Eukaryote DNA is organised into the more compact nucleosome by wrapping 147bp of DNA around a histone octamer core. The N-terminal tails of the histones protrude through the DNA and can be modified by a variety of enzymes. Acetylation of Histone 4 Lysine 16 (H4K16ac) is an important modification associated with an increase in transcription, and in flies is an important component of the doseage compensation system. It is also unique amongst histone modifications in that it has been directly associated with chromatin decompaction. H4K16ac has been linked to development through its Histone Acetyltransferase, MOF. Deletion of MOF in mice leads to mass chromatin defects, and embryonic lethality prior to the blastocyst stage. I set out to understand the role of H4K16ac in differentiating Embryonic Stem cells (ES cells) and chromatin compaction in vivo. I generated a ChIP-seq profile for H4K16ac in undifferentiated ES cells, and after 3 days of retinoic acid (RA) differentiation. This revealed an association of H4K16ac with the promoters of transcribed genes in pluripotent ES cells, followed by loss H4K16ac on ES cell specific genes and gain of the modification on differentiation specific genes. There were some silent genes in ES cells, however, which were acetylated on their promoters. Through this study I also found that H4K16ac and MOF mark active enhancers in ES cells, along with H3K4me1 and H3K27Ac and p300. H4K16ac did not mark a known regulatory region in limb cells, and it is possible that it marks active enhancers only of ES cells. Furthermore, I looked at the compaction state large regions (>100kb) which lost H4K16ac upon differentiation by FISH, to determine if loss of H4K16ac could predict compaction. The regions selected showed no change in compaction state between UD and D3 cells, meaning that loss of H4K16ac does not directly lead to chromatin compaction in vivo. However loss of H4K16ac may be necessary for any subsequent compaction, or the change in compaction may take place at nucleosomal level. Finally, I attempted both to overexpress and reduce the level of MOF in ES cells. I was unable to manipulate the level of MOF in this cell type in either direction; expression of endogenous MOF was silenced after very little time, and stable MOF shRNA cell lines showed no reduction in levels of MOF. Therefore, potentially, dosage of MOF/H4K16ac in this cell type is critical. This study may help to understand the significance of H4K16ac in ES cell differentiation and chromatin compaction.

In S. cerevisiae, the ribosomal DNA locus is silent for RNA polymerase II (Pol II) transcription and recombination (rDNA silencing). Our goal is to understand how histones and histone-modifying enzymes regulate the silent chromatin at the rDNA locus. Sir2, a NAD+-dependent histone deacetylase, is required for rDNA silencing. To understand how Sir2 regulates rDNA silencing, we performed chromatin immunoprecipitation to measure the association of modified histones across the rDNA repeat in wild-type and sir2Δ cells. We found that in sir2Δ cells, histone H3 at the rDNA became hyperacetylated and hypermethylated. High levels of K4-methylated H3 correlate with Pol II transcription. Consistent with this, we found that the nontranscribed spacer (NTS) region was transcribed by Pol II in sir2Δ cells. To investigate if transcription of the NTS region regulates rDNA silencing, we overexpressed this region both in trans and in cis. Our data showed that overexpression of the NTS region in cis caused Pol II silencing defect and hyperrecombination at the rDNA. These data suggest that Sir2 contributes to maintain the silent chromatin at the rDNA by repressing Pol II transcription in the NTS region. We also found that the NTS transcripts could be translated in vitro and that they copurified with polysomes, suggesting that the transcripts may encode proteins or that the transcripts are somehow involved in the process of translation. Additionally, we examined the role of linker histone H1 in regulating rDNA silencing. We found that, unlike Sir2 that represses both Pol II transcription and recombination, histone H1 only represses recombination at the rDNA. The hyperrecombination defect at the rDNA is more severe in sir2Δ hho1Δ double mutant than in either single mutant, suggesting histone H1 and Sir2 act independently. Consistently, hho1Δ cells did not accumulate extrachromosomal rDNA circles (ERCs) or the Holliday junction intermediates, which accumulate in sir2Δ cells. These data suggest that histone H1 and Sir2 regulate different recombination pathways. In summary, my research has provided insight into the mechanism of how silent chromatin at the rDNA locus is regulated, which will help us understand how fundamental components of chromosomes affect gene expression and genome stability.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22746号 / 医博第4664号 / 新制||医||1047(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 武藤 学, 教授 小川 誠司, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Skeletal muscle development and growth involve significant changes in gene expression. The overall objective of this dissertation project was to identify transcription factors, enhancers, and mechanisms that control gene expression during skeletal muscle development and growth on a genome-wide scale. Three independent studies were conducted in this project. The objective of the first study was to identify potentially novel mechanisms that mediate myoblast differentiation, a process whereby the mononuclear muscle precursor cells myoblasts express skeletal muscle-specific genes and fuse with each other to form multinucleated myotubes. Comparing gene expression profiles in C2C12 cells, a widely used model of myoblasts, before and 6 days after induced myogenic differentiation by RNA sequencing (RNA-seq) revealed 11,046 differentially expressed genes, of which 5,615 and 5,431 were upregulated and downregulated, respectively. Functional enrichment analyses revealed that the upregulated genes were associated with biological processes or cellular components such as skeletal muscle contraction, autophagy, and sarcomere. In contrast, the downregulated genes were associated with biological processes or cellular components such as ribonucleoprotein complex biogenesis, mRNA processing, and ribosome. Western blot analyses showed an increased conversion of LC3-I to LC3-II protein during myoblast differentiation, further demonstrating the upregulation of autophagy during myoblast differentiation. Blocking the autophagic flux in C2C12 cells with chloroquine inhibited the expression of skeletal muscle-specific genes and the formation of myotubes, confirming a positive role of autophagy in myoblast differentiation and fusion. The aim of the second study was to identify enhancers and transcription factors that regulate gene expression during the differentiation of bovine satellite cells, which are the myogenic precursor cells in adult skeletal muscle, into myotubes. In this study chromatin immunoprecipitation followed by sequencing (ChIP-seq) was used to identify active enhancers, i.e., genomic regions marked with histone modification H3K27ac (acetylation of lysine 27 of H3 histone protein). 19,027 and 47,669 H3K27ac-marked enhancers were identified from undifferentiated and differentiating bovine satellite cells, respectively. Of these enhancers, 5,882 and 35,723were specific to undifferentiated and differentiating bovine satellite cells, respectively while 13,199 were shared by both undifferentiated and differentiating bovine satellite cells. Many of the H3K27ac-marked enhancers specific to differentiating bovine satellite cells were associated with muscle structure and development genes and were enriched with binding sites for MyoD, AP-1, AP-4, KLF, TEAD, and MEF2 transcription factors. Through siRNA-mediated knockdown, AP-4 was found to be essential for differentiation of bovine satellite cells into myotubes. The objective of the third study was to identify enhancers and transcription factors that control differential gene expression in skeletal muscle between neonatal and adult cattle. First, RNA-seq was performed to compare gene expression profiles in skeletal muscle between neonatal calves and adult steers. This analysis identified 924 genes downregulated and 1,021 upregulated from calf to steer muscle. Among genes downregulated in steer muscle were myosin heavy chain3 (MYH3) and MYH8, and among genes upregulated in steer muscle were MYH7 and myoglobin. Surprisingly, many so-called adult muscle genes, such as MYH1 and MYH2, were not differentially expressed between calf and steer muscle. Gene ontology analyses showed that many genes downregulated in steer muscle are involved in protein synthesis and glycolysis and that many genes upregulated in steer muscle function in blood vessel development and immune cell activation. Next, ChIP-seq was performed to identify genomic regions marked with H3K27ac, i.e., active enhancers, in the skeletal muscle of neonatal calves and adult steers. This experiment led to the finding of 20,163 enhancers specifically active in the calf muscle, 14,909 enhancers specifically active in the steer muscle, and 27,002 enhancers active in both the calf and steer muscle. Motif enrichment analyses revealed the enrichment of binding sites for the KLF family and TEAD family transcription factors in enhancers active specifically in the calf muscle, the enrichment of binding sites for the FOXO family and the SMAD family transcription factors in enhancers specifically active in the steer muscle, and the enrichment of binding sites for the MRF family and MEF2 family transcription factors in enhancers active in both the calf and steer muscle. . These results shed light on the differences in gene expression and biology between newborn calf and adult steer skeletal muscle. These results also shed light on the enhancers and transcription factors that control these differences. / Doctor of Philosophy / Muscle is the central part of meat. So, to improve meat yield, it is essential to know how muscle development is controlled. Muscle development, also called myogenesis, starts with muscle progenitor cells developing into myoblasts. Myoblasts then differentiate and fuse with each other to form myotubes. Myotubes undergo hypertrophy and form functional muscle fibers. During myogenesis, each step involves significant changes in gene expression. Gene expression is controlled mainly by proteins called transcription factors. The overall goal of this project was to identify transcription factors and DNA sequences bound by these factors that control gene expression during muscle development. This project consisted of three studies. In the first study, we used the RNA sequencing (RNA-seq) technique to find genes differentially expressed in myoblasts between before and after terminal differentiation. Analyzing the RNA-seq data led to the discovery that autophagy, a 'self-eating' biological process, is required for myoblast differentiation. In the second study, we used a technique called chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify genomic regions called active enhancers in differentiating bovine myoblasts. This work led to the identification of thousands of active enhancers and dozens of transcription factors binding to these genomic regions that control the differentiation of bovine myoblasts. In the third study, we combined RNA-seq and ChIP-seq to explore the genes and genomic regions controlling muscle transition from newborn calves to adult cattle. This part of the project led to the finding of thousands of genes differentially expressed and thousands of genomic regions differentially activated between newborn calf and adult steer muscle.

No description available.

During spermatogenesis, histones are largely replaced by transition proteins and protamines in normal stallions. Incomplete nucleoprotein exchange results in the abnormal retention of histones and transition proteins, which is an indicator of poor sperm quality. Equine nucleoprotein exchange has not previously been investigated in detail, so that equine sperm chromatin quality problems, which are often responsible for poor breeding performance of stallions, are not well understood. In order to characterize chromatin remodeling events in stallion spermatogenesis and to identify proteins indicative of sperm chromatin defects, such as excessive amounts of histones, we identified antibodies that recognize equine testis-specific proteins of interest. Immunoblotting of testis and sperm protein lysates and immunofluorescence staining of histological tissue sections were used to identify candidate marker proteins of incomplete sperm chromatin maturation. Results of the study, which represents the first comprehensive characterization of the nucleoprotein exchange during spermatogenesis in the stallion, challenge the paradigm that the main function of histone H4 lysine (hyper-) acetylation (concomitant H4K5 and H4K8 acetylation) is to facilitate nucleosome ejection during spermatid nuclear elongation to allow for transition protein and protamine insertion into the chromatin. That paradigm was based on observations in mice and rats where H4 acetylation in several lysine residues occurs just prior to or during nuclear elongation. In contrast, the equine data presented here show strong acetylation of H4 in K5, K8 and K12 positions immediately after meiosis in round spermatids, independent of nuclear transition protein 1 deposition. Furthermore, results of H4K16 acetylation analyses underline the importance of this mark, which is likely mediated by DNA damage signaling pathways, emphasizing the importance of DNA repair processes for the exchange of nucleoprotein exchange in spermiogenesis and therefore, in extension, for male fertility. In addition, a revised description of the equine spermatogenic cycle is proposed here that is better aligned with human, mouse and rat spermatogenesis. Finally, the testis-specific histone variant TH2B was identified as a potential quantitative marker of equine sperm quality.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19891号 / 医博第4140号 / 新制||医||1016(附属図書館) / 32968 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 山田 泰広, 教授 開 祐司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Numerous memory studies have demonstrated that epigenetic-mediated transcriptional regulation, such as post-translational histone modifications, is essential to memory formation and maintenance. Moreover, many studies on the mechanisms of memory have focused on fear memories underlying traumatic events, which helps to understand post-traumatic stress disorder (PTSD). However, these mainly focus on individuals directly experiencing the event, while different species have shown the ability to learn fear indirectly by observing a conspecific experiencing a trauma. Thus, our understanding of indirect fear learning (IFL)'s characteristics is very limited. The trimethylation of histone 3 lysine 4 (H3K4me3) is an essential regulator of active gene transcription in cells and has been shown to be critical for memory formation in the hippocampus, a major site of memory storage. However, it is unknown how H3K4me3 is coordinated to target genes during memory formation. Monoubiquitination of histone H2B (H2Bubi) is critical for recruiting H3K4me3 to DNA in a gene-specific manner during memory formation in the hippocampus. Furthermore, there is a great overlap between H3K4me3 and phosphorylation of histone H2A.X at serine 139 (H2A.XpS139), a marker to study DNA double-strand break (DSB) loci. DSB is a critical mechanism for solving DNA-related topological issues during transcription and replication, which could be triggered in some immediate early genes (IEGs) by neuronal activity, such as memory consolidation.Here, we used rat fear conditioning paradigms in combination with quantitative molecular assays, such as chromatin immunoprecipitation (ChIP), and gene editing techniques, like siRNAs and CRISPR-dCas9 manipulations, to study the role of hippocampal 1) H2Bubi and 2) DSBs in contextual fear memory consolidation and reconsolidation, respectively. Additionally, we behaviorally and molecularly characterized IFL and compared it to directly acquired fear subjects. We found that contextual fear conditioning changed the expression of 86 genes in the hippocampus one hour after training. Remarkably, siRNA knockdown of the H2Bubi ligase, Rnf20, abolished changes in all but one of these genes, Per1. Additionally, we report that the loss of Rnf20 in neurons, but not astrocytes, of the hippocampus impaired long-term memory formation. We next found an increase in H2A.XpS139 and H3K4me3 levels in the Npas4, an IEG important for contextual fear memory, promoter region 5 minutes after retrieval. In vivo siRNAmediated knockdown of the enzyme responsible for DSB, topoisomerase II β, prior to retrieval, decreased Npas4 promoter-specific H3K4me3 and H2A.XpS139 levels and impaired long-term memory. Lastly, our data show that both sexes can indirectly acquire fear from either sex using the auditory-cued IFL model. Moreover, our data show that molecular profiles in the amygdala are largely unique to direct or indirect fear learning and vary by sex. Collectively, this data reveals novel roles for histone phosphorylation and ubiquitination in regulating H3K4me3 and memory formation and shows behavioral and molecular differences in each sex based on the way they acquire fear. / Doctor of Philosophy / Changes in epigenetic mechanisms, processes that control the expression of genes without changing the original sequences, play a crucial role in the formation and maintenance of memory. Moreover, many studies on the mechanisms of memory have focused on fear memories underlying traumatic events, helping to understand post-traumatic stress disorder (PTSD). However, these majorly focus on individuals directly experiencing the event, while different species have shown the ability to learn fear indirectly by observing a conspecific experiencing a trauma. Thus, our understanding of indirect fear learning (IFL)'s characteristics is very limited. In the present study, we investigated some of these epigenetic mechanisms called histone modifications. In the brain, histone 3 lysine 4 trimethylation (H3K4me3), a histone modification, is critical for memory formation in the hippocampus, a key area for memory storage. However, it is still not fully understood how H3K4me3 is coordinated during memory formation. Another histone modification called H2B monoubiquitination (H2Bubi) helps recruit H3K4me3 to DNA and so is also crucial for memory formation. Here, using rat models, we found that the expression of 86 genes is changed during memory formation in the hippocampus and that this result is almost entirely dependent on the presence of H2Bubi. We also discovered that H2Bubi is critical for longterm memory formation only in neurons of the hippocampus, and not astrocytes (another type of brain cells). Additionally, there is a connection between H3K4me3 and the phosphorylation of histone H2A.X, another epigenetic mechanism that co-occurs with DNA breaks and may serve as a markerfor studying these breaks. DNA breaks play a vital role during gene expression and could be triggered by neuronal activity during memory formation. We observed an increase in H2A.X phosphorylation and H3K4me3 levels in a memory-permissive gene five minutes after memory retrieval. Inhibition of DSBs, prior to retrieval abolished these changes, and impaired long-term memory. This suggests a critical role for DSBs in memory maintenance and that H2A.X phosphorylation is necessary for the recruitment of H3K4me3 to DNA. Lastly, our data demonstrated that both males and females could learn fear indirectly from either sex by observing them undergoing auditory-cued fear conditioning. Additionally, we found distinct molecular patterns in the amygdala, a brain region involved in fear processing, depending on whether fear was directly or indirectly acquired, and it varied between sexes. Collectively, data from this dissertation reveals novel roles for histone modifications in memory formation and shows behavioral and molecular differences in each sex based on the way they acquire fear.

Polycomb-group (Pc-G) proteins regulate homeotic gene silencing associated with the repressive covalent histone modification, trimethylation of histone H3 lysine 27 (H3K27me3). Pc-G mediated silencing is believed to remodel chromatin, rendering target genes inaccessible to transcription factors. Pc-G mediated silencing might result in irreversible changes in chromatin structure, however, there has been little analysis addressing whether Pc-G mediated silencing is reversible. In this work we focused on CURLY LEAF (CLF), the first Pc-G homologue discovered in Arabidopsis. CLF mediated repression of the floral homeotic gene AGAMOUS (AG) was challenged during early and late leaf development. AG was activated by the late leaf promoter, revealing that Pc-G mediated silencing can be overcome in old leaves in the presence of CLF. AG was also activated in young leaf primordia, yet did not persist in older leaves, revealing that transient activation of a Pc-G target is not epigenetically stable. To address the mechanism of Pc-G action within an endogenous environment, the histone dynamics at the APETALA1 (AP1) locus were characterized by Chromatin Immunoprecipitation. Unexpectedly, we found that the activation of AP1 in leaves did not require the removal of H3K27me3, questioning whether H3K27me3 is sufficient to silence. The roles of CLF in leaf and flower development are masked due to partial redundancy with SWINGER (SWN). clf- swn- mutants form a callus-like mass on sterile-tissue culture with no distinguishable plant organs. The role of CLF in regulating flowering time in natural populations of A. thaliana was investigated by complementing clf- mutants with CLF alleles from two accessions. We found that natural variation in CLF did not affect flowering time. To dissect the roles of CLF and SWN in late leaf and flower development, two approaches were developed for targeted expression. Firstly, CLF was introduced into the LhG4/ pOp transactivation system to provide CLF during early plant development. For mosaic analysis, CLF was introduced into the CRE lox recombination system in order to create clf- sectors surrounded by CLF+ SWN+ and CLF+ swn- cells.

The human genome consists of over 3 billion nucleotides and would be around 2 meters long if uncoiled and laid out. Each human somatic cell contains all this in their nucleus which is only around 5 µm across. This extreme compaction is largely achieved by wrapping the DNA around a histone octamer, the nucleosome. Still, the DNA is accessible to the transcriptional machinery and this regulation is highly dynamic and change rapidly with, e.g. exposure to drugs. The individual histone proteins can carry specific modifications such as methylations and acetylations. These modifications are a major part of the epigenetic status of the DNA which contributes significantly to the transcriptional status of a gene - certain modifications repress transcription and others are necessary for transcription to occur. Specific histone methylations and acetylations have also been implicated in more detailed regulation such as inclusion/exclusion of individual exons, i.e. splicing. Thus, the nucleosome is involved in chromatin remodeling and transcriptional regulation – both directly from steric hindrance but also as a signaling platform via the epigenetic modifications. In this work, we have developed tools for storage (Paper I) and normalization (Paper II) of next generation sequencing data in general, and analyzed nucleosome locations and histone modification in particular (Paper I, III and IV). The computational tools developed allowed us as one of the first groups to discover well positioned nucleosomes over internal exons in such wide spread organisms as worm, mouse and human. We have also provided biological insight into how the epigenetic histone modifications can control exon expression in a combinatorial way. This was achieved by applying a Monte Carlo feature selection system in combination with rule based modeling of exon expression. The constructed model was validated on data generated in three additional cell types suggesting a general mechanism.