Michael Grunstein - US grants

While the structure of the chromosome is being slowly unravelled, the function of the chromosomal proteins is largely unknown. The focus of our laboratory is the determination of histone function in yeast through genetic and biochemical means. Histone acetylation is a means by which chromosomal folding may be regulated during the cell cycle. Therefore, we propose to analyze this process in detail. We have developed an assay for enzymatic acetylation in yeast and have used this assay to identify a temperature sensitive mutant (MCS-1147) showing decreased acetylase activity at the non-permissive temperature. We propose to a) purify, and characterize the specificity of yeast histone acetylases, b) determine whether enzymes acetylation is cell cycle specific, and c) obtain mutations in other histone acetylase. These experiments would allow us to determine the biological function of acetylation and will be accomplished by classical enzymological techniques as well as in vitro mutagenesis and yeast transformation of yeast histone acetylase genes. The results of these experiments should provide important insight into the in vivo biological function of histone acetylation and have a profound influence on our understanding of the eukaryotic chromosome.

Chromatin undergoes reversible folding in order to allow genes to be transcribed, replicated and separated during mitosis. Histones are involved in the compaction of chromatin and the reversible histone modifications that affect the core histone N-termini and histone H1 have been implicated in these functions. In the yeast, Saccharyomyces cerevisiae histone deletions and point mutations will be made to alter core histone domains and sites of acetylation which may be involved in folding. We propose to obtain conditional mutations in histones and develop and inducible gene system which will allow the results of deleterious mutations to be analyzed biochemically. Assays will be set up utilizing electron microscopy, sedimentation and nuclease hypersensitivity to establish which histone domains and sequences are invloved in folding. The gene for a histone H1-like protein in yeast will be characterized by sequence and genetic analysis and the protein by chromatin reconstitution to determine the function of this protein. Finally, a distantly related yeast, Schizosaccharomyces pombe, will be studied in order to determine the generality of our key findings in S. cerevisiae.

The yeast, Saccharomyces cerevisiae, will be used to determine how the chromosome assembles and is topologically altered during the cell cycle. The following distinct but related steps in formation of the dynamic chromosome will be studied. a) The cis- and trans-acting factors involved in controlling histone synthesis and stoichiometry in S phase will be identified. b) Strains in which individual core histones H2B and H4 are repressible by GAL promoters will be used to determine the steps in histone assembly, how nucleosomes segregate during DNA replication and whether gross changes in chromosome structure affect transcription. c) Deletions in the charged histone ends will also be analyzed by conditional synthesis to determine their effects on assembly of the nucleosome and changes in chromosome structure. d) Since N-terminal acetylation of histones has been implicated in assembly and structural changes of the chromosome, we will study this process by site-directed mutagenesis of acetylated lysines. Also, we will use a cloned gene (HAT-c), whose mutation results in decreased histone acetylase activity, to probe the function of acetylation during the yeast cell cycle.

In the yeast, Saccharomyces cerevisiae, the silent mating loci, HMLalpha and HMRa, are permanently inactivated in normal cells. This repression is part of the mechanism which generates different cell types (a, alpha, diploid) and allows mating to occur. Silencing occurs between specific DNA boundaries, termed 'silencers', and requires the function of at least the four proteins Sir1, Sir2, Sir3 and Sir4. Recently, we found that deletions in the extremely conserved histone H4 N-terminus of yeast (but not similar deletions in the N-termini of histones H2A or H2B) activate the silent mating loci specifically. We propose here experiments which will help uncover many of the details in the mechanism of chromosomal repression of the silent mating loci. Using directed mutagenesis we intend to determine a) which sequences in histone H4 are required for silencing and whether there is special significance to H4 N-terminal acetylation in modifying repression of the silent mating loci. b) We will determine whether histones are involved in other mating locus specific functions such as gene conversion, autonomous replication and segregation. c) Antibodies to Sir proteins will be used to determine whether these repressor proteins interact directly with silencer chromatin isolated as episomes and in particular whether they interact with the H4 N-terminus. d) We will ask whether there are other, as yet undefined factors which interact with H4 to repress the silent mating loci. This will be done by the identification of genes whose products suppress histone gene mutations. e) We will determine whether it is H4 alone or the H3- H4 complex which is involved in repression of HMLalpha and HMRa. The chromosomal repression of the silent mating loci in yeast has analogies to the inactivation of single copy genes seen in heterochromatin of flies and mammals. This too is accompanied by changes in chromatin structure and results in long-term genetic inactivation. The understanding of the molecular basis of silencing, is likely to have a profound effect on our understanding of terminal repression, and therefore cellular differentiation, in all eukaryotes. This knowledge should impact our ability to deal with major illness, such as cancer, in which cellular differentiation is abnormal.

The histone H3 and H4 termini help regulate cell cycle progression, gene activity and nucleosome assembly. These functions are likely to be mediated by acetylation and deacetylation of these N terminal tails which may not only affect their interactions with underlying DNA but with other proteins. We have recently discovered the family of five histone deacetylases in yeast which have different and overlapping roles in H3 and H4 acetylation, cell cycle progression and gene activity. Our goal now is to define these roles at a molecular level. We proposed to identify catalytic and regulatory non-catalytic subunits of each of these complexes since these factors may determine deacetylase specificity to different sites in histones, different genes and different stages of the cell cycle. We wish to know the extent to which deacetylase regulates un-induced and induced gene activity and whether this occurs through effects on nucleosome positioning, DNA topology or chromosomal condensation. Since nucleosome structure is disrupted at promoters by activators even before transcription and since histone acetylation provides a logical mechanism for nucleosome disruption, we propose to determine whether a known acetyltransferase (TAF130), that is part of the transcription machinery, disrupts nucleosome at promoters. Finally, we will determine which sites of acetylation in H3 and H4 are important for nucleosome assembly. Understanding gene activity and the cell cycle is central to our knowledge of human disease processes. Chromatin modifications affect both. Moreover, disruption of a histone deacetylase by a drug has been identified as a means of selectively inactivating parasites involved in diseases such as malaria and toxoplasmosis. The molecular characterization of the multiple histone deacetylases in yeast will allow the design of rational approaches to combat these disease states.

DESCRIPTION (Adapted from Applicant's Abstract): The mechanism of genetic repression by heterochromatin is a major unsolved problem in cellular and developmental gene regulation. Similar chromosomal regions with the features of heterochromatin are found at the telomeres in both complex and simple eukaryotes. Recently, an important clue to the understanding of this form of silencing has come from the discovery that certain histone N termini interact genetically and biochemically with specific Sir repressor proteins to repress and localize heterochromatin at the nuclear periphery in yeast. The goal of this proposal is to use genetic and biochemical means to determine how histones and non-histone proteins interact to allow silencing. Individual sites in histones H3 and H4 at both N terminal and non-N terminal regions involved in silencing will be mapped. These will be used genetically to identify non-histone factors which interact with thee sites to allow silencing. These sites will also be investigated as to their effect on the localization of telomeres to the nuclear periphery in order to determine whether the repression of heterochromatin is linked to its peripheral localization. It has been shown that histones H3 and H4 interact with the Sir3p and Sir4p repressors in vitro. In addition, Sir3p and Sir4p interact with each other. These interactions will be investigated to determine the sites of interaction and how they relate to the sites recognized by other silencing factors including Rap1, Sir3 and Sir4 proteins. This information will be used to correlate the interaction in vitro with defects in silencing in vivo. Another goal of this project is to determine biochemically, with crosslinking experiments, whether Sir3p and Sir4p interact directly with chromatin and specific histones in silenced regions. Factors responsible for these interactions will be identified in these experiments by using mutations in proteins known to be involved in silencing. Finally, histone acetyltransferases and deacetylases will be purified and their sequences will be used to clone the genes coding for these enzymes to determine genetically and biochemically whether acetylation of specific histone lysine residues is a means for regulating heterochromatin structure. These studies, which are designed to define the components which interact to form the heterochromatic complex in yeast, may lead to a novel understanding of the molecular nature of heterochromatin in eukaryotic organisms.

The Chemistry Department at the University of California-Los Angeles will acquire a stimulated emission depletion confocal laser scanning microscope (STED-CLSM) with this award from the Major Research Instrumentation (MRI) program. The requested microscope displays resolution down to 28 nm in the focal plane, a 10x improvement over conventional light microscopes. The instrument will be used to develop multi-color inorganic, stable, quantum rods as novel STED probes, decipher the structure of chromatin and its packaging into chromosomes in the cell, study cell signaling, viral and bacterial infection pathways, neural plasticity and many other important biological questions.

STED microscopy provides an alternative to electron microscopy because it capitalizes on the well-established advantages of fluorescence microscopy (sensitivity, molecular specificity, genetically encoded probes, live cells, ease of operation). The STED concept relies on a purely physical phenomenon, stimulated emission, coupled with smart optics, to sharpen the confocal excitation spot, resulting in much more detailed, nanometer resolved images. Bridging the gap between electron and diffraction-limited light microscopy, a STED nanoscope should be a powerful tool for unraveling the relationship between structure and function in cell biology. Indeed, many outstanding problems lay in the nanometer scale, such as the organization of chromosomes, the assembly of large protein complexes and viral structures, organelle structures, as well as applications to non-biological nano-scale devices.

DESCRIPTION (provided by applicant): Histone post-translational modifications control the access of transcription factors to the underlying DNA enabling the regulation of eukaryotic genes that are wrapped in nucleosomes. How this occurs is largely unclear and we have little knowledge of the mechanisms by which histones are modified during replication and how such modifications are altered during transcription to enable nucleosome loss and gene activity. This proposal will address the molecular mechanisms by which acetylation of histones takes place during yeast DNA replication and subsequently, how not only histone acetyltransferases (HATs) but also histone deacetylases (HDACs) such as Hos2 and Hda1 are involved in gene activation. Also, the pathway between nucleosome displacement and replacement is a focus of this proposal since our findings argue that `naked' DNA devoid of nucleosomes is not actually naked and proteins, such as topoisomerase II, that coat `naked'DNA help regulate nucleosome assembly and gene activity. Finally, this proposal addresses the unique functions of a novel acetylation site (histone H3 K56) that regulates yeast histone genes and is most often methylated in human cells. Of special interest is not only the mechanism by which acetylation of this site regulates histone genes but how acetylation and methylation of K56 control the master regulators of pluripotency in human embryonic development. It is already known that defects in histone deacetylase mediated pathways and in topoisomerases can influence the progression of cancers. Our basic studies should allow a better understanding of the cancer state and of the very nature of pluripotency in humans leading to novel therapeutic approaches to disease.