In embryonic stem cells (ESCs), the SWI/SNF, CHD, and INO80 families of ATP-dependent chromatin remodellers have been implicated in maintaining pluripotency-associated gene expression, however the involvement of ISWI family remodellers has yet to be defined. Here, we explore the importance of the mammalian ISWI homologue SNF2H (Smarca5) by deriving a conditional knockout mouse ESC line and observing the consequences of SNF2H depletion on the pluripotent state. Cre-mediated deletion of Snf2h disrupts hallmark characteristics of pluripotency, resulting in distinct morphological changes; reduced expression of the master transcription factors Oct4, Sox2, and Nanog; and reduced alkaline phosphatase activity. To understand the mechanisms of SNF2H-mediated regulation, we mapped SNF2H-bound nucleosomes genome-wide. SNF2H is broadly distributed across the genome, but is preferentially enriched at active regulatory regions and transcription factor binding sites. These findings demonstrate the importance of SNF2H in ESCs and shed light on genome-wide mechanisms of transcriptional regulation.

The innate immune system is the first line of host defense against external exposures. During these initial encounters, antigen presenting cells - specifically monocytes and macrophages - modulate further inflammatory responses. Macrophages exist along a spectrum of phenotypic programs; on the inflammatory M1 end they enhance immune activity while on the anti-inflammatory M2 end they suppress further immune activation. Furthermore, within M2 macrophages there exist many subpopulations, namely M2a and M2d, each with specific roles during infection or exposure. We sought to compare the epigenetic profiles of these subpopulations of macrophages to determine key regulatory gene networks and factors that could be exploited for therapeutic benefit.While traditionally viewed as primitive and nonspecific, a growing body of clinical and experimental evidence argues the innate immune system develops memory as a result of previous exposures, allowing the innate system to respond with enhanced and broad immunological protection upon exposure to a secondary stimulus. This biological process of innate immunity has been termed trained immunity. Trained immunity shares many phenotypic and epigenetic characteristics with adaptive immune memory; however, one of the starkest distinctions is the propensity of trained immunity to develop against heterologous stimuli. Innate memory is not antigen specific, frequently protecting the host against unrelated organisms.

La sénescence cellulaire est une réponse à un stress des cellules de mammifères se caractérisant entre autres par un arrêt durable du cycle cellulaire, la production d’un sécrétome particulier (SASP) et un remaniement de la chromatine. Celle-ci peut être déclenchée par des stress génotoxiques, l’activation d’oncogènes ou un raccourcissement trop important des télomères. Des analyses en spectrométrie de masse ont permis de mettre en évidence l’accumulation d’un variant d’histone dans le cas de sénescence cellulaire induite par des dommages à l’ADN. Ce variant, nommé H2A.J, représente 1% de la quantité totale d’histones H2A mais atteint près de 20% en sénescence longue durée. Cette thèse se concentre sur l’étude de la fonction de cette protéine H2A.J qui semblerait impliquer dans l’activation transcriptionnelle du SASP. Afin d'étudier le rôle de cette protéine, j'utilise des lignées stables de fibroblastes humains exprimant ou non un shARN ciblant le gène de H2A.J et je cherche à mettre en évidence des différences entre ces lignées. Du fait de son accumulation lié à l’activation des voies de réparation des dommages à l’ADN, ce variant présente un potentiel comme biomarqueur du vieillissement in vivo. A ce jour, aucun biomarqueur spécifique de cet état n’a été identifié. Enfin, L’expression constitutive du gène codant pour cette protéine suggère une régulation post-transcriptionnelle de sa déposition. L’un des objectifs du projet est de mettre en évidence les voies de régulation permettant l’accumulation dans la chromatine de H2A.J. / In mammalian cells, cellular senescence has been defined as a stress response. It is characterized by a stable cell cycle arrest, morphological transformation, a secretion of pro-inflammatory factors termed the SASP (senescence associated secretory phenotype) and the alteration of the chromatin structure. Originally, telomere loss or dysfunction was shown to trigger the onset of senescence. However, the senescence state can also result from inadequate culture conditions, oncogene induction or genotoxic stresses. Work in the lab focuses on mechanisms governing the onset and maintenance of senescence and on the search for new markers of senescence. We have recently identified chromatin modifications and epigenetic regulations during cellular senescence, such as post-translational modifications of histones and changes in the histone variants composition of nucleosomes. Mass spectrometry revealed the accumulation of a specific histone variant in DNA-damage induced senescence. This variant, H2A.J, makes up to 1% of the H2A histone content during proliferation, but reaches 20% of H2A species during deep senescence. The goal of my thesis work was to determine the function of this histone variant. We produced stable human fibroblast cell lines expressing shRNAs silencing the H2A.J gene. Microarray and RNA-sequencing analyses have shown that H2AJ-depleted fibroblasts have an altered transcriptome. In particular, such cells show a greatly delayed derepression in senescence of several SASP genes coding for some key cytokines and chemokines. This result indicates that accumulation of H2A.J in senescence is important for efficient expression of the SASP phenotype. Finally, the accumulation of senescent cells in aged tissues has often been inferred using surrogate markers (DNA damage, SA-B-Galactosidase, etc.). Our data suggest that H2AJ accumulation may be a novel in-vivo biomarker of aging for certain cell types.

Plants are sessile organisms and constantly encounter a myriad of pathogens; therefore, they rely on highly effective defense system for their survival. Our understanding of how plant immunity is triggered and regulated has seen tremendous progress over the last two decades, with many important players identified in the model systems, Arabidopsis thaliana. Mitogen activated protein kinases play a central role in signal transduction in biotic and abiotic stresses. MAPK pathways are regulated by three-interlinked protein kinases (MAPKKK, MAPKK, MAPK), which are sequentially activated by phosphorylation. The activation of the three MAPKs MPK3, MPK4 and MPK6 is one of the earliest cellular responses following pathogen attack leading to the phosphorylation of appropriate cytosolic or nuclear targets to regulate cellular processes. However, only few targets of MPK3, MPK4 and MPK6 have been identified and validated so far and many MAPK substrates remain to be discovered. We performed largescale phosphoproteomics on mock treated and flg22 treated WT and the three loss-of-function mutants mpk3, mpk4 and mpk6 to identify novel MAPKs substrates and their cellular functions in response to pathogen attack. We identify and validated some of the differentially phosphorylated cytosolic and chromatin targets of MPK3, MPK4 and MPK6. DEK2, a nuclear protein involved in multiple chromatin-related processes, was identified in the phosphoproteomics screen as an in vivo target of MPK6 and it interacts in planta and is phosphorylated in vitro by the three immune MAPKs. dek2 loss-of-function mutants were susceptible to bacterial as well as fungal pathogens. Additionally, transcriptome data of the dek2-1 mutant show that DEK2 is a transcriptional repressor inclusive of defense related genes and hormone synthesis and signaling genes. We determined that DEK2 is a reader of the histone mark, H3K9me1, by Microscale thermophoresis. From ChIP-Seq analysis, DEK2 was found to be enriched at class I TCP binding motif regions. We further need to determine whether DEK2 binds to TCP transcription factors directly or indirectly. Finally, based on our data we postulate a hypothetical working model for the function of DEK2 as a transcriptional repressor and a reader of H3K9me1 mark.

UHRF1 is a critical epigenetic regulator which serves as a molecular model for understanding the crosstalk between histone modification and DNA methylation. It is integrated in the process of DNA maintenance methylation through its histone ubiquitylation activity, ultimately functioning as a recruiter of DNA methyltransferase 1 (DNMT1). As the faithful propagation of DNA methylation patterns during cell division is a common molecular phenomenon among vertebrates, understanding the underlying conserved mechanism of UHRF1 for executing such a key process is important. Here, I present a broad-range evolutionary comparison of UHRF1 binding behavior and enzymatic activity of six species spanning across the vertebrata subphylum. According to their distinct binding modes to differentially methylated histone H3, a pattern is emerging which separates between mammalian and nonmammalian orthologs. H. sapiens, P. troglodytes and M. musculus UHRF1 orthologs utilize the functionality of both TTD and PHD domains to interact with histone H3 peptides, while G. gallus, X. laevis, and D. rerio employ either TTD or PHD. Further, UHRF1 allosteric regulation by 16:0 PI5P is a unique case to primate orthologs where H3K9me3 peptide binding is enhanced upon hUHRF1 and pUHRF1 interacting with 16:0 PI5P. This is due to their closed and autoinhibited conformation wherein TTD is blocked by the PBR region in linker 4. 16:0 PI5P outcompetes TTD for PBR binding resulting in a release of TTD blockage, hence, enhanced H3K9me3 binding. However, owing to the lack of phosphatidylinositol binding specificity and reduced sequence conservation of linker 4, the regulatory impact of 16:0 PI5P in avian and lower vertebrate orthologs could not be detected. Additionally, all UHRF1 orthologs exert their ubiquitylation enzymatic activity on histone H3 substrates, supporting the notion that the overall functionality of UHRF1 orthologs is conserved, despite their divergent molecular approaches. Taken together, my findings suggest that UHRF1 orthologs adopt distinct conformational states with a differential response to the allosteric regulators 16:0 PI5P and hemi-methylated DNA.

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

In C. elegans, the germline is a tightly regulated tissue where silencing pathways regulate genes, allowing expression of “self” while silencing “non-self.” Doublestranded RNAs (dsRNAs), short interfering RNAs (siRNAs), and piwi-associated RNAs (piRNAs) can transmit this regulation across generations via transgenerational epigenetic inheritance (TEI) mechanisms (Bošković and Rando, 2018). Analogously, some pathways can counteract gene silencing to allow sustained expression in the germline. One such example is a non-coding DNA structure called Periodic An/Tn clusters that can prevent the silencing of transgenes in the germline (Frøkjær-Jensen et al., 2016). In this thesis, I developed a novel piRNA-based tool called piRNA interference (piRNAi), where target-specific short “guide” piRNAs (sg-piRNAs) can robustly silence endogenous genes and transgenes. I have used piRNAi to understand the rules for licensing gene expression and transgenerational epigenetic inheritance in the C. elegans germline. Initially, I describe design rules for generating transgenes with PATC-rich introns that resist germline silencing and are robustly expressed from extrachromosomal arrays. PATC-rich transgenes showed more accurate gene expression patterns and did not prevent germline regulation by 3’ untranslated regions (3’ UTRs). Next, I developed the piRNAi technique to understand the role of PATCs in licensing transgene expression and the rules for how endogenous genes can be targeted for piRNA-mediated silencing and TEI. I demonstrate that a PATC-rich gfp transgene and endogenous genes are not resistant to piRNA-mediated silencing. Finally, I used piRNAi to define rules for TEI: 1. I identified two new endogenous targets for TEI (him-5 and him-8) that can inherit silencing for four and six generations respectively, after transient exposure to sg-piRNAs. 2. I demonstrate that an endogenous gene (him-5) can be semi-permanently silenced in the absence of the piRNA/PRG-1 pathway. 3. The duration of TEI was significantly shortened in a transgene that contained PATC-rich introns. Altogether, my thesis shows that an endogenous small RNA pathway can be reprogrammed to silence endogenous genes and transgenes in the germline, which enables novel experimental paradigms for studying inherited and semipermanent silencing.

DNA methylation is an epigenetic mechanism with roles that range from the fine tuning of transcription to genome wide dynamic acclimation to changing environments and regulation of developmental processes. While recent work has confirmed the presence and regulatory functions of DNA methylation in non-bilaterians, its role and distribution in Porifera has never been addressed. In this study, we performed whole genome bisulfite sequencing of the demosponge Amphimedon queenslandica and show that DNA methylation occurs mostly in CpG dinucleotides of coding regions. While high levels of gene-body methylation correlate positively with high expression and co-occur with the histone modification H3K36me3, they are not associated with amelioration of spurious transcription as found in other metazoans; nonetheless, per-exon methylation levels are predictive for exon retention suggesting a role in mRNA splicing. Additionally, analyses of Amphimedon and other sponges genomic data consistently revealed biased dinucleotide frequencies that suggest a long history of methylation-driven CpG conversion. Despite a genome wide loss of CpG dinucleotides, these are positively selected in exons and in methylated genes. These results indicate DNA methylation as a component of early metazoans regulome and challenge hypothesis on CpG methylation acting as a means for codon usage optimization.

No description available.

京都大学 / マギル大学 / 新制・課程博士 / 博士(ゲノム医学) / 甲第24204号 / 医博JD第2号 / 新制||医||JD1(附属図書館) / 京都大学大学院医学研究科京都大学マギル大学ゲノム医学国際連携専攻 / (主査)准教授 Wilson Michael (トロント大学), 教授 竹内 理, 准教授 Bailey Swneke (マギル大学), 教授 伊藤 貴浩, 教授 Shoubridge Eric (マギル大学) / 学位規則第4条第1項該当 / Doctor of Philosophy in Human Genetics / Kyoto University / McGill University / DFAM