The process of gene expression requires that the DNA that encodes a gene be transcribed to make a messenger RNA (mRNA) copy, which is then translated to produce a protein. For decades, it was believed that the process of transcription occurred primarily across genes to produce mRNAs. However, over the past few years, with the advent of new technologies, experiments have demonstrated that transcription occurs in a much more widespread fashion. For example, only 1.5% of the human genome encodes proteins, while over 70% is transcribed. While there is little knowledge about the functions of this previously unknown transcription, emerging evidence has pointed towards regulatory roles that are important in human biology and disease. However, many questions remain. This proposal focuses on understanding some of these classes of transcription. The experiments focus around a protein, Spt6, that controls transcription genome-wide. Spt6 is found in organisms from yeast to humans and, in all these organisms it has been shown to play crucial roles. In human cells, Spt6 broadly controls transcription and it has been implicated in cancer. Spt6 interacts with both nucleosomes and RNA polymerase II (RNAPII). Nucleosomes are the main repeating structural units of chromosomes, in which 150 base pairs of DNA are wrapped around an octamer of eight histone proteins. In the absence of Spt6, nucleosomes are not positioned correctly and transcription by RNAPII is greatly altered. This proposal will study Spt6 and transcription in the yeast, Saccharomyces cerevisiae, where transcription is highly conserved with humans. S. cerevisiae is a powerful system as it can be studied by genetic and genome-wide approaches at high resolution. The broad objectives of this proposal are to understand the mechanism by which Spt6 functions, to characterize its role in regulation of transcription across the genome, and to elucidate the roles of two classes of recently identified classes of transcripts that it regulates.
Specific Aim 1 will address Spt6 function by studying its interactions with histones by crosslinking purified Spt6 to purified histone octamers and identifying the cross linked amino acids by mass spectrometry. Then, mutations will be created that alter those amino acids in order to study the consequences of impairing the Spt6-histone interaction in living cells.
Specific Aim 2 will study intragenic transcription, a poorly understood form of transcription that is normally repressed by Spt6. Experiments will comprehensively identify all intragenic transcripts, determine if they are expressed in response to environmental changes, and study candidates for their possible biological roles.
Specific Aim 3 will use a recently developed method, NET-seq, to measure the level of active transcription by Spt6 at high resolution. Spt6 regulation of antisense transcription will be characterized by NET-seq analysis of spt6 mutants, and the regulatory roles of antisense transcription will be studied. As intragenic and antisense transcription, and Spt6 have been associated with human disease, what is learned from these studies will be directly relevant to human health.

Public Health Relevance

The correct regulation of gene expression is crucial to the normal growth and health of humans;when it goes awry, it can lead to several diseases, including cancer. Recently, previously unknown aspects of gene expression have been discovered, but they are still poorly understood. The proposed experiments will study a protein that is required for these aspects of regulation;what is learned will expand our understanding of fundamental aspects of gene expression and, likely, human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM032967-31
Application #
8628624
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
1983-12-01
Project End
2017-11-30
Budget Start
2014-01-01
Budget End
2014-11-30
Support Year
31
Fiscal Year
2014
Total Cost
$518,647
Indirect Cost
$209,726
Name
Harvard University
Department
Genetics
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Chang, Jennifer S; Winston, Fred (2013) Cell-cycle perturbations suppress the slow-growth defect of spt10ýý mutants in Saccharomyces cerevisiae. G3 (Bethesda) 3:573-83
Chang, Jennifer S; Winston, Fred (2011) Spt10 and Spt21 are required for transcriptional silencing in Saccharomyces cerevisiae. Eukaryot Cell 10:118-29
Kiely, Christine M; Marguerat, Samuel; Garcia, Jennifer F et al. (2011) Spt6 is required for heterochromatic silencing in the fission yeast Schizosaccharomyces pombe. Mol Cell Biol 31:4193-204
Ivanovska, Iva; Jacques, Pierre-Etienne; Rando, Oliver J et al. (2011) Control of chromatin structure by spt6: different consequences in coding and regulatory regions. Mol Cell Biol 31:531-41
Libuda, Diana E; Winston, Fred (2010) Alterations in DNA replication and histone levels promote histone gene amplification in Saccharomyces cerevisiae. Genetics 184:985-97
Diebold, Marie-Laure; Koch, Michael; Loeliger, Erin et al. (2010) The structure of an Iws1/Spt6 complex reveals an interaction domain conserved in TFIIS, Elongin A and Med26. EMBO J 29:3979-91
Diebold, Marie-Laure; Loeliger, Erin; Koch, Michael et al. (2010) Noncanonical tandem SH2 enables interaction of elongation factor Spt6 with RNA polymerase II. J Biol Chem 285:38389-98
Winston, Fred (2009) A transcription switch toggled by noncoding RNAs. Proc Natl Acad Sci U S A 106:18049-50
Cheung, Vanessa; Chua, Gordon; Batada, Nizar N et al. (2008) Chromatin- and transcription-related factors repress transcription from within coding regions throughout the Saccharomyces cerevisiae genome. PLoS Biol 6:e277
Zhang, Lei; Fletcher, Aaron G L; Cheung, Vanessa et al. (2008) Spn1 regulates the recruitment of Spt6 and the Swi/Snf complex during transcriptional activation by RNA polymerase II. Mol Cell Biol 28:1393-403

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