67 results on '"Paul D. Kaufman"'
Search Results
2. Novel genetic tools for probing individual H3 molecules in each nucleosome
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Yuichi Ichikawa and Paul D. Kaufman
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Transcription, Genetic ,Centromere ,Computational biology ,Article ,Histones ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Histone H3 ,Transcription (biology) ,Genetics ,Nucleosome ,030304 developmental biology ,0303 health sciences ,biology ,030302 biochemistry & molecular biology ,Genomics ,General Medicine ,Nucleosomes ,Chromatin ,genomic DNA ,Histone ,Gene Expression Regulation ,chemistry ,Mutation ,biology.protein ,Protein Multimerization ,DNA - Abstract
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.
- Published
- 2018
3. Ki-67: more than a proliferation marker
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Paul D. Kaufman and Xiaoming Sun
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Cyclin-Dependent Kinase Inhibitor p21 ,0301 basic medicine ,Heterochromatin ,Mitosis ,Retinoblastoma Protein ,Article ,03 medical and health sciences ,Protein Domains ,Cell Line, Tumor ,Genetics ,Humans ,Protein Isoforms ,Proliferation Marker ,Amino Acid Sequence ,Interphase ,Genetics (clinical) ,Cell Proliferation ,Ribonucleoprotein ,biology ,Cell cycle ,Cell biology ,Ki-67 Antigen ,030104 developmental biology ,Gene Expression Regulation ,Ribonucleoproteins ,Ki-67 ,biology.protein ,Tumor Suppressor Protein p53 ,Developmental biology ,Cell Nucleolus - Abstract
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.
- Published
- 2018
4. Distinct features of nucleolus-associated domains in mouse embryonic stem cells
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Paul D. Kaufman, Lihua Julie Zhu, Aimin Yan, Aizhan Bizhanova, and Jun Yu
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Heterochromatin ,Nucleolus ,Gene Expression ,Genomics ,Article ,Histones ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,Animals ,Constitutive heterochromatin ,In Situ Hybridization, Fluorescence ,Genetics (clinical) ,030304 developmental biology ,0303 health sciences ,Replication timing ,Nuclear Lamina ,biology ,Chromosome Mapping ,Computational Biology ,Cell Differentiation ,Mouse Embryonic Stem Cells ,Fibroblasts ,Embryonic stem cell ,Chromatin ,Cell biology ,Gene Ontology ,Histone ,biology.protein ,Nuclear lamina ,Biomarkers ,Cell Nucleolus ,030217 neurology & neurosurgery - Abstract
BackgroundHeterochromatin 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.ResultsHere, 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.ConclusionsThese studies implicate association with the nucleolar periphery as a mechanism for developmentally-regulated gene silencing, and will facilitate future studies of NADs during mESC differentiation.
- Published
- 2019
5. Two Contrasting Classes of Nucleolus-Associated Domains in Mouse Fibroblast Heterochromatin
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Anastassiia Vertii, Hervé Pagès, Lihua Julie Zhu, Jianhong Ou, Aimin Yan, Haibo Liu, Paul D. Kaufman, and Jun Yu
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DNA Replication ,Nucleolus ,Heterochromatin ,Biology ,Histones ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Gene expression ,Genetics ,Animals ,Constitutive heterochromatin ,Gene ,Genetics (clinical) ,Cells, Cultured ,In Situ Hybridization, Fluorescence ,030304 developmental biology ,0303 health sciences ,Genome ,Nuclear Lamina ,Research ,DNA replication ,Chromosome Mapping ,Gene Expression Regulation, Developmental ,Fibroblasts ,Embryo, Mammalian ,Chromatin ,Cell biology ,Histone ,biology.protein ,Nuclear lamina ,Cell Nucleolus ,030217 neurology & neurosurgery - Abstract
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 NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, due 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 with 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. Interestingly, Type II NADs are enriched in distinct gene classes, notably factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, present in MEFs but not in undifferentiated embryonic stem (ES) cells.
- Published
- 2018
- Full Text
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6. Close to the edge: Heterochromatin at the nucleolar and nuclear peripheries
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Paul D. Kaufman and Aizhan Bizhanova
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Heterochromatin ,Cellular differentiation ,Biophysics ,Biology ,Biochemistry ,Genome ,Genomic Instability ,Article ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,Genetics ,Animals ,Humans ,Molecular Biology ,Mitosis ,Gene ,030304 developmental biology ,Genomic organization ,0303 health sciences ,Genome, Human ,Chromosome ,Chromatin ,Cell biology ,Cell Nucleolus ,030217 neurology & neurosurgery - Abstract
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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- 2021
7. Author response: An asymmetric centromeric nucleosome
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Noriko Saitoh, Yuichi Ichikawa, and Paul D. Kaufman
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Physics ,Centromeric nucleosome ,Cell biology - Published
- 2018
8. An asymmetric centromeric nucleosome
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Noriko Saitoh, Yuichi Ichikawa, and Paul D. Kaufman
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0301 basic medicine ,Gene isoform ,Saccharomyces cerevisiae Proteins ,QH301-705.5 ,Science ,Centromere ,Static Electricity ,S. cerevisiae ,Saccharomyces cerevisiae ,Computational biology ,histone ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Protein Domains ,Nucleosome ,Amino Acid Sequence ,Biology (General) ,Microbial Viability ,Centromeric nucleosome ,Base Sequence ,General Immunology and Microbiology ,biology ,Kinetochore ,General Neuroscience ,nucleosome ,Temperature ,General Medicine ,Chromosomes and Gene Expression ,Budding yeast ,Nucleosomes ,Chromatin ,kinetochore ,030104 developmental biology ,Histone ,Mutation ,biology.protein ,Medicine ,Protein Multimerization ,Research Advance ,Function (biology) ,Plasmids - Abstract
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.
- Published
- 2018
9. Histone chaperones and chromatin assembly
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Paul D. Kaufman
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Chemistry ,Biophysics ,Biochemistry ,Chromatin remodeling ,Chromatin ,Cell biology ,Histone H1 ,Structural Biology ,Histone methyltransferase ,Histone H2A ,Histone methylation ,Genetics ,Nucleosome ,Histone code ,Molecular Biology - Published
- 2018
10. Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes
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Paul D. Kaufman and Yuichi Ichikawa
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0301 basic medicine ,Streptavidin affinity chromatography ,Strategy and Management ,Dimethyl Suberimidate ,Crosslink ,Axis of symmetry ,Industrial and Manufacturing Engineering ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Protein-protein interaction ,Nucleosome ,Molecule ,biology ,Mechanical Engineering ,Metals and Alloys ,Yeast ,Chromatin ,Histone ,030104 developmental biology ,chemistry ,Eukaryotic chromosome fine structure ,biology.protein ,Biophysics ,030217 neurology & neurosurgery ,DNA - Abstract
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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- 2018
11. Correction to: Novel genetic tools for probing individual H3 molecules in each nucleosome
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Paul D. Kaufman and Yuichi Ichikawa
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Rest (physics) ,Published Erratum ,Diagram ,Genetics ,Nucleosome ,General Medicine ,Computational biology ,Biology ,Proteomics ,Peptide sequence - Abstract
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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- 2018
12. Grabbing the genome by the NADs
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Paul D. Kaufman and Timothy D. Matheson
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0301 basic medicine ,Genetics ,Chromosomes, Human, X ,Genome, Human ,Nucleolus ,Heterochromatin ,Biology ,Chromatin Assembly and Disassembly ,Genome ,Article ,Cell biology ,Chromatin ,03 medical and health sciences ,030104 developmental biology ,X Chromosome Inactivation ,CTCF ,Animals ,Humans ,Human genome ,Gene ,Cell Nucleolus ,Genetics (clinical) ,Genomic organization - Abstract
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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- 2015
13. Author response: A synthetic biology approach to probing nucleosome symmetry
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Neil L. Kelleher, Christoph W. Müller, Caitlin F. Connelly, Yuanyuan Chen, Hsin-Jung Chou, Vineeta Bajaj, Oliver J. Rando, Thomas C. Miller, Daniel N. Bolon, Upasna Sharma, Paul M. Thomas, Nir Friedman, Hadas Jacobi, Hsuiyi V Chen, Yuichi Ichikawa, Paul D. Kaufman, Alon Appleboim, Yupeng Zheng, and Nebiyu Abshiru
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Physics ,Synthetic biology ,Theoretical physics ,Nucleosome ,Symmetry (geometry) - Published
- 2017
14. Ki-67 contributes to normal cell cycle progression and inactive X heterochromatin in p21 checkpoint-proficient human cells
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Jun Yu, Xiaoming Sun, Paul D. Kaufman, Timothy D. Matheson, Aizhan Bizhanova, and Lihua Julie Zhu
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0301 basic medicine ,Cyclin-Dependent Kinase Inhibitor p21 ,DNA Replication ,Cell type ,Cell division ,Heterochromatin ,HEK 293 cells ,Cell Cycle ,Cell Biology ,Biology ,Cell cycle ,Chromatin ,Cell biology ,Cell Line ,03 medical and health sciences ,030104 developmental biology ,Ki-67 Antigen ,Cell culture ,Nuclear lamina ,Humans ,Heterochromatin maintenance ,Molecular Biology ,Cell Division ,Research Article - Abstract
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, 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 co-depletion of the Rb checkpoint protein, but more thorough suppression of the transcriptional and cell cycle defects was observed upon depletion of 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 × (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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- 2017
- Full Text
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15. A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli
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Paul D. Kaufman, Xiaoming Sun, Timothy D. Matheson, Xuemei Han, Carolyn L. Smith, Eric Campeau, John R. Yates, and Daniel J. Trombly
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NPM1 ,Nucleolus ,Protein subunit ,Biology ,SAP30 ,Chromosomes, Human ,Humans ,Protein Interaction Domains and Motifs ,Chromatin Assembly Factor-1 ,Molecular Biology ,chemistry.chemical_classification ,DNA ligase ,Nuclear Functions ,Articles ,Cell Biology ,Molecular biology ,Transport protein ,Cell biology ,Protein Transport ,Ki-67 Antigen ,Histone ,chemistry ,biology.protein ,Cell Nucleolus ,HeLa Cells ,Protein Binding ,Transcription Factors - Abstract
Chromatin assembly factor-1 contains a separable domain unrelated to histone deposition, which provides a previously unrecognized ability to maintain nucleolar protein and chromosome associations., 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.
- Published
- 2014
16. Want reprogramming? Cut back on the chromatin assembly!
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Paul D. Kaufman
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Nucleosome assembly ,Cellular differentiation ,Totipotent ,Biology ,Embryonic stem cell ,Chromatin ,Cell biology ,chemistry.chemical_compound ,chemistry ,Structural Biology ,Stem cell ,Molecular Biology ,Reprogramming ,DNA - Abstract
Totipotency, the ability of early embryonic cells to generate a complete adult organism as well as extraembryonic tissue, is a fleeting property found only in very early embryonic cells. A breakthrough study now shows that inhibition of DNA replication–linked nucleosome assembly causes embryonic stem cells to resemble totipotent cells. Notably, inhibition of chromatin assembly stimulates reprogramming during somatic-cell nuclear transfer experiments.
- Published
- 2015
17. The p150N domain of Chromatin Assembly Factor-1 regulates Ki-67 accumulation on the mitotic perichromosomal layer
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Timothy D. Matheson and Paul D. Kaufman
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0301 basic medicine ,Nucleolus ,Protein subunit ,SUMO protein ,Mitosis ,Chromosomes ,Histones ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Chromatin Assembly Factor-1 ,Interphase ,Molecular Biology ,Transcription factor ,030304 developmental biology ,0303 health sciences ,DNA synthesis ,biology ,Chemistry ,Cell Cycle ,Cell Biology ,Chromatin ,Cell biology ,Ki-67 Antigen ,030104 developmental biology ,Histone ,Chromosome Structures ,030220 oncology & carcinogenesis ,Premature chromosome condensation ,biology.protein ,Brief Reports ,Cell Nucleolus ,HeLa Cells ,Transcription Factors - Abstract
Human chromatin assembly factor-1 contains a domain (p150N) dispensable for histone deposition but required for normal levels of Ki-67 accumulation on the mitotic perichromosomal layer. This activity requires the sumoylation-interacting motif within p150N. Thus p150N coordinates both interphase and mitotic nuclear structures via Ki67., 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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- 2016
- Full Text
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18. Chromatin-mediated Candida albicans virulence
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Paul D. Kaufman and Jessica Lopes da Rosa
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Genome instability ,biology ,DNA damage ,Biophysics ,Virulence ,biology.organism_classification ,Biochemistry ,Article ,Corpus albicans ,Microbiology ,Chromatin ,Immune system ,Structural Biology ,Genetics ,Candida albicans ,Molecular Biology ,Gene - Abstract
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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- 2012
19. Overlapping Regulation of CenH3 Localization and Histone H3 Turnover by CAF-1 and HIR Proteins in Saccharomyces cerevisiae
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Oliver J. Rando, Erin M. Green, Paul D. Kaufman, John Holik, and Jessica Lopes da Rosa
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Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Cell Survival ,Chromosomal Proteins, Non-Histone ,Centromere ,Mutant ,Saccharomyces cerevisiae ,Histone exchange ,Investigations ,Histones ,Histone H3 ,Ribonucleases ,Histone H1 ,Histone H2A ,Genetics ,Animals ,Humans ,Kinetochores ,CAF-1 ,biology ,Protein Stability ,Temperature ,Nuclear Proteins ,Genomics ,Chromatin Assembly and Disassembly ,Molecular biology ,DNA-Binding Proteins ,Enzyme Activation ,Repressor Proteins ,Protein Transport ,Histone ,Mutation ,biology.protein ,RNA Polymerase II ,Chromatin immunoprecipitation ,Half-Life - Abstract
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.
- Published
- 2011
20. Proliferating Cell Nuclear Antigen (PCNA) Is Required for Cell Cycle-regulated Silent Chromatin on Replicated and Nonreplicated Genes
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Joseph Irudayaraj, Ann L. Kirchmaier, Jiji Chen, Andrew P. Miller, Jennifer L. Jacobi, Paul D. Kaufman, and Taichi E. Takasuka
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DNA Replication ,Saccharomyces cerevisiae Proteins ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Biochemistry ,S Phase ,Histones ,Ribonucleases ,Proliferating Cell Nuclear Antigen ,Gene Regulation ,Gene Silencing ,Epigenetics ,DNA, Fungal ,Molecular Biology ,Gene ,Histone Acetyltransferases ,Genetics ,biology ,DNA replication ,Acetylation ,Antigens, Nuclear ,Cell Biology ,Cell cycle ,Chromatin ,Proliferating cell nuclear antigen ,Histone ,Genetic Loci ,Mutation ,biology.protein ,Origin recognition complex ,Chromosomes, Fungal ,Molecular Chaperones - Abstract
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.
- Published
- 2010
21. Cover Image, Volume 75, Issue 3
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Anastassiia Vertii, Paul D. Kaufman, Heidi Hehnly, and Stephen Doxsey
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Structural Biology ,Cell Biology - Published
- 2018
22. A negatively charged residue in place of histone H3K56 supports chromatin assembly factor association but not genotoxic stress resistance
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Paul D. Kaufman and Judith A. Erkmann
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Saccharomyces cerevisiae Proteins ,Saccharomyces cerevisiae ,Cell Biology ,Biology ,Chromatin Assembly and Disassembly ,Biochemistry ,Article ,Chromatin remodeling ,Histones ,Histone H3 ,Phenotype ,Histone H1 ,Gene Expression Regulation, Fungal ,Histone methyltransferase ,Mutation ,Histone H2A ,Histone code ,Histone octamer ,Molecular Biology ,Alleles ,DNA Damage ,Histone Acetyltransferases ,Histone binding - Abstract
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.
- Published
- 2009
23. Molecular functions of the histone acetyltransferase chaperone complex Rtt109–Vps75
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Christopher E. Berndsen, James L. Keck, Paul D. Kaufman, John M. Denu, Toshiaki Tsubota, Scott E. Lindner, Susan Lee, and James M. Holton
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Models, Molecular ,Saccharomyces cerevisiae Proteins ,Molecular Sequence Data ,Static Electricity ,Saccharomyces cerevisiae ,In Vitro Techniques ,Xenopus Proteins ,Biology ,SAP30 ,Crystallography, X-Ray ,Article ,Substrate Specificity ,Histones ,Xenopus laevis ,03 medical and health sciences ,Histone H3 ,Histone H1 ,Structural Biology ,Catalytic Domain ,Histone H2A ,Animals ,Histone code ,Amino Acid Sequence ,Histone octamer ,Molecular Biology ,Histone Acetyltransferases ,030304 developmental biology ,0303 health sciences ,030302 biochemistry & molecular biology ,Histone acetyltransferase ,Recombinant Proteins ,Protein Structure, Tertiary ,Enzyme Activation ,Kinetics ,Biochemistry ,Multiprotein Complexes ,Histone methyltransferase ,biology.protein ,Molecular Chaperones - Abstract
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.
- Published
- 2008
24. Histone H3-K56 Acetylation Is Catalyzed by Histone Chaperone-Dependent Complexes
- Author
-
Judith A. Erkmann, John M. Denu, Carolyn L. Smith, Lanhao Yang, Toshiaki Tsubota, Michael A. Freitas, Paul D. Kaufman, and Christopher E. Berndsen
- Subjects
Saccharomyces cerevisiae Proteins ,Molecular Sequence Data ,Coenzymes ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Biology ,SAP30 ,Catalysis ,Mass Spectrometry ,Article ,Substrate Specificity ,Histones ,Histone H3 ,Histone H1 ,Histone H2A ,Animals ,Histone code ,Amino Acid Sequence ,Histone octamer ,Amino Acids ,DNA, Fungal ,Molecular Biology ,Histone Acetyltransferases ,Histone binding ,Lysine ,Acetylation ,Cell Biology ,Histone acetyltransferase ,Recombinant Proteins ,Kinetics ,Protein Subunits ,Biochemistry ,Multiprotein Complexes ,biology.protein ,Chickens ,Molecular Chaperones ,Protein Binding - Abstract
Acetylation of histone H3 on lysine 56 occurs during mitotic and meiotic S phase in fungal species. This acetylation blocks a direct electrostatic interaction between histone H3 and nucleosomal DNA, and the absence of this modification is associated with extreme sensitivity to genotoxic agents. We show here that H3-K56 acetylation is catalyzed when Rtt109, a protein that lacks significant homology to known acetyltransferases, forms an active complex with either of two histone binding proteins, Asf1 or Vps75. Rtt109 binds to both these cofactors, but not to histones alone, forming enzyme complexes with kinetic parameters similar to those of known histone acetyltransferase (HAT) enzymes. Therefore, H3-K56 acetylation is catalyzed by a previously unknown mechanism that requires a complex of two proteins: Rtt109 and a histone chaperone. Additionally, these complexes are functionally distinct, with the Rtt109/Asf1 complex, but not the Rtt109/Vps75 complex, being critical for resistance to genotoxic agents.
- Published
- 2007
25. Regulation of Histone Deposition Proteins Asf1/Hir1 by Multiple DNA Damage Checkpoint Kinases in Saccharomyces cerevisiae
- Author
-
Paul D. Kaufman, Judith A. Sharp, and Gizem Rizki
- Subjects
Saccharomyces cerevisiae Proteins ,Saccharomyces cerevisiae ,Cell Cycle Proteins ,Investigations ,Protein Serine-Threonine Kinases ,medicine.disease_cause ,Histones ,Histone H3 ,HIR complex ,Genetics ,medicine ,Gene silencing ,Gene Silencing ,Mutation ,biology ,Kinase ,Intracellular Signaling Peptides and Proteins ,Nuclear Proteins ,Epistasis, Genetic ,G2-M DNA damage checkpoint ,biology.organism_classification ,Molecular biology ,Protein Structure, Tertiary ,Cell biology ,Repressor Proteins ,Checkpoint Kinase 2 ,Histone ,biology.protein ,Protein Kinases ,DNA Damage ,Molecular Chaperones - Abstract
CAF-1, Hir proteins, and Asf1 are histone H3/H4 binding proteins important for chromatin-mediated transcriptional silencing. We explored genetic and physical interactions between these proteins and S-phase/DNA damage checkpoint kinases in the budding yeast Saccharomyces cerevisiae. Although cells lacking checkpoint kinase Mec1 do not display defects in telomeric gene silencing, silencing was dramatically reduced in cells lacking both Mec1 and the Cac1 subunit of CAF-1. Silencing was restored in cac1Δ and cac1Δ mec1Δ cells upon deletion of Rad53, the kinase downstream of Mec1. Restoration of silencing to cac1Δ cells required both Hir1 and Asf1, suggesting that Mec1 counteracts functional sequestration of the Asf1/Hir1 complex by Rad53. Consistent with this idea, the degree of suppression of silencing defects by rad53 alleles correlated with effects on Asf1 binding. Furthermore, deletion of the Dun1 kinase, a downstream target of Rad53, also suppressed the silencing defects of cac1Δ cells and reduced the levels of Asf1 associated with Rad53 in vivo. Loss of Mec1 and Rad53 did not alter telomere lengths or Asf1 protein levels, nuclear localization, or chromosome association. We conclude that the Mec1 and Dun1 checkpoint kinases regulate the Asf1-Rad53 interaction and therefore affect the activity of the Asf1/Hir complex in vivo.
- Published
- 2005
26. Sas4 and Sas5 Are Required for the Histone Acetyltransferase Activity of Sas2 in the SAS Complex
- Author
-
Wei-Jong Shia, Jerry L. Workman, David A. Band, Paul D. Kaufman, Rolf Sternglanz, Shigehiro Osada, and Ann Sutton
- Subjects
Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Saccharomyces cerevisiae ,Lysine ,Biochemistry ,law.invention ,chemistry.chemical_compound ,Acetyltransferases ,law ,Histone acetyltransferase activity ,Gene Silencing ,Molecular Biology ,Histone Acetyltransferases ,biology ,Acetylation ,Cell Biology ,biology.organism_classification ,Enzyme Activation ,Histone ,chemistry ,biology.protein ,Recombinant DNA ,DNA - Abstract
The SAS2 gene is involved in transcriptional silencing in Saccharomyces cerevisiae. Based on its primary sequence, the Sas2 protein is predicted to be a member of the MYST family of histone acetyltransferases (HATs). Sas2 forms a complex with Sas4 and Sas5, which are required for its silencing function. Here we show that recombinant Sas2 has HAT activity that absolutely requires Sas4 and is stimulated by Sas5. The recombinant SAS complex acetylates H4 lysine 16 and H3 lysine 14. Furthermore, a purified SAS complex from yeast shows similar activity and specificity. In contrast to other MYST HATs, neither the recombinant nor the native SAS complex acetylated nucleosomal histones under conditions that were optimum for acetylating free histones. Finally, although the SAS subunits interact genetically and physically with Asf1, a histone deposition factor, association of H3 and H4 with Asf1 blocks their acetylation by the SAS complex, raising the possibility that the SAS HAT complex may acetylate free histones prior to their deposition onto DNA by Asf1 or CAF-I.
- Published
- 2003
27. Defects in SPT16 or POB3 (yFACT) in Saccharomyces cerevisiae Cause Dependence on the Hir/Hpc Pathway: Polymerase Passage May Degrade Chromatin Structure
- Author
-
Susan Ruone, Peter Eriksson, Tim Formosa, Paul D. Kaufman, David J. Stillman, Alison R. Rhoades, Melissa D. Adams, Aileen E. Olsen, and Yaxin Yu
- Subjects
Transcriptional Activation ,Saccharomyces cerevisiae Proteins ,Chromosomal Proteins, Non-Histone ,Genes, Fungal ,Mutant ,Gene Dosage ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Models, Biological ,DNA-binding protein ,Histones ,Acetyltransferases ,Genetics ,Nucleosome ,Chromatin Assembly Factor-1 ,Transcription factor ,Histone Acetyltransferases ,biology ,Nuclear Proteins ,FACT complex ,Chromatin ,DNA-Binding Proteins ,Repressor Proteins ,Phenotype ,Histone ,Mutation ,Trans-Activators ,biology.protein ,HMGN Proteins ,Transcriptional Elongation Factors ,Carrier Proteins ,Transcription Factors ,Research Article - Abstract
Spt16/Cdc68, Pob3, and Nhp6 collaborate in vitro and in vivo as the yeast factor SPN, which is homologous to human FACT. SPN/FACT complexes mediate passage of polymerases through nucleosomes and are important for both transcription and replication. An spt16 mutation was found to be intolerable when combined with a mutation in any member of the set of functionally related genes HIR1, HIR2/SPT1, HIR3/HPC1, or HPC2. Mutations in POB3, but not in NHP6A/B, also display strong synthetic defects with hir/hpc mutations. A screen for other mutations that cause dependence on HIR/HPC genes revealed genes encoding members of the Paf1 complex, which also promotes transcriptional elongation. The Hir/Hpc proteins affect the expression of histone genes and also promote normal deposition of nucleosomes; either role could explain an interaction with elongation factors. We show that both spt16 and pob3 mutants respond to changes in histone gene numbers, but in opposite ways, suggesting that Spt16 and Pob3 each interact with histones but perhaps with different subsets of these proteins. Supporting this, spt16 and pob3 mutants also display different sensitivities to mutations in the N-terminal tails of histones H3 and H4 and to mutations in enzymes that modulate acetylation of these tails. Our results support a model in which SPN/FACT has two functions: it disrupts nucleosomes to allow polymerases to access DNA, and it reassembles the nucleosomes afterward. Mutations that impair the reassembly activity cause chromatin to accumulate in an abnormally disrupted state, imposing a requirement for a nucleosome reassembly function that we propose is provided by Hir/Hpc proteins.
- Published
- 2002
28. Chromatin Assembly Factor I Mutants Defective for PCNA Binding Require Asf1/Hir Proteins for Silencing
- Author
-
Tamar Kama, Denise C. Krawitz, and Paul D. Kaufman
- Subjects
Saccharomyces cerevisiae Proteins ,Nucleosome assembly ,Chromosomal Proteins, Non-Histone ,Genes, Fungal ,Molecular Sequence Data ,Mutant ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Biology ,Fungal Proteins ,Histone H3 ,Proliferating Cell Nuclear Antigen ,Amino Acid Sequence ,Gene Silencing ,Chromatin Assembly Factor-1 ,Molecular Biology ,Alleles ,CAF-1 ,Binding Sites ,Base Sequence ,Sequence Homology, Amino Acid ,Chromatin Assembly Factor I ,Nuclear Proteins ,Cell Biology ,Telomere ,DNA Dynamics and Chromosome Structure ,Molecular biology ,Chromatin ,DNA-Binding Proteins ,Repressor Proteins ,Protein Subunits ,Phenotype ,Histone ,Mutation ,biology.protein ,Molecular Chaperones ,Plasmids - Abstract
Chromatin assembly factor I (CAF-I) is a conserved histone H3/H4 deposition complex. Saccharomyces cerevisiae mutants lacking CAF-I subunit genes (CAC1 to CAC3) display reduced heterochromatic gene silencing. In a screen for silencing-impaired cac1 alleles, we isolated a mutation that reduced binding to the Cac3p subunit and another that impaired binding to the DNA replication protein PCNA. Surprisingly, mutations in Cac1p that abolished PCNA binding resulted in very minor telomeric silencing defects but caused silencing to be largely dependent on Hir proteins and Asf1p, which together comprise an alternative silencing pathway. Consistent with these phenotypes, mutant CAF-I complexes defective for PCNA binding displayed reduced nucleosome assembly activity in vitro but were stimulated by Asf1p-histone complexes. Furthermore, these mutant CAF-I complexes displayed a reduced preference for depositing histones onto newly replicated DNA. We also observed a weak interaction between Asf1p and Cac2p in vitro, and we hypothesize that this interaction underlies the functional synergy between these histone deposition proteins.
- Published
- 2002
29. Chromatin-mediated Candida albicans virulence
- Author
-
Jessica, Lopes da Rosa and Paul D, Kaufman
- Subjects
DNA Repair ,Transcription, Genetic ,Virulence ,Candida albicans ,Host-Pathogen Interactions ,Candidiasis ,Humans ,Chromatin ,Genomic Instability - Abstract
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.
- Published
- 2014
30. Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing
- Author
-
Erik T. Fouts, Paul D. Kaufman, Judith A. Sharp, and Denise C. Krawitz
- Subjects
Saccharomyces cerevisiae Proteins ,Nucleosome assembly ,Chromosomal Proteins, Non-Histone ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,General Biochemistry, Genetics and Molecular Biology ,Fungal Proteins ,Histones ,03 medical and health sciences ,Ribonucleases ,Gene Expression Regulation, Fungal ,Proliferating Cell Nuclear Antigen ,Gene silencing ,Nucleosome ,Gene Silencing ,Chromatin Assembly Factor-1 ,Gene ,DNA Polymerase III ,030304 developmental biology ,0303 health sciences ,Agricultural and Biological Sciences(all) ,biology ,Biochemistry, Genetics and Molecular Biology(all) ,Chromatin Assembly Factor I ,030302 biochemistry & molecular biology ,DNA replication ,Nuclear Proteins ,Molecular biology ,Nucleosomes ,DNA-Binding Proteins ,Repressor Proteins ,Histone ,biology.protein ,Carrier Proteins ,General Agricultural and Biological Sciences ,Molecular Chaperones ,Transcription Factors - Abstract
Background: Position-dependent gene silencing in yeast involves many factors, including the four HIR genes and nucleosome assembly proteins Asf1p and chromatin assembly factor I (CAF-I, encoded by the CAC1–3 genes). Both cac Δ asf1 Δ and cac Δ hir Δ double mutants display synergistic reductions in heterochromatic gene silencing. However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously been explored. Results: Our biochemical and genetic studies of yeast Asf1p revealed links to Hir protein function. In vitro, an active histone deposition complex was formed from recombinant yeast Asf1p and histones H3 and H4 that lack a newly synthesized acetylation pattern. This Asf1p/H3/H4 complex generated micrococcal nuclease–resistant DNA in the absence of DNA replication and stimulated nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis. Also, Asf1p bound to the Hir1p and Hir2p proteins in vitro and in cell extracts. In vivo, the HIR1 and ASF1 genes contributed to silencing the heterochromatic HML locus via the same genetic pathway. Deletion of either HIR1 or ASF1 eliminated telomeric gene silencing in combination with pol30–8 , encoding an altered form of the DNA polymerase processivity factor PCNA that prevents CAF-I from contributing to silencing. Conversely, other pol30 alleles prevented Asf1/Hir proteins from contributing to silencing. Conclusions: Yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity.
- Published
- 2001
31. Chemical screening identifies filastatin, a small molecule inhibitor of Candida albicans adhesion, morphogenesis, and pathogenesis
- Author
-
Hong Cao, Elizabeth A. Lilly, Paul D. Kaufman, Charu Jain, Luca Issi, Amie C. Dehner, Paul L. Fidel, Ahmed Fazly, Reeta P. Rao, and Akbar Ali
- Subjects
Nematoda ,Phenotypic screening ,Morphogenesis ,Hyphae ,Piperazines ,Microbiology ,Pathogenesis ,Small Molecule Libraries ,Mice ,Candida albicans ,Cell Adhesion ,Animals ,Humans ,Cell adhesion ,Cells, Cultured ,Multidisciplinary ,biology ,Biofilm ,Epithelial Cells ,Adhesion ,Biological Sciences ,biology.organism_classification ,Corpus albicans ,Cell biology ,High-Throughput Screening Assays ,Polystyrenes - Abstract
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.
- Published
- 2013
32. A small molecule inhibitor of fungal histone acetyltransferase Rtt109
- Author
-
Vineeta Bajaj, Paul D. Kaufman, Jessica Lopes da Rosa, and James Spoonamore
- Subjects
Saccharomyces cerevisiae Proteins ,Clinical Biochemistry ,Pharmaceutical Science ,SAP30 ,Biochemistry ,Article ,Small Molecule Libraries ,Histone H3 ,Saccharomyces ,Structure-Activity Relationship ,Drug Discovery ,Histone H2A ,Enzyme Inhibitors ,Molecular Biology ,Histone Acetyltransferases ,Histone deacetylase 5 ,biology ,Dose-Response Relationship, Drug ,Molecular Structure ,Chemistry ,Histone deacetylase 2 ,Organic Chemistry ,Histone acetyltransferase ,Histone methyltransferase ,Acetyltransferase ,biology.protein ,Molecular Medicine - Abstract
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.
- Published
- 2013
33. Nucleosome Assembly by a Complex of CAF-1 and Acetylated Histones H3/H4
- Author
-
Paul D. Kaufman, Bruce Stillman, Alain Verreault, and Ryuji Kobayashi
- Subjects
Cell Extracts ,DNA Replication ,Cytoplasm ,DNA, Complementary ,Saccharomyces cerevisiae Proteins ,Nucleosome assembly ,Chromosomal Proteins, Non-Histone ,Molecular Sequence Data ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Histones ,Histone H4 ,Histone H1 ,Acetyltransferases ,Histone H2A ,Humans ,Histone code ,Amino Acid Sequence ,Histone octamer ,Chromatin Assembly Factor-1 ,Cloning, Molecular ,Histone Acetyltransferases ,Cell Nucleus ,Biochemistry, Genetics and Molecular Biology(all) ,Lysine ,Chromatin Assembly Factor I ,Acetylation ,Molecular biology ,Chromatin ,Nucleosomes ,Cell biology ,DNA-Binding Proteins ,Molecular Weight ,Transcription Factors - Abstract
Chromatin assembly factor 1 (CAF-1) assembles nucleosomes in a replication-dependent manner. The small subunit of CAF-1 (p48) is a member of a highly conserved subfamily of WD-repeat proteins. There are at least two members of this subfamily in both human (p46 and p48) and yeast cells (Hat2p, a subunit of the B-type H4 acetyltransferase, and Msi1p). Human p48 can bind to histone H4 in the absence of CAF-1 p150 and p60. p48, also a known subunit of a histone deacetylase, copurifies with a chromatin assembly complex (CAC), which contains the three subunits of CAF-1 (p150, p60, p48) and H3 and H4, and promotes DNA replication-dependent chromatin assembly. CAC histone H4 exhibits a novel pattern of lysine acetylation that overlaps with, but is distinct from, that reported for newly synthesized H4 isolated from nascent chromatin. Our data suggest that CAC is a key intermediate of the de novo nucleosome assembly pathway and that the p48 subunit participates in other aspects of histone metabolism.
- Published
- 1996
34. Nucleosome assembly: the CAF and the HAT
- Author
-
Paul D. Kaufman
- Subjects
Cell Nucleus ,Saccharomyces cerevisiae Proteins ,biology ,Nucleosome assembly ,Chromosomal Proteins, Non-Histone ,Cell Biology ,Solenoid (DNA) ,Molecular biology ,Linker DNA ,Nucleosomes ,Cell biology ,DNA-Binding Proteins ,Chromatin Assembly Factor-1 ,Histone ,Acetyltransferases ,Chromatosome ,biology.protein ,Humans ,Nucleosome ,Histone code ,Histone octamer ,Histone Acetyltransferases - Abstract
Recent data argue strongly that a protein complex termed chromatin assembly factor-I (CAF-I) plays a major role in de novo nucleosome assembly during DNA replication. Human CAF-1 deposits newly synthesized, acetylated histones onto replicated DNA in vitro and localizes to sites of DNA replication in S-phase cells. Specific lysines of the histones used for nucleosome assembly are acetylated; in the past year the first gene encoding a histone acetyltransferase was cloned. However, mechanistic links between histone acetylation and nucleosome assembly have not been established in vivo or in vitro.
- Published
- 1996
35. The p150 and p60 subunits of chromatin assemblyfactor I: A molecular link between newly synthesized histories and DNA replication
- Author
-
Ryuji Kobayashi, Bruce Stillman, Naama Kessler, and Paul D. Kaufman
- Subjects
DNA Replication ,Chromosomal Proteins, Non-Histone ,Macromolecular Substances ,Molecular Sequence Data ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Histones ,03 medical and health sciences ,0302 clinical medicine ,Histone H2A ,Humans ,Histone code ,Nucleosome ,Amino Acid Sequence ,Chromatin Assembly Factor-1 ,Cloning, Molecular ,Repetitive Sequences, Nucleic Acid ,Sequence Deletion ,030304 developmental biology ,CAF-1 ,Cell Nucleus ,0303 health sciences ,Base Sequence ,Biochemistry, Genetics and Molecular Biology(all) ,Chromatin Assembly Factor I ,Molecular biology ,Recombinant Proteins ,3. Good health ,Chromatin ,Cell biology ,DNA-Binding Proteins ,Oligodeoxyribonucleotides ,Protein Biosynthesis ,030220 oncology & carcinogenesis ,Origin recognition complex ,Protein Binding ,Transcription Factors - Abstract
SummaryChromatin assembly factor I (CAF-I) from human cellnuclei is a three-subunit protein complex that assembles histone octamers onto replicating DNA in a cell-free system. Sequences of cDNAs encoding the two largest CAF-I subunits reveal that the p150 protein contains large clusters of charged residues, whereas p60 contains WD repeats. p150 and p60 directly interact and are both required for DNA replication-dependent assembly of nucleosomes. Deletion of the p60-binding domain from the p150 protein prevents chromatin assembly. p150 and p60 form complexes with newly synthesized histories H3 and acetylated H4 in human cell extracts, suggesting that such complexes are intermediates between histone synthesis and assembly onto replicating DNA.
- Published
- 1995
36. Simian virus 40 origin- and T-antigen-dependent DNA replication with Drosophila factors in vitro
- Author
-
P G Mitsis, Bruce Stillman, James T. Kadonaga, Paul D. Kaufman, and Rohinton T. Kamakaka
- Subjects
DNA polymerase II ,fungi ,DNA replication ,Eukaryotic DNA replication ,Cell Biology ,Biology ,Origin of replication ,DNA polymerase delta ,Molecular biology ,Replication factor C ,Control of chromosome duplication ,biology.protein ,Origin recognition complex ,Molecular Biology - Abstract
DNA replication of double-stranded simian virus 40 (SV40) origin-containing plasmids, which has been previously thought to be a species-specific process that occurs only with factors derived from primate cells, is catalyzed with an extract derived from embryos of the fruit fly Drosophila melanogaster. This reaction is dependent upon both large T antigen, the SV40-encoded replication initiator protein and DNA helicase, and a functional T-antigen binding site at the origin of DNA replication. The efficiency of replication with extracts derived from Drosophila embryos is approximately 10% of that observed with extracts prepared from human 293 cells. This activity is not a unique property of embryonic extracts, as cytoplasmic extracts from Drosophila tissue culture cells also support T-antigen-mediated replication of SV40 DNA. By using highly purified proteins, DNA synthesis is initiated by Drosophila polymerase alpha-primase in a T-antigen-dependent manner in the presence of Drosophila replication protein A (RP-A; also known as single-stranded DNA-binding protein), but neither human RP-A nor Escherichia coli single-stranded DNA-binding protein could substitute for Drosophila RP-A. In reciprocal experiments, however, Drosophila RP-A was able to substitute for human RP-A in reactions carried out with human polymerase alpha-primase. These results collectively indicate that many of the specific functional interactions among T antigen, polymerase alpha-primase, and RP-A are conserved from primates to Drosophila species. Moreover, the observation that SV40 DNA replication can be performed with Drosophila factors provides a useful assay for the study of bidirectional DNA replication in Drosophila species in the context of a complete replication reaction.
- Published
- 1994
37. New partners for HP1 in transcriptional gene silencing
- Author
-
Paul D. Kaufman
- Subjects
Heterochromatin ,Chromosomal Proteins, Non-Histone ,Cell ,Transcriptional gene silencing ,Cell Cycle Proteins ,Biology ,Chromatin Assembly ,Article ,Epigenesis, Genetic ,Histones ,Schizosaccharomyces ,medicine ,RNA, Antisense ,Gene Silencing ,Molecular Biology ,Genetics ,Recombination, Genetic ,Acetylation ,Cell Biology ,Chromatin Assembly and Disassembly ,Cell biology ,Nucleosomes ,medicine.anatomical_structure ,Histone deacetylase complex ,Heterochromatin protein 1 ,Schizosaccharomyces pombe Proteins ,Molecular Chaperones ,Transcription Factors - Abstract
Heterochromatin impacts various nuclear processes by providing a recruiting platform for diverse chromosomal proteins. In fission yeast, HP1 proteins Chp2 and Swi6, which bind to methylated histone H3 lysine 9, associate with SHREC (Snf2/HDAC repressor complex) and Clr6 histone deacetylases (HDACs) involved in heterochromatic silencing. However, heterochromatic silencing machinery is not fully defined. We describe a histone chaperone complex containing Asf1 and HIRA that spreads across silenced domains via its association with Swi6 to enforce transcriptional silencing. Asf1 functions in concert with a Clr6 HDAC complex to silence heterochromatic repeats, and it suppresses antisense transcription by promoting histone deacetylation. Furthermore, we demonstrate that Asf1 and SHREC facilitate nucleosome occupancy at heterochromatic regions but TFIIIC transcription factor binding sites within boundary elements are refractory to these factors. These analyses uncover a role for Asf1 in global histone deacetylation and suggest that HP1-associated histone chaperone promotes nucleosome occupancy to assemble repressive heterochromatin.
- Published
- 2011
38. Catalytic activation of histone acetyltransferase Rtt109 by a histone chaperone
- Author
-
Brittany N. Albaugh, Scott E. Lindner, Kevin M. Arnold, John M. Denu, Kenneth A. Satyshur, Erin M. Kolonko, Paul D. Kaufman, Yuanyuan Chen, and James L. Keck
- Subjects
Models, Molecular ,Saccharomyces cerevisiae Proteins ,Blotting, Western ,Static Electricity ,Cell Cycle Proteins ,Catalysis ,Mass Spectrometry ,Histones ,Histone H1 ,X-Ray Diffraction ,Histone H2A ,Histone octamer ,Histone Acetyltransferases ,Multidisciplinary ,biology ,Chemistry ,Histone acetyltransferase ,Biological Sciences ,Histone ,Biochemistry ,Acetylation ,Histone methyltransferase ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Crystallization ,Dimerization ,Molecular Chaperones - Abstract
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 1∶2 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.
- Published
- 2010
39. Histone acetyltransferase Rtt109 is required for Candida albicans pathogenesis
- Author
-
Jessica Lopes da Rosa, Lihua Julie Zhu, Victor L. Boyartchuk, and Paul D. Kaufman
- Subjects
DNA Repair ,DNA damage ,DNA repair ,Cell Survival ,Microbiology ,Histone H3 ,Mice ,Gene Expression Regulation, Fungal ,Candida albicans ,Animals ,DNA, Fungal ,Histone Acetyltransferases ,Mice, Inbred BALB C ,Multidisciplinary ,biology ,Macrophages ,Candidiasis ,Histone acetyltransferase ,Biological Sciences ,biology.organism_classification ,Molecular biology ,Corpus albicans ,Chromatin ,Mice, Inbred C57BL ,Acetylation ,biology.protein ,Female ,Reactive Oxygen Species - Abstract
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.
- Published
- 2010
40. P element transposition in vitro proceeds by a cut-and-paste mechanism and uses GTP as a cofactor
- Author
-
Donald C. Rio and Paul D. Kaufman
- Subjects
Transposable element ,GTP' ,Stereochemistry ,Molecular Sequence Data ,Transposases ,General Biochemistry, Genetics and Molecular Biology ,Cofactor ,Cell Line ,Transposition (music) ,P element ,chemistry.chemical_compound ,Drosophilidae ,Escherichia coli ,Animals ,Magnesium ,Transposase ,Base Sequence ,biology ,Temperature ,biology.organism_classification ,Nucleotidyltransferases ,Molecular biology ,Drosophila melanogaster ,chemistry ,DNA Transposable Elements ,biology.protein ,Guanosine Triphosphate ,DNA ,Plasmids - Abstract
We have developed an in vitro reaction system for Drosophila P element transposition. Transposition products were recovered by selection in E. coli, and contained simple P element insertions flanked by 8 bp target site duplications as observed in vivo. Transposition required Mg +2 and partially purified P element transposase. Unlike other DNA rearrangement reactions, P element transposition in vitro used GTP as a cofactor; deoxyGTP, dideoxyGTP, or the nonhydrolyzable GTP analogs GMP-PNP or GMP-PCP were also used. Transposon DNA molecules cleaved at the P element termini were able to transpose, but those lacking 3′-hydroxyl groups were inactive. These biochemical data are consistent with genetic data suggesting that P element transposition occurs via a "cut-and-paste" mechanism.
- Published
- 1992
41. Regulation of histone acetylation and turnover by histone chaperones
- Author
-
Paul D. Kaufman
- Subjects
Histone deacetylase 5 ,Chemistry ,Histone deacetylase 2 ,HDAC11 ,Biochemistry ,Cell biology ,Histone H1 ,Histone methyltransferase ,Histone H2A ,Histone methylation ,Genetics ,Histone code ,Molecular Biology ,Biotechnology - Published
- 2009
42. Drosophila P-element transposase is a transcriptional repressor in vitro
- Author
-
Paul D. Kaufman and Donald C. Rio
- Subjects
Transcription, Genetic ,Molecular Sequence Data ,Transposases ,RNA polymerase II ,P element ,Animals ,Promoter Regions, Genetic ,Binding Sites ,Multidisciplinary ,Base Sequence ,biology ,General transcription factor ,Nucleotidyltransferases ,TATA Box ,Molecular biology ,Repressor Proteins ,Drosophila melanogaster ,DNA Transposable Elements ,biology.protein ,Transcription factor II F ,RNA Polymerase II ,Transcription factor II E ,Transcription factor II D ,Transcription factor II B ,Transcription factor II A ,Research Article - Abstract
Mobility of P transposable elements in Drosophila melanogaster depends on the 87-kDa transposase protein encoded by the P element. Transposase recognizes a 10-base-pair DNA sequence that overlaps an A + T-rich region essential for transcription from the P-element promoter. We report here that transposase represses transcription from the P-element promoter in vitro. This transcriptional repression is blocked by prior formation of an RNA polymerase II transcription complex on the template DNA. Binding of transposase on the P-element promoter is blocked by prior binding of either the Drosophila RNA polymerase II complex or the yeast transcription factor TFIID. These data suggest that transposase represses transcription by preventing assembly of an RNA polymerase II complex at the P-element promoter.
- Published
- 1991
43. Cell Cycle– and Chaperone-Mediated Regulation of H3K56ac Incorporation in Yeast
- Author
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Judith A. Erkmann, Oliver J. Rando, Paul D. Kaufman, Tommy Kaplan, Chih Long Liu, John Holik, Nir Friedman, and Michael Grunstein
- Subjects
DNA Replication ,Cancer Research ,lcsh:QH426-470 ,Saccharomyces cerevisiae ,Biology ,Histones ,03 medical and health sciences ,Histone H3 ,0302 clinical medicine ,Histone H1 ,Genetics and Genomics/Epigenetics ,Histone H2A ,Histone methylation ,Genetics ,Nucleosome ,Histone code ,Histone octamer ,Genetics and Genomics/Genomics ,Molecular Biology/Chromatin Structure ,DNA, Fungal ,Molecular Biology ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Histone Acetyltransferases ,0303 health sciences ,Lysine ,Cell Cycle ,Molecular Biology/Chromosome Structure ,Acetylation ,Cell biology ,Genetics and Genomics/Chromosome Biology ,lcsh:Genetics ,Biochemistry ,Histone methyltransferase ,030217 neurology & neurosurgery ,Research Article ,Molecular Chaperones - Abstract
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., Author Summary Wrapping of eukaryotic genomes by the histone proteins impacts virtually all known DNA templates processes. Covalent modification of the histone proteins has emerged as a key mechanism for regulation of transcription and other processes. Here, we report high-resolution genomic mapping of a relatively recently described modification, H3K56 acetylation. We find that H3K56ac is localized at all known sites of nucleosome incorporation, not only during genomic replication, but also during replication-independent nucleosome replacement. By measuring changes in histone replacement during M phase (when K56ac is erased by deacetylases), we find evidence suggesting that histone replacement may help control genomic replication programs. Finally, analysis of histone replacement in mutants in the K56 acetylation pathway provide evidence for self-reinforcing patterns of histone replacement, where replacement of a fresh nucleosome is slower than replacement of a nucleosome that has already been replaced once. These results provide a broad perspective on histone incorporation throughout the cell cycle and deepen our understanding of the relationship between histone turnover and histone modification.
- Published
- 2008
44. Structural and biochemical investigations of the Rtt109‐histone chaperone complexes
- Author
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Toshiaki Tsubota, James L. Keck, John M. Denu, Paul D. Kaufman, and Christopher E. Berndsen
- Subjects
Histone ,biology ,Chemistry ,Chaperone (protein) ,Genetics ,biology.protein ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2008
45. Structure of the yeast histone H3-ASF1 interaction: implications for chaperone mechanism, species-specific interactions, and epigenetics
- Author
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Toshiaki Tsubota, James M. Berger, Andrew J. Antczak, and Paul D. Kaufman
- Subjects
Cell Cycle Proteins ,Saccharomyces cerevisiae ,Biology ,Epigenesis, Genetic ,03 medical and health sciences ,Histone H3 ,0302 clinical medicine ,Species Specificity ,Histone H1 ,Structural Biology ,Histone H2A ,Histone methylation ,Animals ,Humans ,Histone code ,Gene Silencing ,Histone octamer ,lcsh:QH301-705.5 ,Silent Information Regulator Proteins, Saccharomyces cerevisiae ,030304 developmental biology ,Histone binding ,Genetics ,0303 health sciences ,Cell biology ,lcsh:Biology (General) ,Histone methyltransferase ,Dimerization ,030217 neurology & neurosurgery ,Research Article ,Molecular Chaperones ,Protein Binding - Abstract
Background The histone H3/H4 chaperone Asf1 (anti-silencing function 1) is required for the establishment and maintenance of proper chromatin structure, as well as for genome stability in eukaryotes. Asf1 participates in both DNA replication-coupled (RC) and replication-independent (RI) histone deposition reactions in vitro and interacts with complexes responsible for both pathways in vivo. Asf1 is known to directly bind histone H3, however, high-resolution structural information about the geometry of this interaction was previously unknown. Results Here we report the structure of a histone/histone chaperone interaction. We have solved the 2.2 Å crystal structure of the conserved N-terminal immunoglobulin fold domain of yeast Asf1 (residues 2–155) bound to the C-terminal helix of yeast histone H3 (residues 121–134). The structure defines a histone-binding patch on Asf1 consisting of both conserved and yeast-specific residues; mutation of these residues abrogates H3/H4 binding affinity. The geometry of the interaction indicates that Asf1 binds to histones H3/H4 in a manner that likely blocks sterically the H3/H3 interface of the nucleosomal four-helix bundle. Conclusion These data clarify how Asf1 regulates histone stoichiometry to modulate epigenetic inheritance. The structure further suggests a physical model in which Asf1 contributes to interpretation of a "histone H3 barcode" for sorting H3 isoforms into different deposition pathways.
- Published
- 2006
46. Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis
- Author
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C. D. Allis, James M. Berger, Judith Recht, Alma L. Burlingame, Robert L. Diaz, Toshiaki Tsubota, Paul D. Kaufman, Donald F. Hunt, Xiaoli Zhang, Jason C. Tanny, Jeffrey Shabanowitz, and Benjamin A. Garcia
- Subjects
Models, Molecular ,Saccharomyces cerevisiae Proteins ,Protein Conformation ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Biology ,S Phase ,Histones ,Histone H3 ,Histone H1 ,Histone H2A ,Histone methylation ,Histone code ,Histone octamer ,Histone binding ,Multidisciplinary ,Lysine ,Acetylation ,Spores, Fungal ,Biological Sciences ,Meiosis ,Phenotype ,Biochemistry ,Histone methyltransferase ,DNA Damage ,Molecular Chaperones - Abstract
Histone acetylation affects many nuclear processes including transcription, chromatin assembly, and DNA damage repair. Acetylation of histone H3 lysine 56 (H3 K56ac) in budding yeast occurs during mitotic S phase and persists during DNA damage repair. Here, we show that H3 K56ac is also present during premeiotic S phase and is conserved in fission yeast. Furthermore, the H3 K56ac modification is not observed in the absence of the histone chaperone Asf1. asf1 Δ and H3 K56R mutants exhibit similar sensitivity to DNA damaging agents. Mutational analysis of Asf1 demonstrates that DNA damage sensitivity correlates with ( i ) decreased levels of H3 K56ac and ( ii ) a region implicated in histone binding. In contrast, multiple asf1 mutants that are resistant to DNA damage display WT levels of K56ac. These data suggest that maintenance of H3 K56 acetylation is a primary contribution of Asf1 to genome stability in yeast.
- Published
- 2006
47. Replication-independent histone deposition by the HIR complex and Asf1
- Author
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Alexa A. Franco, Paul D. Kaufman, John R. Yates, Kevin J. Wu, Aaron O. Bailey, Erin M. Green, and Andrew J. Antczak
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Chromatin Immunoprecipitation ,Saccharomyces cerevisiae Proteins ,Chromosomal Proteins, Non-Histone ,Immunoblotting ,Cell Cycle Proteins ,Electrophoretic Mobility Shift Assay ,DNA-binding protein ,General Biochemistry, Genetics and Molecular Biology ,Mass Spectrometry ,Article ,Histones ,03 medical and health sciences ,Histone H3 ,HIR complex ,Yeasts ,Histone H2A ,Histone code ,Chromatin Assembly Factor-1 ,030304 developmental biology ,0303 health sciences ,biology ,Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,030302 biochemistry & molecular biology ,Nuclear Proteins ,DNA ,Molecular biology ,3. Good health ,Cell biology ,DNA-Binding Proteins ,Repressor Proteins ,Histone ,Multiprotein Complexes ,Mutation ,biology.protein ,General Agricultural and Biological Sciences ,Chromatin immunoprecipitation ,Molecular Chaperones - Abstract
Summary The orderly deposition of histones onto DNA is mediated by conserved assembly complexes, including chromatin assembly factor-1 (CAF-1) and the Hir proteins [1–4]. CAF-1 and the Hir proteins operate in distinct but functionally overlapping histone deposition pathways in vivo [5, 6]. The Hir proteins and CAF-1 share a common partner, the highly conserved histone H3/H4 binding protein Asf1, which binds the middle subunit of CAF-1 as well as to Hir proteins [7–11]. Asf1 binds to newly synthesized histones H3/H4 [12], and this complex stimulates histone deposition by CAF-1 [7, 11, 12]. In yeast, Asf1 is required for the contribution of the Hir proteins to gene silencing [7, 13]. Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 comprise the HIR complex, which copurifies with the histone deposition protein Asf1. Together, the HIR complex and Asf1 deposit histones onto DNA in a replication-independent manner. Histone deposition by the HIR complex and Asf1 is impaired by a mutation in Asf1 that inhibits HIR binding. These data indicate that the HIR complex and Asf1 proteins function together as a conserved eukaryotic pathway for histone replacement throughout the cell cycle.
- Published
- 2005
48. Histone deposition proteins: links between the DNA replication machinery and epigenetic gene silencing
- Author
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Paul D. Kaufman and Alexa A. Franco
- Subjects
Genetics ,DNA Replication ,Eukaryotic DNA replication ,Saccharomyces cerevisiae ,Biology ,Biochemistry ,Models, Biological ,Epigenesis, Genetic ,Nucleosomes ,DNA replication factor CDT1 ,Histones ,Control of chromosome duplication ,Histone H2A ,biology.protein ,Histone code ,Humans ,Cancer epigenetics ,Epigenetics ,Gene Silencing ,Molecular Biology ,Epigenomics - Published
- 2005
49. Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C
- Author
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Paul D. Kaufman, Wendy Lam, Peter M. J. Burgers, and Alexa A. Franco
- Subjects
DNA Replication ,biology ,DNA replication ,Eukaryotic DNA replication ,Cell Cycle Proteins ,DNA polymerase delta ,Molecular biology ,Research Papers ,S Phase ,DNA replication factor CDT1 ,Histones ,Replication factor C ,Licensing factor ,Control of chromosome duplication ,Genetics ,biology.protein ,Origin recognition complex ,Developmental Biology ,Protein Binding - Abstract
Chromatin assembly and DNA replication are temporally coupled, and DNA replication in the absence of histone synthesis causes inviability. Here we demonstrate that chromatin assembly factor Asf1 also affects DNA replication. In budding yeast cells lacking Asf1, the amounts of several DNA replication proteins, including replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase ϵ (Pol ϵ), are reduced at stalled replication forks. In contrast, DNA polymerase α (Pol α) accumulates to higher than normal levels at stalled forks in asf1Δ cells. Using purified, recombinant proteins, we demonstrate that RFC directly binds Asf1 and can recruit Asf1 to DNA molecules in vitro. We conclude that histone chaperone protein Asf1 maintains a subset of replication elongation factors at stalled replication forks and directly interacts with the replication machinery.
- Published
- 2005
50. Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA
- Author
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Paul D. Kaufman, Peter D. Adams, James M. Berger, Sally M. Daganzo, Rugang Zhang, Maxim Poustovoitov, Wei Chen, Jan P. Erzberger, Adrian A. Canutescu, Hidelita Santos, Ilya G. Serebriiskii, Xiaofen Ye, John R. Pehrson, and Roland L. Dunbrack
- Subjects
Senescence ,Indoles ,Time Factors ,Heterochromatin ,Chromosomal Proteins, Non-Histone ,Recombinant Fusion Proteins ,Blotting, Western ,Regulator ,Cell Count ,Cell Cycle Proteins ,Biology ,Transfection ,General Biochemistry, Genetics and Molecular Biology ,Cell Line ,Histones ,03 medical and health sciences ,0302 clinical medicine ,Dosage Compensation, Genetic ,Histone H2A ,Immunoprecipitation ,Amino Acid Sequence ,Molecular Biology ,Cellular Senescence ,030304 developmental biology ,0303 health sciences ,Tumor Suppressor Proteins ,Cell Cycle ,Nuclear Proteins ,Cell Biology ,Cell cycle ,Senescence-associated heterochromatin focus ,Immunohistochemistry ,Chromatin ,Cell biology ,Neoplasm Proteins ,Repressor Proteins ,Gene Expression Regulation ,Chromobox Protein Homolog 5 ,030220 oncology & carcinogenesis ,ras Proteins ,Heterochromatin protein 1 ,Developmental Biology ,Molecular Chaperones ,Transcription Factors - Abstract
In senescent cells, specialized domains of transcriptionally silent senescence-associated heterochromatic foci (SAHF), containing heterochromatin proteins such as HP1, are thought to repress expression of proliferation-promoting genes. We have investigated the composition and mode of assembly of SAHF and its contribution to cell cycle exit. SAHF is enriched in a transcription-silencing histone H2A variant, macroH2A. As cells approach senescence, a known chromatin regulator, HIRA, enters PML nuclear bodies, where it transiently colocalizes with HP1 proteins, prior to incorporation of HP1 proteins into SAHF. A physical complex containing HIRA and another chromatin regulator, ASF1a, is rate limiting for formation of SAHF and onset of senescence, and ASF1a is required for formation of SAHF and efficient senescence-associated cell cycle exit. These data indicate that HIRA and ASF1a drive formation of macroH2A-containing SAHF and senescence-associated cell cycle exit, via a pathway that appears to depend on flux of heterochromatic proteins through PML bodies.
- Published
- 2004
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