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Program in Molecular Medicine

Program in Gene Function and Expression

interdisciplinary graduate program : faculty

Section: Research

Paul D. Kaufman, Ph.D.

Academic Role: Associate Professor

Faculty Appointment(s) In:
Program in Gene Function and Expression
Program in Molecular Medicine

Assembly and Function of Eukaryotic Chromosomes

Photo: Paul D. Kaufman, Ph.D. 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.

Current Projects

Introduction

In 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 stability

In 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 acetylation

Acetylation 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 Vps75

Our 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 proteins

We 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.

Office: LRB 506
Phone: 508-856-5016
E-mail: Paul.Kaufman1@umassmed.edu
Keywords: Organisms - vertebrate/human cell lines, Chromosome Structure & Dynamics, Protein Acetylation and Deacetylation, Biochemistry, Organisms - yeast

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