Rep 16:3195-207
Bedard, Lynn Glowczewski; Dronamraju, Raghuvar; Kerschner, Jenny L et al. (2016) Quantitative Analysis of Dynamic Protein Interactions during Transcription Reveals a Role for Casein Kinase II in Polymerase-associated Factor (PAF) Complex Phosphorylation and Regulation of Histone H2B Monoubiquitylation. J Biol Chem 291:13410-20
Andrews, Forest H; Strahl, Brian D; Kutateladze, Tatiana G (2016) Insights into newly discovered marks and readers of epigenetic information. Nat Chem Biol 12:662-8
Sorenson, Matthew R; Jha, Deepak K; Ucles, Stefanie A et al. (2016) Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae. RNA Biol 13:412-26
Chen, Hongfeng; Workman, Jason J; Strahl, Brian D et al. (2016) Histone H3 and TORC1 prevent organelle dysfunction and cell death by promoting nuclear retention of HMGB proteins. Epigenetics Chromatin 9:34
McDaniel, Stephen L; Fligor, Jennifer E; Ruan, Chun et al. (2016) Combinatorial Histone Readout by the Dual Plant Homeodomain (PHD) Fingers of Rco1 Mediates Rpd3S Chromatin Recruitment and the Maintenance of Transcriptional Fidelity. J Biol Chem 291:14796-802
Andrews, Forest H; Shanle, Erin K; Strahl, Brian D et al. (2016) The essential role of acetyllysine binding by the YEATS domain in transcriptional regulation. Transcription 7:14-20
Andrews, Forest H; Gatchalian, Jovylyn; Krajewski, Krzysztof et al. (2016) Regulation of Methyllysine Readers through Phosphorylation. ACS Chem Biol 11:547-53
Chen, Shoudeng; Yang, Ze; Wilkinson, Alex W et al. (2015) The PZP Domain of AF10 Senses Unmodified H3K27 to Regulate DOT1L-Mediated Methylation of H3K79. Mol Cell 60:319-27

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