M Mitchell Smith
University Of Virginia Charlottesville
Project start date: 2000-01-01
Project end date: 2014-01-31
Sponsored Links Excellgen http://Excellgen.com
THE ROLE OF HISTONE H4 IN GENOME STABILITY
M Mitchell Smith, Professor
University Of Virginia Charlottesville Box 400195 Charlottesville, Va 229044195
Grant 5R01GM060444-04 from National Institute Of General Medical Sciences IRG: CDF
Abstract: The maintenance of genome integrity is essential for the accurate transmission of genetic information and to prevent tumorigenesis. Indeed, both DNA sequence and chromosome instabilities are associated with a high percentage and wide variety of human cancers. The repair of chromosomal damage must take place in the context of chromatin structure, but virtually nothing is known about the interactions of the repair machinery with the components of chromatin. We have discovered that the N-terminal domain, or "tail", of histone H4 is required for efficient DNA damage repair and that this function requires the activity of an acetylatable lysine residue. On the basis of genetic and biochemical observations, we hypothesize that H4 facilitates DNA damage repair at double-strand chromosome breaks by interacting with components of the repair machinery through protein-protein interactions signaled by lysine acetylation. We further hypothesize that specific histone acetyl transferases and/or histone deacetylases participate directly in DNA repair via reversible H4 acetylation. To test these models we will focus on three major research questions. First, we will examine the structure, processing and fidelity of repair at double-strand breaks in histone H4 mutants defective for reversible acetylation. We will test the role of H4 acetylation in both nonhomologous DNA end joining and homologous double-strand break repair. Second, we will examine the genetic and biochemical interactions between histone H4 acetylation and DNA repair proteins. We will exploit our finding that histone H4 acetylation site mutants are hypersensitivity to the radiomimetic drug camptothecin to screen for interacting genes, and we will purify yeast Ku70-associated proteins directly to identify new components of the H4-dependent repair pathway. Finally, we will use genetic screens and in vivo cross-linking coupled with chromatin immunoprecipitation to characterize a specific histone acetyl transferase for its role in DNA damage repair and its influence on the state of histone H4 acetylation at a defined double-strand break. Our observation that reversible histone H4 acetylation is required for DNA repair represents a previously unrecognized role for this important chromatin modification.
Keywords: DNA repair, acetylation, acyltransferase, enzyme activity, gene interaction, genome, histone, protein structure function, chromatin, gene expression, gene mutation, genetic regulation, lysine, Saccharomyces cerevisiae, crosslink, fungal genetics, immunoprecipitation, polymerase chain reaction, site directed mutagenesis, yeast two hybrid system
Project start date: 2000-01-01
Project end date: 2003-12-31
5R01GM060444-04 (2003): $276586
5R01GM060444-03 (2002): $268762
5R01GM060444-02 (2001): $261165
5R01GM060444-08 (2007): $298191
5R01GM060444-07 (2006): $307131
5R01GM060444-06 (2005): $314044
Grants awarded to M Mitchell Smith
HISTONE GENE EXPRESSION IN YEAST
M Mitchell Smith, Professor
University Of Virginia Charlottesville Box 400195 Charlottesville, Va 229044195
Grant 5R01GM028920-11 from National Institute Of General Medical Sciences IRG: GEN
Abstract: We have been engaged in a multifaceted investigation of the expression of the histone H3 and H4 genes in the yeast Saccharomyces cerevisiae. During the last period our experiments on regulation of the genes identified an upstream activation site (UAS) in the promoter region of the H3-H4 genes responsible for cell cycle control at the level of transcription. Gene deletions demonstrated a lack of dosage compensation among the H3-H4 gene sets indicating post-transcriptional regulation pathways as well. During the next period we will search for additional regulatory sites in the promoter region by linker substitution mutagenesis. We will attempt to identify and characterize the putative cell cycle activation protein and its gene by biochemical assays and genetic mutational analysis of the UAS. Post-transcriptional regulatory mechanisms will be examined by placing expression of histone mRNA under constitutive transcription control. Also during the last period nuclease mapping experiments demonstrated a set of cell cycle stage specific changes in the chromatin structure of the H3-H4 genes. The replication dependence of these changes will be investigated in cell division cycle mutants blocked in DNA replication. Future experiments will examine changes in hypersensitive sites at the sequence level by analysis of partial chromatin digestions and by in vivo methylation protection assays. We will also examine the relationship between hypersensitive sites and regulatory sequences such as the UAS by mutational analysis. Our previous genetic analyses of histone H4 have generated mutants with distinct phenotypes including increased mitotic chromosome loss, phenotypic sterility, and loss of function. During the next project period we plan to construct a tagged histone H4 by inserting the coding sequence for an antigenic oligopeptide. This will permit us to follow mutant H4 proteins by immunological techniques and determine expression levels of the protein, cellular localization, and interaction with chromatin. We have developed a rapid genetic screen for the efficient detection of functional H4 derivatives. A mutational map of permissible amino acid substitutions in the histone H4 will be derived from the sequence of these clones. Our analysis of mitotic stability H4 mutants will be extended by defining the amino acid substitutions that produce the defect. We plan to investigate the molecular basis for the phenotypic sterility histone H4 mutants. The hypothesis that these H4 mutants disrupt chromatin structure and prevent repression of silent mating type information will be tested by constructing double mutants with other genes involved in mating phenotype. Both the transcription of these related genes and the chromatin structure of the mating type genes will be assayed.
Keywords: fungal genetics, gene expression, genetic regulation, histone, nucleic acid sequence, DNA biosynthesis, cell division, cell growth regulation, chromatin, gene dosage, genetic manipulation, genetic promoter element, genetic transcription, messenger RNA, molecular biology, mutagen testing, posttranscriptional RNA processing, protein biosynthesis, structural gene, Saccharomyces, autoradiography, chemical fingerprinting, diethylaminoethyl cellulose chromatography, endonuclease, gel electrophoresis, gene deletion mutation, growth media, molecular cloning, nucleic acid hybridization, nucleic acid probe, paper electrophoresis, radioactive phosphorus, radiotracer, temperature sensitive mutant
Project start date: 1981-04-01
Project end date: 1992-03-31
5R01GM028920-17 (1997): $268414
REGULATION OF HISTONE GENE EXPRESSION IN YEAST
M Mitchell Smith, Professor
University Of Virginia Charlottesville Box 400195 Charlottesville, Va 229044195
Grant 5R01GM028920-03 from National Institute Of General Medical Sciences IRG: GEN
Abstract: The special gene organization and low copy number of the yeast histone genes make them an excellent system for the study of histone gene regulation and control. With recent advances in DNA transformation in yeast, the yeast histone genes provide a possible model for the generation of histone gene mutants and genetic analysis of histone gene control, histone protein function, and nucleosome assembly. Specific areas of study include (1) the structure of the histone gene mRNAs and their relationship to DNA sequence of the genes. The mRNAs will be analyzed by Northern blot experiments, and by sequence analysis of the individual histone gene mRNAs. (2) The cell division cycle control of histone gene transcription. The rate of synthesis and degradation will be measured as a function of the cell cycle. Cell fractions from cultures in balanced growth and cells in synchronously dividing cultures will be assayed for transcription by pulse-label and pulse-chase experiments. (3) Mutant derivatives of the histone genes will be constructed using the cloned genes. These mutants will be used to transform yeast cells and the phenotypic properties of the strains will be examined. In particular an attempt to delete copies of the histone genes from the genome will be made. Mutants will be shuttled into the genome and screened for conditional mutations. Multicopy episomal plasmids will be used to manipulate the gene dosage of the histone genes as a way of selecting mutants. The regulation of the mutant genes will be examined at the transcriptional level.
Keywords: GENETICS STUDY SECTION, GENETICS, GENES, GENE EXPRESSION, GENETICS, GENETIC REGULATION, NUCLEIC ACIDS CLONING, NUCLEIC ACIDS STRUCTURE, NUCLEOSIDES (TIDES) SEQUENCE, NUCLEOPROTEINS, HISTONES, GENETICS, GENES, GENE DOSAGE, GENETICS, GENES, OPERON, STRUCTURAL GENE, GENETICS, GENETIC REGULATION, GENETIC INDUCTION-REPRESSION-DEREPRESSION, TRANSCRIPTION, GENETICS, MUTATION, MUTANTS, TEMPERATURE SENSITIVE MUTANTS, MOLECULAR BIOLOGY (GENERAL), NUCLEIC ACIDS SYNTHESIS, DNA, NUCLEIC ACIDS, MRNA, NUCLEIC ACIDS, SYNTHETIC NUCLEIC ACIDS, HYBRID NUCLEIC ACIDS, PROTEINS BIOSYNTHESIS, cell division, genetic manipulation, BACTERIA, ENTEROBACTERIACEAE, ESCHERICHIA COLI, CHEMISTRY, ANALYTICAL METHODS, FINGERPRINTING, FUNGI, YEASTS, SACCHAROMYCES, GENETICS, MUTATION, CHROMOSOME MUTATION, DELETION, NUCLEASES, ENDONUCLEASES (GENERAL), PHYSICAL SEPARATION, CHROMATOGRAPHY, DEAE CELLULOSE, PHYSICAL SEPARATION, ELECTROPHORESIS, GEL, PHYSICAL SEPARATION, ELECTROPHORESIS, PAPER, RADIOAUTOGRAPHY, RADIOISOTOPES, PHOSPHORUS, RADIOTRACERS, growth media
Project start date: 1981-04-01
Project end date: 1984-03-31
HISTONE GENE EXPRESSION IN YEAST
M Mitchell Smith, Professor
University Of Virginia Charlottesville Box 400195 Charlottesville, Va 229044195
Grant 5R01GM028920-15 from National Institute Of General Medical Sciences IRG: GEN
Abstract: The research proposed in this application is a genetic investigation of the genes that encode the histones H3 and H4 in the budding yeast Saccharomyces cerevisiae. The experiments are divided into two major project areas (1) the molecular genetics of histone protein structure and function, and (2) the cell division cycle control of histone gene transcription. The N-terminal protein domains of the histones H3 and H4 are critical for cellular functions, such as nuclear division and transcription, and the lysines subject to reversible acetylation play an important role in these functions. A mutational study of the H3 and H4 acetylation sites will be conducted to test the involvement of positive charge density in N-terminal domain function. A scanning point mutant analysis will be conducted to identify functional peptide sites within the terminal domains. Specific histone H4 N-terminal domain mutants are temperature sensitive for growth. Extragenic suppressors of this lethality will be identified, cloned, and characterized to understand the steps and pathways dependent on N-terminal domain function. Double mutants containing both the histone H3 and H4 N- terminal domain deletions are inviable. A screen will be carried out to identify other mutants that are also synthetic lethals in combination with either of the individual H3 or H4 N-terminal domain deletions. These genes will be cloned and characterized to examine the pathways and hierarchy of interactions involved. In a previous mutational screen for temperature sensitive lethal mutants, a histone H4 mutant was identified in which an early step in chromosome segregation is blocked at the restrictive temperature. Genetic and biochemical experiments will be conducted to determine if this block is caused by centromere disfunction or a global disruption of chromatid structure. A mutational screen will be carried out to identify extragenic suppressors of this temperature sensitive lethality. The new genes will be cloned and characterized to determine what steps in mitotic chromosome segregation depend on histone H4 function. Several other histone H3 and H4 mutants also have a delay during the G2 phase of the division cycle. A genetic screen will be conducted to identify new G2 checkpoint mutants predicted to monitor chromosome integrity. Transcription of the histone H3 and H4 genes is tightly regulated within the cell division cycle with expression restricted to the late G1 and S phases of the cycle. This control is mediated by upstream activation site (UAS) elements and upstream repressor site (URS) elements. Saturation mutagenesis will be used to define the precise DNA sequence requirements for these cis-acting regulatory sites. There are multiple copies of the UAS element arranged as inverted repeats in each of the three histone promoters examined to date. The importance of the orientation and spacing of these elements will be tested. A DNA binding protein specific for the histone UAS element has been identified. To understand the mechanism of cell cycle control at the histone UAS, the gene encoding this binding protein will be cloned, sequenced, and characterized. A mutational analysis of the UAS binding protein will be carried out to determine its molecular function, and define the signal transduction pathway for cell cycle position information.
Keywords: cell cycle, gene expression, genetic regulation, histone, protein structure function, DNA binding protein, acetylation, allele, binding protein, biological signal transduction, fungal genetics, genetic promoter element, genetic regulatory element, genetic transcription, nucleic acid sequence, Saccharomyces cerevisiae, endonuclease, gene deletion mutation, molecular cloning, nucleic acid probe, site directed mutagenesis
Project start date: 1981-04-01
Project end date: 1996-03-31
5R01GM028920-15 (1995): $232331
5R01GM028920-14 (1994): $222607
Sponsored Links Excellgen http://Excellgen.com
5R01GM028920-13 (1993): $213293
2R01GM028920-12 (1992): $209214
THE ROLE OF HISTONE H4 IN GENOME STABILITY
M Mitchell Smith, Professor
Microbiologyuniversity Of Virginia Charlottesville
box 400195
charlottesville, Va 229044195
Grant 1R01GM060444-01 from National Institute Of General Medical Sciences IRG: CDF
Abstract: The maintenance of genome integrity is essential for the accurate transmission of genetic information and to prevent tumorigenesis. Indeed, both DNA sequence and chromosome instabilities are associated with a high percentage and wide variety of human cancers. The repair of chromosomal damage must take place in the context of chromatin structure, but virtually nothing is known about the interactions of the repair machinery with the components of chromatin. We have discovered that the N-terminal domain, or "tail", of histone H4 is required for efficient DNA damage repair and that this function requires the activity of an acetylatable lysine residue. On the basis of genetic and biochemical observations, we hypothesize that H4 facilitates DNA damage repair at double-strand chromosome breaks by interacting with components of the repair machinery through protein-protein interactions signaled by lysine acetylation. We further hypothesize that specific histone acetyl transferases and/or histone deacetylases participate directly in DNA repair via reversible H4 acetylation. To test these models we will focus on three major research questions. First, we will examine the structure, processing and fidelity of repair at double-strand breaks in histone H4 mutants defective for reversible acetylation. We will test the role of H4 acetylation in both nonhomologous DNA end joining and homologous double-strand break repair. Second, we will examine the genetic and biochemical interactions between histone H4 acetylation and DNA repair proteins. We will exploit our finding that histone H4 acetylation site mutants are hypersensitivity to the radiomimetic drug camptothecin to screen for interacting genes, and we will purify yeast Ku70-associated proteins directly to identify new components of the H4-dependent repair pathway. Finally, we will use genetic screens and in vivo cross-linking coupled with chromatin immunoprecipitation to characterize a specific histone acetyl transferase for its role in DNA damage repair and its influence on the state of histone H4 acetylation at a defined double-strand break. Our observation that reversible histone H4 acetylation is required for DNA repair represents a previously unrecognized role for this important chromatin modification
Keywords: DNA repair, acetylation, acyltransferase, enzyme activity, gene interaction, genome, histone, protein structure /function chromatin, gene expression, gene mutation, genetic regulation, lysine Saccharomyces cerevisiae, crosslink, fungal genetics, immunoprecipitation, polymerase chain reaction, site directed mutagenesis, yeast two hybrid system
Project start date: 2000-01-01
Project end date: 2003-12-31
1R01GM060444-01 (2000): $280189
HISTONE GENE EXPRESSION IN YEAST
M Mitchell Smith, Professor
University Of Virginia Charlottesville Box 400195 Charlottesville, Va 229044195
Grant 5R01GM028920-19 from National Institute Of General Medical Sciences IRG: GEN
Abstract: The histone proteins are essential components of eukaryotic chromatin. Two molecules each of histones H2A, H2B, H3, and H4 from the core octamer of the nucleosome, and provide the first level of compaction of the DNA in the nucleus. Research over the last five yeast has provided mounting evidence that the histones play essential roles in many aspects of nuclear function, including transcription, replication, recombination, and nuclear division. Thus, determining the functional roles of the histones in vivo is critical for understanding mechanisms of gene expression, genome stability, and chromosome dynamics-processes in which defects are intimately related to causes of cancer, and chromosome abnormalities such as Down Syndrome. This project presents a molecular genetic investigation into the roles of the histone proteins in vivo. It focuses on three major research questions (1) the role of the histone N-terminal domains in the maintenance of genome integrity; 92) the structure and function of protein interactions within the nucleosomes; and (3) the structure and function of centromeric chromatin and the kinetochore. The N-terminal domain of histone H4 is required to maintain genome integrity. Mutations in this domain cause increased intrinsic DNA damage, activation of DNA damage-inducible gene expression, and DNA damage check- point arrest at mitosis. We will determine how and why this increased DNA damage is incurred by studying mutants in the process. We will ask the N- terminal domains of other histone proteins play a role in this maintenance function. We will test whether the N-terminal domains determine higher- order chromatin compaction and the extent to which this function is related to the maintenance function. We will continue a successful site-directed mutational analysis histone interactions within the nucleosome. Temperature sensitive mutations in H2B-H4 contact sites lead to aberrant gene transcription, a failure to activate transcription of the Gl cyclin genes, and arrest at start. We will determine the genetic basis of this transcription inhibition through further mutational analyses, and the molecular basis through in vitro reconstitution experiments. Genetic evidence from our laboratory showed that proper chromosome segregation requires histone H4 interaction with a novel histone H3 variant related to the mammalian kinetochore antigen CENP-A. We propose that histone H4 and CENP-A form a specialized nucleosome at the centromere and interact with other kinetochore proteins. This model will be tested both genetically and biochemically.
Keywords: Saccharomyces cerevisiae, fungal genetics, gene induction /repression, histone, protein structure /function, biological signal transduction, cell cycle, endonuclease, gene deletion mutation, genetic promoter element, nucleic acid probe, nucleic acid sequence, transcription factor, molecular cloning, site directed mutagenesis
Project start date: 1981-04-01
Project end date: 2000-03-31
5R01GM028920-19 (1999): $290040
5R01GM028920-18 (1998): $279076
5R01GM028920-27 (2007): $344500
5R01GM028920-26 (2006): $354839
Sponsored Links Excellgen http://Excellgen.com
5R01GM028920-25 (2005): $363430
2R01GM028920-24 (2004): $363186
Epigenetic Regulation Of Gene Expression During Early Mouse Embryogenesis
M Mitchell Smith, Professor
Jackson Laboratory
600 Main St
bar Harbor, Me 046091500
Grant 1F33HD056741-01 from National Institute Of Child Health And Human Development IRG: ZRG1
Abstract: One of the most remarkable gene reprogramming senarios in all of biology is the transformation of the transcriptionally silent fully grown oocyte into the totipotent embryonic stem cell. Epigenetic regulatory factors, including histone modifications and nucleosome remodeling complexes, play essential roles in this oocyte-to-embryo transition (OET). However, little is known about the specific factors involved, the program of histone modifications required for normal repropramming, or the modifications that occur at individual down-stream target genes. For over 25 years, my laboratory has used the budding yeast, Saccharomyces cerevisiae, as a model organism for dissecting the roles of histone modifications and variants in mRNA transcription, DNA replication and repair, and centromere function. Recently, our studies have expanded to focus on histone H4 acetylation by Myst2 in the preimplantation mouse embryo. Results from these preliminary experiments suggest that Myst2 is the enzyme responsible for H4 acetylation in reprogramming and zygotic gene activation. This is a major change in my research direction and success will require advanced expertise in mouse molecular genetics. To accomplish this goal, I have arranged to take a sabbatical in the laboratory of Dr. Barbara Knowles at the Jackson Laboratory. This Kirschstein-NRSA Senior Fellowship will enable a full year´s residency, rather than six months, and ensure a complete training program in techniques and approaches that cannot be completed in a short tenure. During my sabbatical, I propose to address three specific aims. First, we will determine the pattern of histone modifications during OET, globally by high resolution 4Pi microscopy, and specifically at key reporter genes identified by the transcriptome analysis of the Knowles group. Second, we will engineer transgenic and knockout mouse strains that permit maternal depletion of Myst2 expression in the oocyte. We will use these strains to test directly for the requirement of Myst2 during OET. Third, we will examine the fate of H4 acetylation in Myst2- depleted embryos during OET, specifically at reporter genes and globally using tiled promoter microarrays. The results of these studies will have a significant impact on how we think about, and manage, a wide range of human health issues including assisted reproductive techniques, somatic nuclear transfer, and stem cell therapy
Project start date: 2007-08-22
Project end date: 2008-08-21
1F33HD056741-01 (2007): $58886
THE ROLE OF MYST HISTONE ACETYLTRANSFERASE IN GENOME STABILITY
M Mitchell Smith
University Of Virginia Charlottesville, Box 400195, Charlottesville, Va 22904-4195
Grant 2R01GM060444-09A1 from National Institute Of General Medical Sciences
Abstract: Dynamic protein acetylation is essential for normal cell physiology and development. Indeed, defects in acetyltransferases are associated with a wide variety of human diseases. The MYST family of histone acetyltransferases are highly conserved, from yeast to man, and they serve as the catalytic subunits of large multi-protein complexes whose structural compositions are also conserved. MYST acetyltransferases are required for early mammalian embryonic development and aberrant rearrangements or regulation of MYST genes are associated with human cancers. We are investigating the molecular genetics of two signature members of the MYST family the Esa1 enzyme of budding yeast, and the Myst2 enzyme of mouse and humans. ESA1 encodes the only essential histone acetyltransferase in budding yeast. It is the catalytic subunit of two multi-protein complexes, NuA4 and picNuA4. We recently made the surprising discovery that catalysis is not the essential function of Esa1, as previously believed. Our data argue, instead, that Esa1 is a "molecular switch" that uses the binding of Cofactor A to control currently uncharacterized essential functions. We will carry out experiments designed to understand what the essential function of Esa1 is doing, and how it is executed. We will study conditional mutants of ESA1 that specifically expose its essential function, and characterize phenotypes, gene expression patterns, and promoter chromatin structure under nonpermissive conditions. We will specifically challenge the molecular switch model using genetic suppressors and biochemical assays of protein conformation in recombinant picNuA4. The results of these experiments are poised to completely change the way we think about MYST family proteins. Myst2 (Hbo1) is a mammalian MYST family enzyme that serves as the catalytic subunit of multi-protein complexes that include members of the Ing and Jade tumor suppressor families. Based on our work and that of others, it is clear that Myst2 is required for DNA replication licensing, interacts with p53 to mediate stress signaling, and has roles in transcription. We recently discovered that homozygous Myst2 knockout mouse embryos arrest development at embryonic day E7.5, a stage at which rapid proliferation and extensive gene reprogramming are about to occur for gastrulation. Myst2 is the only MYST gene with this knockout phenotype and we propose that it is required for the burst of DNA replication, or gene reprogramming at this stage. We will characterize the gene expression pattern of wild type, heterozygous, and homozygous Myst2 knockout embryos to identify the genes and pathways dependent on Myst2 for development. We will characterize DNA replication licensing, S phase progression, DNA damage response, and protein occupancy at DNA replication origins to define the involvement of Myst2 is proliferation at this critical stage of development. Finally, we will use our knowledge of ESA1 to construct conditional knockout mice carrying mutant Myst2 alleles that will reveal if it also has essential non-enzymatic functions during development. These experiments will greatly expand our understanding of Myst2 function in regulating DNA replication and gene expression in early mammalian development. MYST family protein complexes carry out functions that are essential for proper gene expression, DNA replication, DNA damage repair, and embryonic development. Failures in the function of MYST genes are associated with genome instability and many diseases including human cancers. Little is known about their range of functions and target pathways. The research proposed in this application is designed to uncover new principles in how these enzymes work, what they do, and how they do it
Keywords: No Project Terms available
Relevance: MYST family protein complexes carry out functions that are essential for proper gene expression, DNA replication, DNA damage repair, and embryonic development. Failures in the function of MYST genes are associated with genome instability and many diseases including human cancers. Little is known about their range of functions and target pathways. The research proposed in this application is designed to uncover new principles in how these enzymes work, what they do, and how they do it
Project start date: 2000-01-01
Project end date: 2014-01-31
Budget start date: 1-FEB-2010
Budget end date: 31-JAN-2011
PFA/PA: PA-07-070
2R01GM060444-09A1 (2010): $369600
The Role Of MYST Histone Acetyltransferases In Genome Stability
M Mitchell Smith, Professor
Microbiologyuniversity Of Virginia Charlottesville
box 400195
charlottesville, Va 229044195
Grant 2R56GM060444-09 from National Institute Of General Medical Sciences IRG: MGC
Abstract: Dynamic protein acetylation is essential for normal cell physiology and defects in the enzymes involved are associated with a wide variety of human diseases. The MYST family of histone acetyltransferases are highly conserved, from yeast to man, and they serve as the catalytic subunits of large multi-protein complexes whose structural compositions are also conserved. Aberrant rearrangements or regulation of MYST acetyltransferases are associated with human cancers. However, we know relatively little about their critical protein substrates, the roles they play in the larger protein complexes, or the genetic regulatory pathways that control their function. Recent advances now make tackling these problems feasible and exciting. We are investigating the molecular genetics of two signature members of the MYST family the Esa1 enzyme of budding yeast, and the Hbo1 enzyme of humans. ESA1 encodes the only essential histone acetyltransferase in budding yeast. It is the catalytic subunit of two multi-protein complexes, NuA4 and picNuA4. We recently made the surprising discovery that catalysis is not the essential function of Esa1, as previously thought. Instead, we propose that Esa1 is a regulatory subunit of NuA4 complexes that uses its catalytic domain as a sensor of physiological signals. We will carry out experiments designed to identify the initiating signals detected by Esa1, and to understand the mechanism of signal transduction through NuA4. We will dissect the molecular genetics of suppressors of esa1 mutations and examine the chromatin changes at promoters of regulated genes in response to esa1 mutations and suppressors. The results of these experiments are poised to completely change the way we think about Esa1 and NuA4 function. Hbo1 is a human MYST family enzyme that serves as the catalytic subunit of multi-protein complexes that include members of the Ing and Jade tumor suppressor families. We discovered that Hbo1 has a causal role in the assembly of the pre-replicative complex for DNA replication licensing. Our results predict that pre-RC proteins may be direct substrates for Hbo1 acetylation. We will carry out experiments to identify sites of lysine acetylation and the molecular mechanism through which they facilitate licensing. We also recently found that tumor suppressor p53 and Hbo1 interact physically and functionally. We propose that this interaction is part of the mechanism that decides between cell division cycle arrest and apoptosis in response to physiological stresses. We will test this hypothesis by carrying out experiments to identify a novel protein substrate of Hbo1 implicated in the pathway and examine its role in regulating pro-apoptotic gene transcription. These experiments will greatly expand our understanding of Hbo1 function in regulating DNA replication and cell proliferation
Project start date: 2000-01-01
Project end date: 2009-07-31
2R56GM060444-09 (2008): $348450
READING THE HISTONE CODE:NANOSCALE MORPHOLOGY OF EPIGNEOMIC HISTONE MODIFICATIONS
M Mitchell Smith
University Of Virginia Charlottesville, Box 400195, Charlottesville, Va 22904-4195
Grant 5RC1GM091175-02 from National Institute Of General Medical Sciences
Abstract: Challenge Area 06 Enabling Technologies. Specific Challenge Topic 06-GM-101 Structural Analysis of Macromolecular Complexes. A major challenge in chromatin biology and molecular cytology is how to study the macromolecular structures of specific epigenetic chromatin modifications in single cells at nanoscale resolution. The many post-translational modifications of histone proteins play critical roles in defining the biological functions of chromosomes. There are different sets of modifications associated with transcriptionally active chromatin, with inactive chromatin, with replicating chromatin, with sites of chromosome damage, and with key subnuclear compartments such as centromeres and telomeres. Defects in these epigenetic marks, in the enzymes and proteins that "read", "write", and "erase" them, have been found to occur in many human diseases, including cancer and neural degenerative syndromes. Furthermore, these epigenetic marks are key determinants in stem cell biology, and are important both in maintaining the pluripotent state and in driving differentiation. At present, there are no technologies that can visualize the macromolecular structures of epigenetic histone modifications in single cells at resolutions any better than approximately 200-300 nm. The resolution of light microscopy is limited by diffraction, and the resolution of electron microscopy is limited by lack of contrast. Biochemical techniques including mass spectroscopy, chromatin immunoprecipitation, and chromosome conformation capture, are making great strides in defining the functional combinations of histone marks, but they cannot image those structures within the nucleus or follow their dynamics in live cells. That is the challenge. To meet it, we have designed novel probes of histone modification by fusing multivalent binding domains to photoactivatable fluorophores and expressing these "decoder" constructs in cells. Using recent advances in super-resolution microscopy, the positions of these reporters can be determined with a localization precision of 6-10 nm, reconstructing the image of modifications at a resolution below the width of the 30 nm chromatin fiber. The goal of this project is to exploit this proof of principle and develop the technology to enable researchers to explore the macromolecular structures of chromatin at levels that are an order of magnitude more precise than is currently possible. Over the next two years we will address three major aims. (1) We will construct a high quality, versatile set of decoder constructs that represent all of the known histone modification binding motifs. (2) We will characterize the properties and binding specificities of these decoders by comparing their co-localization with traditional antibody probes, by conducting genome-wide sequencing of bound DNA in chromatin immunoprecipitations, and by imaging the reporters in three-dimensions and in live cells. (3) We will construct decoders that are predicted to have novel new binding specificities through the rational design of chimeric, artificial multivalent, and synthetic binding motif combinations. The results of these efforts will develop the technology to enable the routine visualization of the nanoscape of chromatin epigenetics. This will impact basic research by providing the tools, reagents, and protocols that will marshal a paradigm shift in how chromatin is studied. Moreover, since chromatin modifications have real practical importance for cancer, neural degeneracies, embryonic development, assisted reproductive services, and future stem cell therapies, the ability to image epigenetic marks rapidly, in single live cells, at nanoscale resolutions has the potential to radically improve the diagnosis, classification, and treatment modalities associated with a wide spectrum of human health issues. Complex modifications of proteins on the chromosomes have major roles in regulating cellular physiology and keeping cells normal and healthy. A severe limitation in studying how these modifications work is the fact that we cannot see them, observe their structural organizations, or watch how they come and go. Overcoming this limitation is a formidable challenge that will have enormous impact on both basic and clinical health science, including cancer, neurodegenerative syndromes, and stem cell therapies. This project will exploit breakthroughs in fluorescent light microscopy, and recent insights into the biophysics of the modifications, to develop technology that will enable researchers to see the structures of these modifications for the first time at nanoscale resolution in single live cells
Keywords: 30 nm Chromatin Fiber; 30 nm Fiber; Address; Antibodies; Area; Automobile Driving; Basic Research; Basic Science; Binding; Binding (Molecular Function); Biochemical; Biocompatible Materials; Biological Function; Biological Process; Biomaterials; Biophysics; CHIP assay; Cancers; Cell Function; Cell Nucleus; Cell Process; Cell physiology; Cells; Cellular Function; Cellular Physiology; Cellular Process; Centromere; ChIP (chromatin immunoprecipitation); Chimera; Chimera organism; Chromatin; Chromatin Fiber; Chromosomes; Classification; Clinical; Collaborations; Complex; Consultations; Cytology; DNA Binding; DNA Binding Interaction; DNA Molecular Biology; DNA Sequence; Data; Defect; Development; Diagnosis; Dimensions; Drivings, Automobile; Electron Microscopy; Embryo Development; Embryogenesis; Embryonic Development; Endomycetales; Engineering; Engineerings; Enzymes; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Fluorescence Microscopy; Future; Goals; Health; Health Sciences; Histone Code; Histones; Human; Human, General; Image; Imagery; Instrumentation, Other; Investigators; Label; Life; Macromolecular Complexes; Macromolecular Structure; Maine; Malignant Neoplasms; Malignant Tumor; Mammalian Cell; Man (Taxonomy); Man, Modern; Marshal; Mass Spectrum; Mass Spectrum Analysis; Methods and Techniques; Methods, Other; Microscope; Microscopy; Microscopy, Fluorescence; Microscopy, Light, Fluorescence; Modality; Modification; Molecular; Molecular Biology; Molecular Configuration; Molecular Conformation; Molecular Interaction; Molecular Stereochemistry; Molecular Structure; Morphology; Nature; Nerve Degeneration; Nervous; Neuron Degeneration; Normal Cell; Nuclear; Nucleus; Photometry/Spectrum Analysis, Mass; Play; Position; Positioning Attribute; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Property; Property, LOINC Axis 2; Protein Modification; Protein Modification, Post-Translational; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteins; Protocol; Protocols documentation; Reading; Reagent; Reporter; Research Personnel; Researchers; Resolution; Role; Saccharomycetales; Services; Site; Solutions; Specificity; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Spinal Column; Spine; Structure; Subcellular Process; Syndrome; Systematics; Techniques; Technology; Time; Universities; Validation; Vertebral column; Virginia; Visualization; Width; Work; Writing; Yeast Model System; Yeast, Budding; YeastModel; backbone; base; chromatin immunoprecipitation; chromatin modification; combinatorial; conformation; conformational state; design; designing; driving; fluorophore; gene product; genome-wide; histone modification; human disease; imaging; improved; innovate; innovation; innovative; insight; instrumentation; light microscopy; malignancy; meetings; nano meter scale; nano meter sized; nano scale; nanometer scale; nanometer sized; nanoscale; neoplasm/cancer; neural; neural degeneration; neurodegeneration; neuronal degeneration; new approaches; novel; novel approaches; novel strategies; novel strategy; relating to nervous system; reproductive; social role; stem cell biology; stem cell therapy; telomere; tool; vector
Relevance: Complex modifications of proteins on the chromosomes have major roles in regulating cellular physiology and keeping cells normal and healthy. A severe limitation in studying how these modifications work is the fact that we cannot see them, observe their structural organizations, or watch how they come and go. Overcoming this limitation is a formidable challenge that will have enormous impact on both basic and clinical health science, including cancer, neurodegenerative syndromes, and stem cell therapies. This project will exploit breakthroughs in fluorescent light microscopy, and recent insights into the biophysics of the modifications, to develop technology that will enable researchers to see the stuctures of these modifications for the first time at nanoscale resolution in single live cells
Project start date: 2009-09-30
Project end date: 2011-08-31
Budget start date: 1-SEP-2010
Budget end date: 31-AUG-2011
PFA/PA: RFA-OD-09-003
5RC1GM091175-02 (2010): $442898
1RC1GM091175-01 (2009): $468977
The Role Of Histone H4 In Genome Stability
M Mitchell Smith, Professor
University Of Virginia Charlottesville Box 400195 Charlottesville, Va 229044195
Grant 2R01GM060444-05 from National Institute Of General Medical Sciences IRG: CDF
Abstract: Cells routinely encounter conditions that cause DNA double-strand breaks and defects in DNA replication-coupled events, both from external sources and as a result of normal metabolic activity. Maintaining DNA sequence stability in face of these challenges is essential for normal gene expression, chromosome organization, and faithful transmission of genetic information. Loss of genome stability, through defects in DNA replication or DNA damage repair, is thought to play a causative role in carcinogenesis and tumor progression. Both DNA replication and DNA damage repair must take place in the context of chromatin structure and there is increasing evidence that histone modifications are essential for these processes. We have discovered that acetylation of histone H4 by the NuA4 histone acetyltransferase complex is required for nonhomologous end-joining and replication-coupled double-strand break repair. Furthermore, both human HBO1 and yeast Esa1, members of the MYST family of histone acetyltransferases, play key roles in DNA replication. Little is known about the molecular mechanisms by which histone modifications participate in replication and repair functions. To address these issues we will focus on three major research questions (1) how histone modification functions at the site of a double-strand break and the molecular steps that are defective in histone modification mutants; (2) the molecular role of acetylation in DNA replication initiation and elongation; and (3) the functional genomics of the histone-dependent pathways that maintain genome stability.
Keywords: DNA repair, DNA replication, acetylation, acyltransferase, genome, histone, DNA damage, gene expression, gene interaction, gene mutation, genetic regulation, protein structure function, SDS polyacrylamide gel electrophoresis, flow cytometry, polymerase chain reaction, western blotting
Project start date: 2000-01-01
Project end date: 2007-12-31
2R01GM060444-05 (2004): $313564
HISTONE GENE EXPRESSION IN YEAST
M Mitchell Smith, Professor
University Of Virginia Charlottesville Box 400195 Charlottesville, Va 229044195
Grant 5R01GM028920-23 from National Institute Of General Medical Sciences IRG: GEN
Keywords: centromere, fungal genetics, gene induction /repression, histone, nucleosome, protein structure function, biological signal transduction, cell cycle, gene deletion mutation, genetic promoter element, protein biosynthesis, transcription factor, DNA footprinting, Saccharomyces cerevisiae, immunoprecipitation, mass spectrometry, molecular cloning, nucleic acid sequence, site directed mutagenesis
Project start date: 1981-04-01
Project end date: 2004-03-31
5R01GM028920-23 (2003): $324412
5R01GM028920-22 (2002): $324458
Sponsored Links Excellgen http://Excellgen.com
5R01GM028920-21 (2001): $324502
2R01GM028920-20 (2000): $324544
2R01GM028920-16 (1996): $260668