In eukaryotic cells, chromatin regulates gene expression and maintains genome integrity. The basic repeating unit of chromatin is the nucleosome, consisting of 146 base pairs of DNA wrapped around a histone octamer containing one (H3-H4)2 tetramer and two H2A-H2B dimers. It is believed that the assembly of histones H3-H4 into nucleosomes is the rate-limiting step of nucleosome formation following gene transcription and DNA replication. Mis-regulation of nucleosome assembly results in genome instability and is associated with aging and the development of cancer. We have been addressing how H3-H4 molecules are assembled into nucleosomes, a research area that remains elusive. In mammalian cells, there are two major forms of histone H3, canonical H3 (also called H3.1) and the histone H3 variant H3.3. Though differing only by five amino acids, H3.1 and H3.3 have distinct functions and are assembled into nucleosomes via distinct mechanisms. H3.1-H4 is assembled into nucleosomes by the histone chaperone CAF-1 in a replication-coupled nucleosome assembly process, and H3.3-H4 is assembled into nucleosomes by HIRA through the replication-independent nucleosome assembly pathway. However, it remains unclear how H3.3 and H3.1 are assembled into nucleosomes via distinct pathways despite their limited sequence variation. We have made a remarkable discovery that phosphorylation of H4 serine 47 (H4S47ph), a modification whose function was not known previously, contributes to the association of H3.3-H4 with HIRA. In addition, this modification inhibits the binding of CAF-1 with H3.1-H4. Based on these results, we propose to determine how H4S47ph is regulated by the H4S47 kinase (Pak2) and phosphatases, enzymes involved in cell signaling and how this modification impacts HIRA's role in nucleosome assembly and the formation of senescence-associated heterochromatin foci. These studies will provide a new paradigm connecting nucleosome assembly with cell signaling and reveal a novel form of regulation of senescence, a process important for tumor suppression.

Public Health Relevance

Chromatin is an organized complex of DNA, protein and RNA that maintains epigenetic memory and genome stability. How chromatin structures are maintained during cell division is one of the most fundamental, unanswered biological questions. The basic repeating unit of chromatin is the nucleosome, consisting of 146bp of DNA wrapped around a core histone octamer containing a (H3-H4)2 tetramer and two H2A-H2B dimers. The histone H3 variant H3.3, which differs from the canonical H3, H3.1, by only five amino acid residues, functions distinctly from H3.1 in chromatin regulation. Furthermore, H3.3 and H3.1 are assembled into nucleosomes via distinct mechanisms. In this proposal, we will determine how phosphorylation of histone H4 serine 47 regulates the assembly of H3.3 into nucleosomes and how this modification is regulated by a balance of kinase and phosphatase activities. These studies will not only elucidate a novel regulatory mechanism of nucleosome assembly, but will also shed light on how deregulation of this process contributes to carcinogenesis and aging.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM081838-06
Application #
8420448
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
2008-05-01
Project End
2016-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
6
Fiscal Year
2013
Total Cost
$292,948
Indirect Cost
$107,185
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
Fang, Dong; Gan, Haiyun; Lee, Jeong-Heon et al. (2016) The histone H3.3K36M mutation reprograms the epigenome of chondroblastomas. Science 352:1344-8
Wang, Zhiquan; Zhang, Honglian; Liu, Ji et al. (2016) USP51 deubiquitylates H2AK13,15ub and regulates DNA damage response. Genes Dev 30:946-59
Feng, Jianxun; Gan, Haiyun; Eaton, Matthew L et al. (2016) Noncoding Transcription Is a Driving Force for Nucleosome Instability in spt16 Mutant Cells. Mol Cell Biol 36:1856-67
Dahlin, Jayme L; Chen, Xiaoyue; Walters, Michael A et al. (2015) Histone-modifying enzymes, histone modifications and histone chaperones in nucleosome assembly: Lessons learned from Rtt109 histone acetyltransferases. Crit Rev Biochem Mol Biol 50:31-53
Dahlin, Jayme L; Nissink, J Willem M; Francis, Subhashree et al. (2015) Post-HTS case report and structural alert: Promiscuous 4-aroyl-1,5-disubstituted-3-hydroxy-2H-pyrrol-2-one actives verified by ALARM NMR. Bioorg Med Chem Lett 25:4740-52
Dahlin, Jayme L; Nissink, J Willem M; Strasser, Jessica M et al. (2015) PAINS in the assay: chemical mechanisms of assay interference and promiscuous enzymatic inhibition observed during a sulfhydryl-scavenging HTS. J Med Chem 58:2091-113
Burgess, Rebecca J; Han, Junhong; Zhang, Zhiguo (2014) The Ddc1-Mec3-Rad17 sliding clamp regulates histone-histone chaperone interactions and DNA replication-coupled nucleosome assembly in budding yeast. J Biol Chem 289:10518-29
Yu, Chuanhe; Gan, Haiyun; Han, Junhong et al. (2014) Strand-specific analysis shows protein binding at replication forks and PCNA unloading from lagging strands when forks stall. Mol Cell 56:551-63
Zhang, Hui; Wang, Zhiquan; Zhang, Zhiguo (2013) PP1α, PP1β and Wip-1 regulate H4S47 phosphorylation and deposition of histone H3 variant H3.3. Nucleic Acids Res 41:8085-93
Han, Junhong; Zhang, Hui; Zhang, Honglian et al. (2013) A Cul4 E3 ubiquitin ligase regulates histone hand-off during nucleosome assembly. Cell 155:817-29

Showing the most recent 10 out of 30 publications