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Paul D. Kaufman, Ph.D.Academic Role: Associate Professor
Faculty Appointment(s) In:
Assembly and Function of Eukaryotic Chromosomes
Current ProjectsIntroductionIn eukaryotes, DNA is assembled into a nucleoprotein complex called chromatin. The fundamental repeating unit of chromatin is the nucleosome, which consists of 146 bp of DNA wrapped around an octamer of core histone proteins, comprised of two H2A/H2B dimers flanking an inner (H3/H4)2 tetramer. In addition to their role in compacting the genome, core histone proteins affect all aspects of chromosome function via a wide variety of post-translational modifications.Genome stability in yeast: Asf1 is required for replisome stabilityIn vivo, histones are deposited onto DNA by either DNA replication-coupled or replication-independent mechanisms. In either case, histone deposition is mediated by specialized protein complexes, including the replication-coupled Chromatin Assembly Factor-1 (CAF-1) and the replication-independent HIR complex. Asf1, a highly conserved monomeric histone chaperone, binds newly synthesized histones H3/H4, and in turn binds to and stimulates histone deposition by both the CAF-1 and HIR complexes. In addition to stimulating histone deposition, Asf1 is critical for maintaining genome stability during S phase. Cells lacking Asf1 are highly sensitive to genotoxic agents that perturb S phase and display multiple phenotypes suggesting elevated levels of spontaneous DNA damage. We discovered that these phenotypes result from Asf1 being required for maintenance of DNA replication proteins (the "replisome") at stalled replication forks.Asf1 stimulates histone H3-K56 acetylationAcetylation of H3-K56 is a recently discovered modification that occurs within the core domain of H3 molecules. Lysine 56 interacts with the phospho-backbone of DNA at its entry and exit points of the nucleosome. Two effects of H3-K56 acetylation have been documented. First, acetylation of H3-K56 neutralizes the positive charge on the lysine, disrupting this electrostatic interaction. Accordingly, an H3-K56Q alteration that mimics constitutive acetylation causes reduced superhelicity of plasmid chromatin and more rapid nuclease digestion, suggesting a more flexible wrapping of DNA at the nucleosome edge. Second, acetylation of H3-K56 increases the affinity of histone chaperones like CAF-1 for histones and is required for normal rates of histone turnover.Notably, cells lacking the histone chaperone Asf1 lack H3-K56 acetylation but display unchanged total H3 levels. H3-K56 acetylation is catalyzed by protein complexes containing Rtt109, a histone acetyltransferase (HAT) that has close homologs only in the fungal kingdom, although distant homologs are found in higher eukaryotes. Rtt109 by itself is catalytically very inefficient, and requires the presence of either of two cofactors for robust histone acetylation activity. These two co-factors are both histone-binding proteins, but are otherwise structurally and functionally distinct. One co-factor is Asf1; the other cofactor is Vps75, a homolog of the NAP/SET family of histone transport proteins. Different activities of two histone chaperones: Asf1 and Vps75Our recent data show that Asf1 and Vps75 stimulate the Rtt109 enzyme in fundamentally different ways. First, of all, the lysine substrate specificity of the enzyme is different depending on the cofactor protein. Second, the histone turnover phenotypes of cells lacking either Asf1 or Vps75 are different: cells without Asf1 display reduced turnover rates at rapidly exchanged nucleosomes, but the opposite phenotypes is observed in cells lacking Vps75. Together, these data lead us to hypothesize that Asf1 and Vps75 stimulate nascent histones to enter alternate paths. How these proteins manage these different fates for their histone cargo is an area of active investigation.Genome stability in human cells: cell cycle regulation of chromatin proteinsWe are exploring how the genome wide localization of human chromatin proteins is regulated during the cell cycle. These studies are facilitated by our development of a versatile family of retroviral and lentiviral vectors for protein overexpression and depletion, and by the microscopy, high throughput sequencing and bioinformatics resources at UMMS. Representative PublicationsErkmann JA and Kaufman PD. A negatively charged residue in place of histone H3K56 supports chromatin assembly factor association but not genotoxic stress resistance. DNA Repair (Amst), in press. Campeau E, Ruhl VE, Rodier F, Smith CL, Rahmberg BL, Fuss JO, Campisi J, Yaswen P, Cooper PK and Kaufman PD. A versatile viral system for expression and depletion of proteins in mammalian Cells. PLoS ONE 4: e6529 (2009). Kaplan T, Liu CL, Erkmann JA, Holik J, Grunstein M, Kaufman PD*, Friedman N* and Rando OJ*. Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast. PLoS Genetics 4: e1000270 (2008) (*co-corresponding authors). Berndsen CE, Tsubota T, Lindner SE, Lee S, Holton, JM, Kaufman PD, and Keck JL and Denu JM. Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75. Nat. Struct. Mol. Biol 15: 948-956 (2008). Tsubota T, Berndsen CE, Erkmann JA, Smith CL, Yang L, Freitas MA, Denu JM and Kaufman PD. Histone H3-K56 acetylation is catalyzed by histone chaperone-dependent complexes. Mol. Cell 25: 703-712 (2007). Antczak AJ, Tsubota T, Kaufman PD and Berger JM. Structure of the yeast histone H3-Asf1 interaction: Implications for chaperone mechanism, species-specific interactions, and epigenetics. BMC Struct. Biol. 6: 26 (2006). Recht J, Tsubota T, Tanny JC, Diaz RL, Berger JM, Zhang X, Garcia BA, Shabanowitz J, Burlingame AL, Hunt DF, Kaufman PD, and Allis CD. Histone chaperone Asf1 is required for acetylation of histone H3 lysine 56, a modification associated with S phase in mitosis and meiosis. Proc. Natl. Acad. Sci. USA 103: 6988-6993 (2006). Sharp JA, Rizki G, and Kaufman PD. Regulation of histone deposition proteins Asf1/Hir1 by multiple DNA damage checkpoint kinases in S. cerevisiae. Genetics 171: 885-899 (2005). Green EM, Antczak AJ, Bailey AO, Franco AA, Wu KJ, Yates JR, and Kaufman PD. Replication-independent histone deposition by the HIR complex and Asf1. Curr. Biol. 15: 2044-2049 (2005). Franco AA, Lam WM, Burgers PM, and Kaufman PD. Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with Replication Factor C. Genes Dev. 19: 1365-1375 (2005). Zhang R, Poustovoitov MV, Ye X, Santos HA, Chen W, Daganzo SM, Erzberger JP, Serebriiskii IG, Canutescu AA, Dunbrack RL, Pehrson JR, Berger JM, Kaufman PD and Adams PD. Formation of macroH2A-containing senescence associated heterochromatin foci (SAHF) and senescence driven by ASF1a and HIRA. Dev. Cell 8:19-30 (2005). Franco AA and Kaufman PD. Histone deposition proteins: links between the DNA replication machinery and epigenetic gene silencing. Cold Spring Harbor Symposia on Quantitative Biology 69: 1-8 (2004). Daganzo SM, Erzberger JP, Lam WM, Skordalakes E, Zhang R, Franco AA, Brill SJ, Adams PD, Berger JM, and Kaufman PD. Structure and function of the conserved core of histone deposition protein Asf1. Current Biology 13: 2148-2158 (2003). Sharp JA , Krawitz DC, Gardner KA, Fox CA, and Kaufman PD. The budding yeast silencing protein Sir1 is a functional component of centromeric chromatin. Genes Dev. 17: 2356-61 (2003). Sutton A, Shia W-J, Band D, Kaufman PD, Osada S, Workman JL, and Sternglanz R. Sas4 and Sas5 are required for the histone acetyltransferase activity of Sas2 in the SAS complex. J. Biol. Chem. 278: 16887 - 16892 (2003). Ye X, Franco AA, Santos H, Kaufman PD, and Adams PD. Inhibition of S-phase chromatin assembly causes DNA damage, activation of the S-phase checkpoint and S-phase arrest. Mol. Cell 11: 341-351 (2003). Sharp JA and Kaufman PD. Chromatin proteins are determinants of centromere function. In Current Topics in Microbiology and Immunology: Protein Complexes that Modify Chromatin (ed. J.L. Workman), Vol. 274, p.23-52, Springer-Verlag Press (2003). Formosa T, Ruone S, Adams MD, Olsen AE, Eriksson P, Yu Y, Rhoades AR, Kaufman PD, and Stillman DJ. Defects in SPT16 or POB3 (yFACT) Cause Dependence on the Hir/Hpc Pathway: Accessing DNA May Degrade Chromatin Structure. Genetics 162: 1557-1571 (2002). Sharp JA, Franco AA, Osley MA, and Kaufman PD. Chromatin Assembly Factor-I and Hir proteins contribute to building functional kinetochores in Saccharomyces cerevisiae. Genes Dev. 16: 85-100 (2002). Krawitz DC, Kama T, and Kaufman PD. Chromatin Assembly Factor-I mutants defective for PCNA binding require Asf1/Hir proteins for silencing. Mol. Cell. Biol. 22: 614-625 (2002). Sharp JA, Fouts ET, Krawitz, DC, and Kaufman PD. Yeast Histone Deposition Protein Asf1p Requires Hir Proteins and PCNA for Heterochromatic Silencing. Current Biology 11: 463-473 (2001). Kaufman PD and Almouzni G. DNA replication, nucleotide excision repair, and nucleosome assembly. In Chromatin Structure and Gene Expression (ed. S.C.R. Elgin and J.L. Workman), Vol. 2, p. 24-48, Oxford University Press (2000). Game JC and Kaufman PD. Role of Saccharomyces cerevisiae Chromatin Assembly Factor-I in repair of ultraviolet radiation damage in vivo. Genetics 151: 485-497 (1999). Kaufman PD, Cohen JL, and Osley MA. Hir proteins are required for position-dependent gene silencing in Saccharomyces cerevisiae in the absence of Chromatin Assembly Factor-I. Mol. Cell. Biol. 18: 4793-4806 (1998). Verreault A, Kaufman PD, Kobayashi R, and Stillman B. Nucleosomal DNA regulates the core-histone-binding subunit of the human Hat1 acetyltransferase. Current Biology 8: 96-108 (1998). Kaufman PD, Kobayashi R, and Stillman B. Ultraviolet radiation-sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking Chromatin Assembly Factor-I. Genes Dev. 11: 345-357 (1997). Verreault A, Kaufman PD, Kobayashi R, and Stillman B. Nucleosome assembly by a complex of Chromatin Assembly Factor-I and acetylated histones H3/H4. Cell 87: 95-104 (1996). Gaillard P-HL, Martini EM-D, Kaufman PD, Stillman B, Moustacchi E, and Almouzni G. Chromatin assembly coupled to DNA repair: a new role for Chromatin Assembly Factor-I. Cell 86: 887-896 (1996). Kamakaka RT, Bulger M, Kaufman PD, Stillman B, and Kadonaga JT. Post-replicative chromatin assembly by Drosophila and Human Chromatin Assembly Factor-I. Mol. Cell. Biol. 16: 810-817 (1996). Kaufman PD, Kobayashi R, Kessler N, and Stillman B. The p150 and p60 subunits of Chromatin Assembly Factor-I: A molecular link between newly synthesized histones and DNA replication. Cell 81: 1105-1114 (1995). Rotation Projects1. Genome-wide analysis of chromatin protein occupancy in human cells. 2. Genome-wide analysis of histone protein exchange in yeast. 3. Biochemical analysis of histone deposition proteins. Laboratory PersonnelEric Campeau, Instructor Academic BackgroundPaul Kaufman received his A.B. in Biochemistry from the University of California, Berkeley and his Ph.D. in Biology from MIT. From 1992 to 1996, he was a postdoctoral fellowat Cold Spring Harbor Laboratory, where he was supported by the Life Sciences Research Foundation. He went on to become a Career Staff Scientist at the Lawrence Berkeley National Laboratory and Associate Adjunct Professor of Biochemistry and Molecular Biology at the University of California, Berkeley. Dr. Kaufman joined the Program in Gene Function and Expression at the University of Massachusetts Medical School as an Associate Professor in July of 2005.
Office: LRB 506
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We study several different classes of proteins used by eukaryotic cells to deposit histones onto DNA, as well as enzyme complexes that chemically modify histones in order to alter their function. We study these processes in yeast and human cells, using biochemical, genetic, genomic, and cell biological techniques.
364 Plantation Street Worcester, MA 01605