The bulk of eukaryotic nucleosomes are made up of four core histones: H2A, H2B, H3, and H4. The genes encoding these proteins have been the primary focus of almost two decades of genetic experiments and we have learned a great deal about their roles in gene transcription and silencing. However, all eukaryotes studied to date also express one or more histone variants, less abundant proteins that are related to one of the major histone families but have evolved along separate lineages. Despite the fact that these variants are generally essential for viability in metazoans, little is known about their functions, genetic interactions, or assembly pathways. We have discovered that the histone variant H2A.Z, encoded by the HTZ1 gene in budding yeast, is required for normal gene transcription and chromosome segregation. Thus, histone H2A.Z is a critical player in at least two basic chromosomal functions. We will address three fundamental questions regarding H2A.Z function: (1) How does H2A.Z regulate transcription? Through novel genetic interactions with mutations in RNA polymerase II, we have found that HTZ1 is necessary for transcription elongation, but the mechanisms and molecular pathways involved are currently unknown. We will address this question through genetic screens for new htzl point mutations and for extragenic suppressors of their elongation defects. Using chromatin immunoprecipitation, we will determine the H2A.Z-dependent steps in elongation and their functional consequences. (2) What is the role of H2A.Z in chromosome segregation? We have discovered that htzl is synthetic lethal with mutations in components of the centromere and spindle apparatus. It causes high frequencies of increased chromosome ploidy and is physically located at the centromere of chromosome III. However, we do not know how Htzl functions at the centromere or what proteins it targets. To address these questions, we will further characterize spindle/kinetochore function in htzl mutants. We will use ChIP assays to identify Htzl-dependent components of the kinetochore. We will isolated novel Ipl- alleles of htzl and characterize their interactions with other histone, spindle, and kineotchore mutations. (3) How is Htzl recruited to specific chromosomal locations? We have found that Htz1 is not uniformly distributed throughout the chromatin but is preferentially incorporated at specific loci. Furthermore, these loci include the promoter for some genes, the open reading frame for other genes, and pericentric chromatin at centromeres. To understand the rules and mechanisms for these assembly patterns, we will engineer mutations in specific cis- and trans-acting factors to test alternative models of Htz1 recruitment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028920-26
Application #
7028271
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Carter, Anthony D
Project Start
1981-04-01
Project End
2008-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
26
Fiscal Year
2006
Total Cost
$354,839
Indirect Cost
Name
University of Virginia
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
065391526
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Kim, Jung-Ae; Hsu, Jer-Yuan; Smith, M Mitchell et al. (2012) Mutagenesis of pairwise combinations of histone amino-terminal tails reveals functional redundancy in budding yeast. Proc Natl Acad Sci U S A 109:5779-84
Jensen, Kurt; Santisteban, Maria Soledad; Urekar, Craig et al. (2011) Histone H2A.Z acid patch residues required for deposition and function. Mol Genet Genomics 285:287-96
Hang, Mingda; Smith, M Mitchell (2011) Genetic analysis implicates the Set3/Hos2 histone deacetylase in the deposition and remodeling of nucleosomes containing H2A.Z. Genetics 187:1053-66
Santisteban, Maria Soledad; Hang, Mingda; Smith, M Mitchell (2011) Histone variant H2A.Z and RNA polymerase II transcription elongation. Mol Cell Biol 31:1848-60
Le Masson, Ivan; Yu, David Y; Jensen, Kurt et al. (2003) Yaf9, a novel NuA4 histone acetyltransferase subunit, is required for the cellular response to spindle stress in yeast. Mol Cell Biol 23:6086-102
Sabet, Nevin; Tong, Fumin; Madigan, James P et al. (2003) Global and specific transcriptional repression by the histone H3 amino terminus in yeast. Proc Natl Acad Sci U S A 100:4084-9
Kulesza, Caroline A; Van Buskirk, Heather A; Cole, Michael D et al. (2002) Adenovirus E1A requires the yeast SAGA histone acetyltransferase complex and associates with SAGA components Gcn5 and Tra1. Oncogene 21:1411-22
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Hsu, J Y; Sun, Z W; Li, X et al. (2000) Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 102:279-91
Glowczewski, L; Yang, P; Kalashnikova, T et al. (2000) Histone-histone interactions and centromere function. Mol Cell Biol 20:5700-11

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