京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19405号 / 医科博第65号 / 新制||医科||5(附属図書館) / 32430 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 河本 宏, 教授 斎藤 通紀, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20788号 / 医博第4288号 / 新制||医||1025(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 高橋 淳, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Covalent modifications of histones play a critical role in many important biological processes such as transcription, DNA repair and recombination. Among the major modifications known so far, histone H3 acetylation at lysines 9 and 14 (H3K9/14), monoubiquitination of histone H2B at lysine123 (H2BK123) and H3 lysine 4 methylation (H3K4) are among the more studied ones. The importances of these modifications have been further stressed by its connection to various human diseases including cancers. Previous biochemical studies have shown that H2BK123 ubiquitination is mandatory for methylation at histone H3K4. However, little is known about the regulatory mechanisms of H3K4 methylation by H2B ubiquitination in vivo. Thus, the prime focus of this study is to understand the factors involved in the regulation of H2B ubiquitination, the regulatory mechanisms of the cross-talk between H2BK123 ubiquitination and H3K4 methylation and the role of these covalent modifications in transcriptional regulation under physiological conditions. Here in this study, I have shown that Ubp8p, a histone deubiquitinase, is a bona fide subunit of SAGA (Spt3-Ada-Gcn5 acetyltransferase) co-activator complex and selectively regulates both di and trimethylation of histone H3K4 at the core promoter of a SAGA-dependent gene in vivo. However, over the open reading frames for a subset of constitutive genes H2B ubiquitination selectively upregulates only H3K4 trimethylation but not dimethylation. Moreover, such an upregulation of H3K4 trimethylation has no impact on the RNA Polymerase II (RNAPII) recruitment and hence transcription of the respective genes. Interestingly, at an inducible gene, histone H2B ubiquitination promotes transcription elongation independently of H3K4 methylation. Furthermore, this study also demonstrates for the first time, the molecular mechanism for the cross-talk between H2B ubiquitination and H3K4 methylation in vivo. Evidently a COMPASS subunit, Cps35p, is necessary for the trans-tail cross talk between histones H2B and H3. Finally, this study also shows that Sgf73p, a SAGA subunit, is required for SAGA recruitment at the promoters of several SAGA dependent genes and facilitates transcription in both HAT-dependent and HAT-independent manner. Collectively, the results from this study not only provide deep insights into the regulatory mechanisms of H2B ubiquitination and H3K4 methylation (and their role in transcription) but also give a new functional dimension to SAGA subunit, Sgf73p, under physiological conditions. Given the role of histone acetylation, ubiquitination and methylation in many human diseases, the results from this study is of tremendous clinical value unveiling new therapeutical targets.

Chromatin remodelers utilize the energy derived from ATP hydrolysis to mobilize nucleosomes. ISWI remodelers mobilize and evenly space nucleosomes to regulate gene expression. ISW2, an ISWI remodeler in yeast, has been shown to reposition nucleosome near promoter regions and represses both mRNA and antisense non coding RNA transcription. ISW2 is composed of four subunits and the catalytic Isw2 subunit consists of several conserved domains. The highly conserved ATPase domain is present at the N-terminus whereas the conserved HAND, SANT and SLIDE domain are towards the carboxyl terminal end of Isw2. Nucleosome mobilization by ISW2 requires both extranucleosomal DNA and the N-terminal tail of histone H4. DNA crosslinking and peptide mapping revealed that the ATPase domain contacts nucleosome two helical turns away (SHL2) from dyad to a site close to the H4 tail, whereas the HAND, SANT and SLIDE domain contact a 30bp stretch of DNA comprising the edge of nucleosome and ~20bp of extranucleosomal DNA. The ATPase domain and the C-terminal domains were investigated for their role in regulation of ISW2 activity both in-vitro and in-vivo. It appears that there are distinct modes of ISW2 regulation by these domains. Mutation of a patch of five acidic amino acids on the region of ATPase domain that contact SHL2 was found to be crucial for both ISW2 remodeling and nucleosome stimulated ATPase activity. Acidic patch mutant ISW2 was unable to mobilize nucleosome or hydrolyze ATP in absence of H4 tail. This indicates that the region of ATPase domain contacting nucleosome at SHL2 and H4 tail act in two separate and independent pathways to regulate ISW2 remodeling. Both HAND and SLIDE domain were shown to crosslink entry/exit site and linker DNA respectively. The roles of C-terminal domains were investigated either by deletion of the individual domain or mutation of conserved basic residues on the surface of these domains that are suspected to interact extranucleosomal with DNA. Deletion of HAND domain had minimal effect on in vitro ISW2 activity, however whole genome transcription analysis revealed one key role of this domain in ISW2 regulation. In absence of HAND domain, ISW2 had minimal role on repression of genes that were RPD3 (co-factor) dependent, however significantly derepressed genes that were RPD3 independent. At these loci, nucleosome positions were altered and ISW2 recruitment was reduced in absence of a functional HAND domain. Thus the HAND domain regulates recruitment and remodeling of ISW2 at those genes where ISW2 acts independent of other cofactors. The SANT domain, C-terminal to HAND domain, appears to control the "step size" of nucleosome remodeling and was found to be required for processive nucleosome remodeling by ISW2. Both H4 tail and SANT domain appear to control two distinct stages of ISW2 remodeling. A long alpha helical spacer separates SANT domain from SLIDE domain. SLIDE domain was found to be the protein-protein interaction domain that interacts with accessory Itc1 subunit to maintain ISW2 complex integrity. The two ways by which SLIDE domain regulate ISW2 is by binding or recruitment of ISW2 to promoter regions and additionally by binding independent regulation of both ATPase and remodeling activity. The remodeling mechanism of ISW2 was further compared with another ISWI type remodeler in yeast, Isw1a; using time resolved nucleosome remodeling combined with high resolution site specific histone DNA crosslinking at six different nucleosomal positions to track the movement of the nucleosomes. Nucleosome remodeled by the same remodeler showed discontinuous nucleosome movement between two tracking points indicating formation of small "bulges". One key difference in remodeling mechanism was that although both ISW2 and Isw1a moved nucleosomes towards longer linker DNA, only Isw1a remodeled nucleosomes "backtracked" ~11bp during remodeling. Backtracking of remodeling was prominently observed at nucleosomal regions in close proximity to translocase binding sites suggesting the potentially different mechanisms shared by similar remodeling complexes.

Linker histones (H1) are the basic proteins in higher eukaryotes that are responsible for the final condensation of chromatin. H1 also plays an important role in regulating gene expression. H1 has been described as a transcriptional repressor as it limits the access of transcriptional factors to DNA. Linker histone binds to DNA that enters or exits the nucleosome. Several crystal structures have been published for the nucleosome (histone core/DNA complex), and the interactions of the core histone proteins with DNA are well understood. In contrast the location of the linker histone and its interactions with ds-DNA are poorly understood. In this study we have used isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and CD spectropolarimetry to determine the thermodynamic signatures and structural changes that accompany H1 binding to ds-DNA. The thermodynamic parameters for the binding of intact linker histones (H1.1, H1.4, and H10) to highly polymerized calf-thymus DNA and to short double stranded DNA oligomers have been determined. We have also determined the thermodynamics for binding of H10 C-terminal tail (H10-C) and globular domain (H10-G) to calf-thymus DNA. The real surprise in the energetics is that the enthalpy change for formation of the H1/DNA complex is very unfavorable and that H1/DNA complex formation is driven by very large positive changes in entropy. The binding site sizes for H1.1, H1.4, and H10 were determined to be 36bp, 32bp, and 36bp respectively. CD results indicate that CT-DNA is restructured upon complexation with either the full length H1 protein (H10) or its C-terminal domain (H10-C). In contrast, the structure of H10 is largely unchanged in the DNA complex. Temperature dependence of enthalpy change, osmotic stress and ionic strength dependence of Ka were tested using ITC. These results indicate that the entropy driven H1/DNA complexes are a result primarily from the expulsion of bound water molecules from the binding interface. This study provides new insights into the binding of linker Histone H1 to DNA. A better understanding of the functional properties of H1 and its interactions with DNA could provide new insights in understanding the role H1 in DNA condensation and transcriptional regulation.

The proper packaging of the genome in eukaryotic nuclei is essential for proper gene expression and cell function. Chromatin at the large scale is divided into two major compartments heterochromatin and euchromatin. Heterochromatin compromises the transcriptionally inactive tightly packaged regions of chromatin, while euchromatin is the transcriptionally active region of chromatin. The Heterochromatin Protein family (HP1) proteins are epigenetic hallmarks of constitutive heterochromatin. Recent evidence suggests human HP1α undergoes liquid-liquid phase separation suggesting a role for HP1 phase separation in the formation of compacted heterochromatin within HP1 droplets. Phase separation is a biophysical property of proteins with intrinsically disordered domains which are protein domains that lack a defined secondary structure and have the ability to undertake multiple conformations. In this thesis, I investigated the ability of C. elegans HP1 homologs HPL-2A and HPL-1 to phase separate utilizing directed mutations to elucidate the intermolecular interactions that govern this phenomenon and different assays to assess their phase separation. I concluded that HPL-2A is a bona fide phase separating protein that selectively condenses chromatin. HPL-2A’s phase separation depends on specific interactions, mainly dimerization and the presence of lysine and arginine residues in the hinge region. HPL-2A has a specific IDR that drives its phase separation which is the hinge region as the CTE and NTE are not essential for its phase separation.

Indiana University-Purdue University Indianapolis (IUPUI) / Under normal conditions, smooth muscle cells do not replicate, or proliferate, and provide a means of contraction for many internal organs, including blood vessels and the gut. However, under abnormal or disease conditions, such as congenital heart disease and cancer, smooth muscle cells acquire the ability to replicate, to make extracellular matrix proteins and to migrate. Thus, determining how smooth muscle cells regulate these processes is crucial to understanding how the cells can switch between normal and diseased states. Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation and migration. It is generally accepted that one way that SRF can distinguish between these functions is through pathway-specific co-factor interactions. A novel SRF co-factor, chromodomain helicase DNA binding protein 8 (CHD8), was originally isolated from a yeast two-hybrid assay. CHD8 is widely expressed in adult tissues including smooth muscle. Data from in vitro binding assays indicate that the N-terminus of CHD8 can interact directly with the MADS domain of SRF. Co-immunoprecipitation assays verified the ability of these two proteins to interact within cells. Adenoviral-mediated shRNA knockdown of CHD8 in smooth muscle cells resulted in statistically significant 10-20% attenuation of expression of SRF-dependent, smooth muscle-specific genes. Similar experiments revealed that knockdown of CHD8 did not affect the SRF-dependent induction of immediate early genes required to promote proliferation. In contrast, knockdown of CHD8 in A10 vascular smooth muscle cells resulted in a marked induction in of apoptosis, characterized by increases in apoptotic markers such as phospho-H2A.X, cleaved PARP and activated caspase-3. These data suggest that CHD8 may play a specific role in modulating SRF’s activity toward anti-apoptotic genes, thereby regulating smooth muscle cell survival.

Ocean warming is leading to increased occurrence of coral mass bleaching events, threatening the persistence of these ecosystems and the communities that rely on them. While reef recovery is possible, conservation approaches based purely on transplantation/coral-gardening will not suffice to maintain these ecosystems over the projected environmental changes. Assisted evolution approaches aim to boost acclimatization and adaptation processes. A potential approach could be to harness the naturally occurring mechanism of environmental memory that has been observed in corals and other organisms, where an organism remembers a priming stress event to allow a faster/stronger response when the stress re-occurs. In this thesis I aimed to investigate whether this mechanism exists and how it is regulated on a molecular level in the sea anemone Aiptasia. Aiptasia were primed to heat stress by exposing them to 32 °C water for several years, or for one week. After a recovery period of one week at 25 °C, a naïve and the primed treatments were exposed to lethal thermal stress at 34 °C for three days. Primed treatments performed better than the naïve treatment in survival, photosynthetic efficiency and symbiont density for two days, after which the priming advantage was lost. The difference between the primed treatments indicated that the priming dose may affect priming success. There were clear indications of an epigenetic transcriptional memory mechanism on a transcriptional level. I observed a pronounced difference between control and heat-stressed treatments, indicating that transcription returned to near baseline expression after cessation of the priming exposure. The functional categories of differentially expressed genes in heat stress relative to control were similar between naïve and primed treatments, with the main difference observed in a stronger up- and downregulation of stress response genes in the long-term primed treatment. I optimized a chromatin immunoprecipitation protocol for use with Aiptasia by adjusting fixation, sonication and immunoprecipitation conditions. The enrichment of H3K4me2/me3 and poised RNA Pol II in the promoters of stress response genes will be investigated next to elucidate the mechanism of the observed epigenetic transcriptional memory in Aiptasia, and to ultimately inform conservation strategies for coral reefs globally.

No description available.

Chromatin is decorated by a large array of biochemical modifications made to DNA and histone proteins. These modifications—and the broader organizational structure of chromatin—provide an important additional layer of information that is superimposed upon genome sequence and thus are widely referred to as the epigenome. The epigenome helps control which genes are expressed in a given context to produce the gene expression patterns that underlie the many different cellular phenotypes that arise during an organism’s development, and determine how these gene expression patterns are subsequently maintained for the life of an organism. The epigenetically heritable states are maintained and transmitted by self-propagating epigenetic mechanisms that persist in the absence of an initial stimulus. These epigenetic programs are generally thought to be controlled by core regulatory networks involving molecular writers and readers of chromatin marks. Guided by these principles, in this dissertation, we establish an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system consists of synthetic factors that can write and read m6A, and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we use our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits are able to program epigenetic functions de novo, conceptually validating “read-write” architectures. This dissertation outlines a synthetic framework for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells. / 2021-08-30T00:00:00Z