In eukaryotic cells, DNA is packaged into chromatin that governs gene expression and genome integrity. In order for DNA replication machinery to access DNA, chromatin structure in front of replication forks must temporarily be disassembled. Following DNA replication, parental and newly synthesized histones are deposited onto DNA strands to form nucleosomes, the basic unit of chromatin, and then higher order chromatin structures. This DNA replication-coupled nucleosome assembly process plays an important role in maintenance of genome stability and inheritance of chromatin structures including heterochromatin. Uncoupling of DNA synthesis and chromatin assembly results in genome instability, one of the hallmarks of cancer cells. Moreover, several factors involved in this process have been linked to carcinogenesis and aging. Therefore, it is important to understand how the DNA replication-coupled nucleosome assembly is regulated and how this regulation contributes to transcriptional silencing. Histone chaperones CAF-1, Asf1 and Rtt106 function together to promote nucleosome assembly during S phase of the cell cycle. These factors are also required for transcriptional silencing in budding yeast. Genetic studies indicate that the role of Asf1 and Rtt106 in transcriptional silencing is linked to their ability to bind histones and thereby promote nucleosome assembly. However, it is not known how Rtt106 is recruited to DNA replication forks to promote nucleosome formation during S phase of the cell cycle. Moreover, how histone (H3-H4)2 tetramers are formed through the action of Asf1 remains elusive because Asf1 binds to histone H3 through the same surface involved in (H3-H4)2 tetramer formation. Lastly, through a genetic screen, we have found that Dia2, an F-box containing protein and a component of the SCF E3 ubiquitin ligase, functions in parallel with Rtt106 in transcriptional silencing. However, it is not known how Dia2 impacts transcriptional silencing. In this proposal, we will determine how Rtt106 is recruited to replicating DNA to promote nucleosome assembly;determine the molecular mechanisms by which (H3-H4)2 tetramers are formed from H3-H4 dimers in the Asf1-H3- H4 complex;and elucidate the Dia2's role in transcriptional silencing. These studies will provide novel insight into the molecular mechanism of DNA replication-coupled nucleosome assembly, an important but poorly understood process, that when gone awry, contributes to the development of cancer and aging in humans.

Public Health Relevance

In addition to govern genome integrity, chromatin structure encodes epigenetic information that maintain gene expression states and cell identity. Our long-term goal is to understand how chromatin structure, including heterochromatin that silences transcription, is inherited during S phase of the cell cycle and how inheritance of chromatin structure impacts genome integrity. In the last grant cycle, we made significant progress in understanding how nucleosomes are formed following DNA replication, the first step in the inheritance of higher order chromatin structure. We discovered a novel histone H3-H4 chaperone, Rtt106, which binds new H3 acetylated at lysine 56 (H3K56Ac) and functions in nucleosome assembly and transcriptional silencing. We also found that Cac1, the large subunit of CAF-1 recognizes H3K56Ac by unknown mechanisms. In this proposal, we will determine how Rtt106 is recruited to replication forks to promote nucleosome assembly, how histone (H3-H4)2 tetramers are formed from H3-H4 dimers in the Asf1-H3-H4 complex and how Cac1 recognizes H3K56Ac. Lastly, we will determine how Dia2, a Fox-box containing protein that was previously not known to have a role in transcriptional silencing, functions in parallel with Rtt106 in transcriptional silencing. These studies will provide novel insight into DNA replication-coupled nucleosome assembly, an important but poorly understood process, that when gone awry, contributes to the development of cancer and aging.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM072719-09
Application #
8725178
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
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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
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Dahlin, Jayme L; Sinville, Rondedrick; Solberg, Jonathan et al. (2013) A cell-free fluorometric high-throughput screen for inhibitors of Rtt109-catalyzed histone acetylation. PLoS One 8:e78877
Burgess, Rebecca J; Zhang, Zhiguo (2013) Histone chaperones in nucleosome assembly and human disease. Nat Struct Mol Biol 20:14-22
Burgess, Rebecca J; Zhou, Hui; Han, Junhong et al. (2012) The SCFDia2 ubiquitin E3 ligase ubiquitylates Sir4 and functions in transcriptional silencing. PLoS Genet 8:e1002846
Li, Qing; Zhang, Zhiguo (2012) Linking DNA replication to heterochromatin silencing and epigenetic inheritance. Acta Biochim Biophys Sin (Shanghai) 44:3-13

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