Publications by authors named "Paul D Kaufman"

43 Publications

Close to the edge: Heterochromatin at the nucleolar and nuclear peripheries.

Biochim Biophys Acta Gene Regul Mech 2021 01 8;1864(1):194666. Epub 2020 Dec 8.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

Chromatin is a dynamic structure composed of DNA, RNA, and proteins, regulating storage and expression of the genetic material in the nucleus. Heterochromatin plays a crucial role in driving the three-dimensional arrangement of the interphase genome, and in preserving genome stability by maintaining a subset of the genome in a silent state. Spatial genome organization contributes to normal patterns of gene function and expression, and is therefore of broad interest. Mammalian heterochromatin, the focus of this review, mainly localizes at the nuclear periphery, forming Lamina-associated domains (LADs), and at the nucleolar periphery, forming Nucleolus-associated domains (NADs). Together, these regions comprise approximately one-half of mammalian genomes, and most but not all loci within these domains are stochastically placed at either of these two locations after exit from mitosis at each cell cycle. Excitement about the role of these heterochromatic domains in early development has recently been heightened by the discovery that LADs appear at some loci in the preimplantation mouse embryo prior to other chromosomal features like compartmental identity and topologically-associated domains (TADs). While LADs have been extensively studied and mapped during cellular differentiation and early embryonic development, NADs have been less thoroughly studied. Here, we summarize pioneering studies of NADs and LADs, more recent advances in our understanding of cis/trans-acting factors that mediate these localizations, and discuss the functional significance of these associations.
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http://dx.doi.org/10.1016/j.bbagrm.2020.194666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855492PMC
January 2021

Distinct features of nucleolus-associated domains in mouse embryonic stem cells.

Chromosoma 2020 06 26;129(2):121-139. Epub 2020 Mar 26.

Department of Molecular, Cellular and Cancer Biology, University of Massachusetts Medical School, 364 Plantation St., Worcester, MA, 01605, USA.

Heterochromatin in eukaryotic interphase cells frequently localizes to the nucleolar periphery (nucleolus-associated domains (NADs)) and the nuclear lamina (lamina-associated domains (LADs)). Gene expression in somatic cell NADs is generally low, but NADs have not been characterized in mammalian stem cells. Here, we generated the first genome-wide map of NADs in mouse embryonic stem cells (mESCs) via deep sequencing of chromatin associated with biochemically purified nucleoli. As we had observed in mouse embryonic fibroblasts (MEFs), the large type I subset of NADs overlaps with constitutive LADs and is enriched for features of constitutive heterochromatin, including late replication timing and low gene density and expression levels. Conversely, the type II NAD subset overlaps with loci that are not lamina-associated, but in mESCs, type II NADs are much less abundant than in MEFs. mESC NADs are also much less enriched in H3K27me3 modified regions than are NADs in MEFs. Additionally, comparision of MEF and mESC NADs revealed enrichment of developmentally regulated genes in cell-type-specific NADs. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin. These studies implicate association with the nucleolar periphery as a mechanism for developmentally regulated gene expression and will facilitate future studies of NADs during mESC differentiation.
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http://dx.doi.org/10.1007/s00412-020-00734-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265113PMC
June 2020

Two contrasting classes of nucleolus-associated domains in mouse fibroblast heterochromatin.

Genome Res 2019 08 14;29(8):1235-1249. Epub 2019 Jun 14.

Department of Molecular, Cellular and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining nucleolus-associated domains (NADs) and lamina-associated domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called and demonstrated that it identifies NADs more accurately than other peak-calling tools, owing to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3, and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Type II NADs are enriched in distinct gene classes, including factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, and present in MEFs but not in undifferentiated embryonic stem (ES) cells.
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http://dx.doi.org/10.1101/gr.247072.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6673712PMC
August 2019

Correction to: Novel genetic tools for probing individual H3 molecules in each nucleosome.

Curr Genet 2019 04;65(2):379-380

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, 01605, USA.

In the original publication, Fig. 1 was incorrectly published. The amino acid sequence was shifted to the left relative to the rest of the diagram in the published version and the corrected figure is given here.
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http://dx.doi.org/10.1007/s00294-018-0919-4DOI Listing
April 2019

Novel genetic tools for probing individual H3 molecules in each nucleosome.

Curr Genet 2019 Apr 26;65(2):371-377. Epub 2018 Nov 26.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, 01605, USA.

In eukaryotes, genomic DNA is packaged into the nucleus together with histone proteins, forming chromatin. The fundamental repeating unit of chromatin is the nucleosome, a naturally symmetric structure that wraps DNA and is the substrate for numerous regulatory post-translational modifications. However, the biological significance of nucleosomal symmetry until recently had been unexplored. To investigate this issue, we developed an obligate pair of histone H3 heterodimers, a novel genetic tool that allowed us to modulate modification sites on individual H3 molecules within nucleosomes in vivo. We used these constructs for molecular genetic studies, for example demonstrating that H3K36 methylation on a single H3 molecule per nucleosome in vivo is sufficient to restrain cryptic transcription. We also used asymmetric nucleosomes for mass spectrometric analysis of dependency relationships among histone modifications. Furthermore, we extended this system to the centromeric H3 isoform (Cse4/CENP-A), gaining insights into centromeric nucleosomal symmetry and structure. In this review, we summarize our findings and discuss the utility of this novel approach.
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http://dx.doi.org/10.1007/s00294-018-0910-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421086PMC
April 2019

An asymmetric centromeric nucleosome.

Elife 2018 08 23;7. Epub 2018 Aug 23.

Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, United States.

Nucleosomes contain two copies of each core histone, held together by a naturally symmetric, homodimeric histone H3-H3 interface. This symmetry has complicated efforts to determine the regulatory potential of this architecture. Through molecular design and in vivo selection, we recently generated obligately heterodimeric H3s, providing a powerful tool for discovery of the degree to which nucleosome symmetry regulates chromosomal functions in living cells (Ichikawa et al., 2017). We now have extended this tool to the centromeric H3 isoform (Cse4/CENP-A) in budding yeast. These studies indicate that a single Cse4 N- or C-terminal extension per pair of Cse4 molecules is sufficient for kinetochore function, and validate previous experiments indicating that an octameric centromeric nucleosome is required for viability in this organism. These data also support the generality of the H3 asymmetric interface for probing general questions in chromatin biology.
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http://dx.doi.org/10.7554/eLife.37911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125124PMC
August 2018

Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes.

Bio Protoc 2018 Mar;8(6)

Department of Molecular, Cell, and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, USA.

Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the functional consequences of nucleosome symmetry, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, allowing us to compare cells with single or double H3 alterations. Our biochemical validation of the heterodimeric X-Y interaction included intra-nucleosomal H3 crosslinking using dimethyl suberimidate (DMS). Here, we provide a detailed protocol for the use of DMS to analyze yeast nucleosomes.
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http://dx.doi.org/10.21769/BioProtoc.2770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891137PMC
March 2018

New dimensions of asymmetric division in vertebrates.

Cytoskeleton (Hoboken) 2018 03 8;75(3):87-102. Epub 2018 Feb 8.

Program in Molecular Medicine University of Massachusetts Medical School, Worcester, Massachusetts.

Traditionally, we imagine that cell division gives rise to two identical daughter cells. Nevertheless, all cell divisions, to some degree, display asymmetry. Asymmetric cell division is defined as the generation of two daughter cells with different physical content and/or developmental potential. Several organelles and cellular components including the centrosome, non-coding RNA, chromatin, and recycling endosomes are involved in the process of asymmetric cell division. Disruption of this important process is known to induce profound defects in development, the immune response, regeneration of tissues, aging, and cancer. Here, we discuss recent advances that expand our understanding of the mechanisms and consequences of asymmetric cell division in vertebrate organisms.
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http://dx.doi.org/10.1002/cm.21434DOI Listing
March 2018

Ki-67: more than a proliferation marker.

Chromosoma 2018 06 10;127(2):175-186. Epub 2018 Jan 10.

Department of Molecular, Cell and Cancer Biology, University Massachusetts Medical School, 364 Plantation St. #506, Worcester, MA, 01605, USA.

Ki-67 protein has been widely used as a proliferation marker for human tumor cells for decades. In recent studies, multiple molecular functions of this large protein have become better understood. Ki-67 has roles in both interphase and mitotic cells, and its cellular distribution dramatically changes during cell cycle progression. These localizations correlate with distinct functions. For example, during interphase, Ki-67 is required for normal cellular distribution of heterochromatin antigens and for the nucleolar association of heterochromatin. During mitosis, Ki-67 is essential for formation of the perichromosomal layer (PCL), a ribonucleoprotein sheath coating the condensed chromosomes. In this structure, Ki-67 acts to prevent aggregation of mitotic chromosomes. Here, we present an overview of functional roles of Ki-67 across the cell cycle and also describe recent experiments that clarify its role in regulating cell cycle progression in human cells.
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http://dx.doi.org/10.1007/s00412-018-0659-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945335PMC
June 2018

A synthetic biology approach to probing nucleosome symmetry.

Elife 2017 09 12;6. Epub 2017 Sep 12.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, United States.

The repeating subunit of chromatin, the nucleosome, includes two copies of each of the four core histones, and several recent studies have reported that asymmetrically-modified nucleosomes occur at regulatory elements in vivo. To probe the mechanisms by which histone modifications are read out, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, which we extensively validated genetically and biochemically. Comparing the effects of asymmetric histone tail point mutants with those of symmetric double mutants revealed that a single methylated H3K36 per nucleosome was sufficient to silence cryptic transcription in vivo. We also demonstrate the utility of this system for analysis of histone modification crosstalk, using mass spectrometry to separately identify modifications on each H3 molecule within asymmetric nucleosomes. The ability to generate asymmetric nucleosomes in vivo and in vitro provides a powerful and generalizable tool to probe the mechanisms by which H3 tails are read out by effector proteins in the cell.
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http://dx.doi.org/10.7554/eLife.28836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626479PMC
September 2017

Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells.

Mol Cell Biol 2017 Sep 11;37(17). Epub 2017 Aug 11.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

The Ki-67 protein is widely used as a tumor proliferation marker. However, whether Ki-67 affects cell cycle progression has been controversial. Here we demonstrate that depletion of Ki-67 in human hTERT-RPE1, WI-38, IMR90, and hTERT-BJ cell lines and primary fibroblast cells slowed entry into S phase and coordinately downregulated genes related to DNA replication. Some gene expression changes were partially relieved in Ki-67-depleted hTERT-RPE1 cells by codepletion of the Rb checkpoint protein, but more thorough suppression of the transcriptional and cell cycle defects was observed upon depletion of the cell cycle inhibitor p21. Notably, induction of p21 upon depletion of Ki-67 was a consistent hallmark of cell types in which transcription and cell cycle distribution were sensitive to Ki-67; these responses were absent in cells that did not induce p21. Furthermore, upon Ki-67 depletion, a subset of inactive X (Xi) chromosomes in female hTERT-RPE1 cells displayed several features of compromised heterochromatin maintenance, including decreased H3K27me3 and H4K20me1 labeling. These chromatin alterations were limited to Xi chromosomes localized away from the nuclear lamina and were not observed in checkpoint-deficient 293T cells. Altogether, our results indicate that Ki-67 integrates normal S-phase progression and Xi heterochromatin maintenance in p21 checkpoint-proficient human cells.
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http://dx.doi.org/10.1128/MCB.00569-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559680PMC
September 2017

The p150N domain of chromatin assembly factor-1 regulates Ki-67 accumulation on the mitotic perichromosomal layer.

Mol Biol Cell 2017 01 2;28(1):21-29. Epub 2016 Nov 2.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605

Chromatin assembly factor 1 (CAF-1) deposits histones during DNA synthesis. The p150 subunit of human CAF-1 contains an N-terminal domain (p150N) that is dispensable for histone deposition but promotes the localization of specific loci (nucleolar-associated domains [NADs]) and proteins to the nucleolus during interphase. One of the p150N-regulated proteins is proliferation antigen Ki-67, whose depletion also decreases the nucleolar association of NADs. Ki-67 is also a fundamental component of the perichromosomal layer (PCL), a sheath of proteins surrounding condensed chromosomes during mitosis. We show here that a subset of p150 localizes to the PCL during mitosis and that p150N is required for normal levels of Ki-67 accumulation on the PCL. This activity requires the sumoylation-interacting motif within p150N, which is also required for the nucleolar localization of NADs and Ki-67 during interphase. In this manner, p150N coordinates both interphase and mitotic nuclear structures via Ki67.
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http://dx.doi.org/10.1091/mbc.E16-09-0659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5221625PMC
January 2017

Want reprogramming? Cut back on the chromatin assembly!

Authors:
Paul D Kaufman

Nat Struct Mol Biol 2015 Sep;22(9):648-50

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

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http://dx.doi.org/10.1038/nsmb.3081DOI Listing
September 2015

Grabbing the genome by the NADs.

Chromosoma 2016 06 15;125(3):361-71. Epub 2015 Jul 15.

Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA, 01605, USA.

The regions of the genome that interact frequently with the nucleolus have been termed nucleolar-associated domains (NADs). Deep sequencing and DNA-fluorescence in situ hybridization (FISH) experiments have revealed that these domains are enriched for repetitive elements, regions of the inactive X chromosome (Xi), and several RNA polymerase III-transcribed genes. NADs are often marked by chromatin modifications characteristic of heterochromatin, including H3K27me3, H3K9me3, and H4K20me3, and artificial targeting of genes to this area is correlated with reduced expression. It has therefore been hypothesized that NAD localization to the nucleolar periphery contributes to the establishment and/or maintenance of heterochromatic silencing. Recently published studies from several multicellular eukaryotes have begun to reveal the trans-acting factors involved in NAD localization, including the insulator protein CCCTC-binding factor (CTCF), chromatin assembly factor (CAF)-1 subunit p150, several nucleolar proteins, and two long non-coding RNAs (lncRNAs). The mechanisms by which these factors coordinate with one another in regulating NAD localization and/or silencing are still unknown. This review will summarize recently published studies, discuss where additional research is required, and speculate about the mechanistic and functional implications of genome organization around the nucleolus.
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http://dx.doi.org/10.1007/s00412-015-0527-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4714962PMC
June 2016

A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli.

Mol Biol Cell 2014 Sep 23;25(18):2866-81. Epub 2014 Jul 23.

Program in Gene Function and Expression, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605

Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element-containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.
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http://dx.doi.org/10.1091/mbc.E14-05-1029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161520PMC
September 2014

Chromatin-mediated Candida albicans virulence.

Biochim Biophys Acta 2013 Mar-Apr;1819(3-4):349-55

Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605-2324, USA.

Candida albicans is the most prevalent human fungal pathogen. To successfully propagate an infection, this organism relies on the ability to change morphology, express virulence-associated genes and resist DNA damage caused by the host immune system. Many of these events involve chromatin alterations that are crucial for virulence. This review will focus on the studies that have been conducted on how chromatin function affects pathogenicity of C. albicans and other fungi. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.
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February 2014

Chemical screening identifies filastatin, a small molecule inhibitor of Candida albicans adhesion, morphogenesis, and pathogenesis.

Proc Natl Acad Sci U S A 2013 Aug 31;110(33):13594-9. Epub 2013 Jul 31.

Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Infection by pathogenic fungi, such as Candida albicans, begins with adhesion to host cells or implanted medical devices followed by biofilm formation. By high-throughput phenotypic screening of small molecules, we identified compounds that inhibit adhesion of C. albicans to polystyrene. Our lead candidate compound also inhibits binding of C. albicans to cultured human epithelial cells, the yeast-to-hyphal morphological transition, induction of the hyphal-specific HWP1 promoter, biofilm formation on silicone elastomers, and pathogenesis in a nematode infection model as well as alters fungal morphology in a mouse mucosal infection assay. We term this compound filastatin based on its strong inhibition of filamentation, and we use chemical genetic experiments to show that it acts downstream of multiple signaling pathways. These studies show that high-throughput functional assays targeting fungal adhesion can provide chemical probes for study of multiple aspects of fungal pathogenesis.
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http://dx.doi.org/10.1073/pnas.1305982110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746938PMC
August 2013

A small molecule inhibitor of fungal histone acetyltransferase Rtt109.

Bioorg Med Chem Lett 2013 May 4;23(10):2853-9. Epub 2013 Apr 4.

Program in Gene Function and Expression, Program in Molecular Medicine, University of Massachusetts Medical School, 364 Plantation St., LRB506, Worcester, MA 01605, USA.

The histone acetyltransferase Rtt109 is the sole enzyme responsible for acetylation of histone H3 lysine 56 (H3K56) in fungal organisms. Loss of Rtt109 renders fungal cells extremely sensitive to genotoxic agents, and prevents pathogenesis in several clinically important species. Here, via a high throughput chemical screen of >300,000 compounds, we discovered a chemical inhibitor of Rtt109 that does not inhibit other acetyltransferase enzymes. This compound inhibits Rtt109 regardless of which histone chaperone cofactor protein (Asf1 or Vps75) is present, and appears to inhibit Rtt109 via a tight-binding, uncompetitive mechanism.
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http://dx.doi.org/10.1016/j.bmcl.2013.03.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3654155PMC
May 2013

Chromatin-mediated Candida albicans virulence.

Biochim Biophys Acta 2012 Mar 24;1819(3-4):349-55. Epub 2011 Aug 24.

Candida albicans is the most prevalent human fungal pathogen. To successfully propagate an infection, this organism relies on the ability to change morphology, express virulence-associated genes and resist DNA damage caused by the host immune system. Many of these events involve chromatin alterations that are crucial for virulence. This review will focus on the studies that have been conducted on how chromatin function affects pathogenicity of C. albicans and other fungi. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.
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http://dx.doi.org/10.1016/j.bbagrm.2011.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3243783PMC
March 2012

New partners for HP1 in transcriptional gene silencing.

Authors:
Paul D Kaufman

Mol Cell 2011 Jan;41(1):1-2

Program in Gene Function and Expression, University of Massachusetts Medical School, 364 Plantation Street, LRB506, Worcester, MA 01605, USA.

A new study in this issue of Molecular Cell (Yamane et al., 2010) demonstrates how chromatin assembly proteins HIRA/Asf1 help enforce transcriptional gene silencing in heterochromatin by bridging interactions between HP1 and histone deacetylase complexes.
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http://dx.doi.org/10.1016/j.molcel.2010.12.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038578PMC
January 2011

Catalytic activation of histone acetyltransferase Rtt109 by a histone chaperone.

Proc Natl Acad Sci U S A 2010 Nov 5;107(47):20275-80. Epub 2010 Nov 5.

Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA.

Most histone acetyltransferases (HATs) function as multisubunit complexes in which accessory proteins regulate substrate specificity and catalytic efficiency. Rtt109 is a particularly interesting example of a HAT whose specificity and catalytic activity require association with either of two histone chaperones, Vps75 or Asf1. Here, we utilize biochemical, structural, and genetic analyses to provide the detailed molecular mechanism for activation of a HAT (Rtt109) by its activating subunit Vps75. The rate-determining step of the activated complex is the transfer of the acetyl group from acetyl CoA to the acceptor lysine residue. Vps75 stimulates catalysis (> 250-fold), not by contributing a catalytic base, but by stabilizing the catalytically active conformation of Rtt109. To provide structural insight into the functional complex, we produced a molecular model of Rtt109-Vps75 based on X-ray diffraction of crystals of the complex. This model reveals distinct negative electrostatic surfaces on an Rtt109 molecule that interface with complementary electropositive ends of a symmetrical Vps75 dimer. Rtt109 variants with interface point substitutions lack the ability to be fully activated by Vps75, and one such variant displayed impaired Vps75-dependent histone acetylation functions in yeast, yet these variants showed no adverse effect on Asf1-dependent Rtt109 activities in vitro and in vivo. Finally, we provide evidence for a molecular model in which a 12 complex of Rtt109-Vps75 acetylates a heterodimer of H3-H4. The activation mechanism of Rtt109-Vps75 provides a valuable framework for understanding the molecular regulation of HATs within multisubunit complexes.
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http://dx.doi.org/10.1073/pnas.1009860107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996700PMC
November 2010

Overlapping regulation of CenH3 localization and histone H3 turnover by CAF-1 and HIR proteins in Saccharomyces cerevisiae.

Genetics 2011 Jan 13;187(1):9-19. Epub 2010 Oct 13.

Department of Molecular and Cell Biology, University of California, Berkeley, California 947202, USA.

Accurate chromosome segregation is dependent on the centromere-specific histone H3 isoform known generally as CenH3, or as Cse4 in budding yeast. Cytological experiments have shown that Cse4 appears at extracentromeric loci in yeast cells deficient for both the CAF-1 and HIR histone H3/H4 deposition complexes, consistent with increased nondisjunction in these double mutant cells. Here, we examined molecular aspects of this Cse4 mislocalization. Genome-scale chromatin immunoprecipitation analyses demonstrated broader distribution of Cse4 outside of centromeres in cac1Δ hir1Δ double mutant cells that lack both CAF-1 and HIR complexes than in either single mutant. However, cytological localization showed that the essential inner kinetochore component Mif2 (CENP-C) was not recruited to extracentromeric Cse4 in cac1Δ hir1Δ double mutant cells. We also observed that rpb1-1 mutants displayed a modestly increased Cse4 half-life at nonpermissive temperatures, suggesting that turnover of Cse4 is partially dependent on Pol II transcription. We used genome-scale assays to demonstrate that the CAF-1 and HIR complexes independently stimulate replication-independent histone H3 turnover rates. We discuss ways in which altered histone exchange kinetics may affect eviction of Cse4 from noncentromeric loci.
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http://dx.doi.org/10.1534/genetics.110.123117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3018296PMC
January 2011

Proliferating cell nuclear antigen (PCNA) is required for cell cycle-regulated silent chromatin on replicated and nonreplicated genes.

J Biol Chem 2010 Nov 2;285(45):35142-54. Epub 2010 Sep 2.

Department of Biochemistry, Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, USA.

In Saccharomyces cerevisiae, silent chromatin is formed at HMR upon the passage through S phase, yet neither the initiation of DNA replication at silencers nor the passage of a replication fork through HMR is required for silencing. Paradoxically, mutations in the DNA replication processivity factor, POL30, disrupt silencing despite this lack of requirement for DNA replication in the establishment of silencing. We tested whether pol30 mutants could establish silencing at either replicated or non-replicated HMR loci during S phase and found that pol30 mutants were defective in establishing silencing at HMR regardless of its replication status. Although previous studies tie the silencing defect of pol30 mutants to the chromatin assembly factors Asf1p and CAF-1, we found pol30 mutants did not exhibit a gross defect in packaging HMR into chromatin. Rather, the pol30 mutants exhibited defects in histone modifications linked to ASF1 and CAF-1-dependent pathways, including SAS-I- and Rtt109p-dependent acetylation events at H4-K16 and H3-K9 (plus H3-K56; Miller, A., Yang, B., Foster, T., and Kirchmaier, A. L. (2008) Genetics 179, 793-809). Additional experiments using FLIM-FRET revealed that Pol30p interacted with SAS-I and Rtt109p in the nuclei of living cells. However, these interactions were disrupted in pol30 mutants with defects linked to ASF1- and CAF-1-dependent pathways. Together, these results imply that Pol30p affects epigenetic processes by influencing the composition of chromosomal histone modifications.
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http://dx.doi.org/10.1074/jbc.M110.166918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2966128PMC
November 2010

Chromatin as a potential carrier of heritable information.

Curr Opin Cell Biol 2010 Jun 17;22(3):284-90. Epub 2010 Mar 17.

Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605, USA.

Organisms with the same genome can inherit information in addition to that encoded in the DNA sequence-this is known as epigenetic inheritance. Epigenetic inheritance is responsible for many of the phenotypic differences between different cell types in multicellular organisms. Work by many investigators over the past decades has suggested that a great deal of epigenetic information might be carried in the pattern of post-translational modifications of the histone proteins, although this is not as well established as many believe. For example, it is unclear whether and how the histones, which are displaced from the chromosome during passage of the replication fork and are often exchanged from the DNA template at other times, carry information from one cellular generation to the next. Here, we briefly review the evidence that some chromatin states are indeed heritable, and then focus on the mechanistic challenges that remain in order to understand how this inheritance can be achieved.
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http://dx.doi.org/10.1016/j.ceb.2010.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022377PMC
June 2010

Histone acetyltransferase Rtt109 is required for Candida albicans pathogenesis.

Proc Natl Acad Sci U S A 2010 Jan 4;107(4):1594-9. Epub 2010 Jan 4.

Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA 01605-2324, USA.

Candida albicans is a ubiquitous opportunistic pathogen that is the most prevalent cause of hospital-acquired fungal infections. In mammalian hosts, C. albicans is engulfed by phagocytes that attack the pathogen with DNA-damaging reactive oxygen species (ROS). Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 is important for yeast model organisms to survive DNA damage and maintain genome integrity. To assess the importance of Rtt109 for C. albicans pathogenicity, we deleted the predicted homolog of Rtt109 in the clinical C. albicans isolate, SC5314. C. albicans rtt109(-/-) mutant cells lack acetylated H3K56 (H3K56ac) and are hypersensitive to genotoxic agents. Additionally, rtt109(-/-) mutant cells constitutively display increased H2A S129 phosphorylation and elevated DNA repair gene expression, consistent with endogenous DNA damage. Importantly, C. albicans rtt109(-/-) cells are significantly less pathogenic in mice and more susceptible to killing by macrophages in vitro than are wild-type cells. Via pharmacological inhibition of the host NADPH oxidase enzyme, we show that the increased sensitivity of rtt109(-/-) cells to macrophages depends on the host's ability to generate ROS, providing a mechanistic link between the drug sensitivity, gene expression, and pathogenesis phenotypes. We conclude that Rtt109 is particularly important for fungal pathogenicity, suggesting a unique target for therapeutic antifungal compounds.
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http://dx.doi.org/10.1073/pnas.0912427107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2824404PMC
January 2010

A negatively charged residue in place of histone H3K56 supports chromatin assembly factor association but not genotoxic stress resistance.

DNA Repair (Amst) 2009 Dec 30;8(12):1371-9. Epub 2009 Sep 30.

Program in Gene Function and Expression, University of Massachusetts Medical School, 364 Plantation St. #506, Worcester, MA 01605, USA.

In fungal species, lysine 56 of newly synthesized histone H3 molecules is modified by the acetyltransferase Rtt109, which promotes resistance to genotoxic agents. To further explore how H3 K56ac contributes to genome stability, we conducted screens for suppressors of the DNA damage sensitivity of budding yeast rtt109 Delta mutants. We recovered a single extragenic suppressor mutation that efficiently restored damage resistance. The suppressor is a point mutation in the histone H3 gene HHT2, and converts lysine 56 to glutamic acid. In some ways, K56E mimics K56ac, because it suppresses other mutations that interfere with the production of H3 K56ac and restores histone binding to chromatin assembly proteins CAF-1 and Rtt106. Therefore, we demonstrate that enhanced association with chromatin assembly factors can be accomplished not only by acetylation-mediated charge neutralization of H3K56 but also by the replacement of the positively charged lysine with an acidic residue. These data suggest that removal of the positive charge on lysine 56 is the functionally important consequence of H3K56 acetylation. Additionally, the suppressive function of K56E requires the presence of a second H3 allele, because K56E impairs growth when it is the sole source of histones, even more so than does constitutive H3K56 acetylation. Our studies therefore emphasize how H3 K56ac not only promotes chromatin assembly but also leads to chromosomal malfunction if not removed following histone deposition.
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http://dx.doi.org/10.1016/j.dnarep.2009.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2787813PMC
December 2009

A versatile viral system for expression and depletion of proteins in mammalian cells.

PLoS One 2009 Aug 6;4(8):e6529. Epub 2009 Aug 6.

Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.

The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006529PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2717805PMC
August 2009

Molecular functions of the histone acetyltransferase chaperone complex Rtt109-Vps75.

Nat Struct Mol Biol 2008 Sep;15(9):948-56

Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, 1300 University Avenue, Madison, Wisconsin 53706, USA.

Histone acetylation and nucleosome remodeling regulate DNA damage repair, replication and transcription. Rtt109, a recently discovered histone acetyltransferase (HAT) from Saccharomyces cerevisiae, functions with the histone chaperone Asf1 to acetylate lysine K56 on histone H3 (H3K56), a modification associated with newly synthesized histones. In vitro analysis of Rtt109 revealed that Vps75, a Nap1 family histone chaperone, could also stimulate Rtt109-dependent acetylation of H3K56. However, the molecular function of the Rtt109-Vps75 complex remains elusive. Here we have probed the molecular functions of Vps75 and the Rtt109-Vps75 complex through biochemical, structural and genetic means. We find that Vps75 stimulates the kcat of histone acetylation by approximately 100-fold relative to Rtt109 alone and enhances acetylation of K9 in the H3 histone tail. Consistent with the in vitro evidence, cells lacking Vps75 showed a substantial reduction (60%) in H3K9 acetylation during S phase. X-ray structural, biochemical and genetic analyses of Vps75 indicate a unique, structurally dynamic Nap1-like fold that suggests a potential mechanism of Vps75-dependent activation of Rttl09. Together, these data provide evidence for a multifunctional HAT-chaperone complex that acetylates histone H3 and deposits H3-H4 onto DNA, linking histone modification and nucleosome assembly.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2678805PMC
http://dx.doi.org/10.1038/nsmb.1459DOI Listing
September 2008

Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast.

PLoS Genet 2008 Nov 21;4(11):e1000270. Epub 2008 Nov 21.

School of Computer Science and Engineering, The Hebrew University, Jerusalem, Israel.

Acetylation of histone H3 lysine 56 is a covalent modification best known as a mark of newly replicated chromatin, but it has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase and a further specific decrease in turnover at several early origins of replication, which switch from rapidly replaced in G1 phase to stably bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle and suggest that H3K56 acetylation provides a positive-feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.
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http://dx.doi.org/10.1371/journal.pgen.1000270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581598PMC
November 2008