The DNA in eukaryotic chromosomes is bound by histone proteins. Octamers of histone proteins are organized into nucleosomes, the fundamental building blocks of chromatin. Histones are evicted and replaced during all types of polymerase movement, making histone deposition a critical process for all aspects of chromosome biology throughout the cell cycle. In vivo, nucleosome formation requires histone-binding proteins to prevent uncontrolled aggregation of histones and DMA. This proposal is focused on a conserved eukaryotic protein complex important for histone deposition, Chromatin Assembly Factor-1 (CAF-1). CAF-1 interacts with and is stimulated to deposit histones by another histone-binding protein termed Asf1. CAF-1 deposits histones preferentially onto replicating DNA, and thus represent a paradigm for understanding nucleosome formation during S phase of the cell cycle. We will address the following interrelated questions: What auxiliary proteins are required for histone deposition by CAF-1? How is CAF-1 stimulated by Asf1? How is the deposition of different core histone subcomplexes (H3/H4 versus H2A/H2B) coordinated? Are histones exchanged among these complexes, and do multiple assembly complexes contribute histones to the same nucleosome? We will address these questions using biochemical, biophysical, and molecular genetic approaches: We have developed a new in vitro nucleosome assembly assay to facilitate isolation of accessory factors and characterization of reaction intermediates during histone deposition. We will explore how CAF-1 interacts with histones by mapping of interaction sites and analytical ultracentrifugation analyses of CAF-1/histone complexes. We will generate differentially labeled histones to determine whether different complexes contribute to the same nucleosome. Relevance: Inhibition of human CAF-1 results in S phase arrest, apparently due to replication fork collapse. A human Asf1 protein is required for human cellular senescence, a differentiation pathway important for avoidance of tumorigenesis. Therefore, these highly conserved histone deposition proteins directly impact genome stability and growth control, important aspects of human health. The proposed biochemical studies of these proteins are consequently of high priority for cancer research, and these and new proteins discovered in the course of this work may be good candidates in the future for therapeutic intervention.

Public Health Relevance

Inhibition of human CAF-1 results in S phase arrest, apparently due to replication fork collapse. A human Asf1 protein is required for human cellular senescence, a differentiation pathway important for avoidance of tumorigenesis. Therefore, these highly conserved histone deposition proteins directly impact genome stability and growth control, important aspects of human health. The proposed biochemical studies of these proteins are consequently of high priority for cancer research, and these and new proteins discovered in the course of this work may be good candidates in the future for therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055712-13
Application #
7791446
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Carter, Anthony D
Project Start
1997-06-01
Project End
2011-07-31
Budget Start
2010-05-01
Budget End
2011-07-31
Support Year
13
Fiscal Year
2010
Total Cost
$370,013
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Genetics
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Sun, Xiaoming; Bizhanova, Aizhan; Matheson, Timothy D et al. (2017) Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells. Mol Cell Biol 37:
Matheson, Timothy D; Kaufman, Paul D (2017) The p150N domain of chromatin assembly factor-1 regulates Ki-67 accumulation on the mitotic perichromosomal layer. Mol Biol Cell 28:21-29
Matheson, Timothy D; Kaufman, Paul D (2016) Grabbing the genome by the NADs. Chromosoma 125:361-71
Kaufman, Paul D (2015) Want reprogramming? Cut back on the chromatin assembly! Nat Struct Mol Biol 22:648-50
Lopes da Rosa, Jessica; Bajaj, Vineeta; Spoonamore, James et al. (2013) A small molecule inhibitor of fungal histone acetyltransferase Rtt109. Bioorg Med Chem Lett 23:2853-9
Lopes da Rosa, Jessica; Holik, John; Green, Erin M et al. (2011) Overlapping regulation of CenH3 localization and histone H3 turnover by CAF-1 and HIR proteins in Saccharomyces cerevisiae. Genetics 187:9-19
Kaufman, Paul D (2011) New partners for HP1 in transcriptional gene silencing. Mol Cell 41:1-2
Lopes da Rosa, Jessica; Boyartchuk, Victor L; Zhu, Lihua Julie et al. (2010) Histone acetyltransferase Rtt109 is required for Candida albicans pathogenesis. Proc Natl Acad Sci U S A 107:1594-9
Kaufman, Paul D (2010) Toxicity and lifespan extension: complex outcomes of histone overexpression in budding yeast. Cell Cycle 9:4611-2
Kaufman, Paul D; Rando, Oliver J (2010) Chromatin as a potential carrier of heritable information. Curr Opin Cell Biol 22:284-90

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