Neurodevelopmental disorders encompass a spectrum of different conditions with both genetic and environmental etiologies. Although rapid progress has been made in deciphering the genetic landscape of these disorders, in most cases, it remains unclear how mutations undermine neurodevelopmental mechanisms. However, increasing identification of risk genes suggests chromatin remodelling is frequently impacted. For instance, de novo variants encoding the chromatin remodeller CHD4 causes Sifrim-Hitz-Weiss syndrome, which manifests as an overgrowth-intellectual disability syndrome. To further understand Chd4’s role during cortical development, we excised the ATPase domain of Chd4 in the germline or specifically in the developing telencephalon, creating three mouse models. Germline heterozygotes presented a slight decrease in brain weight, cortex area and Ctip2+ cells, with females displaying more overt impairments in learning and memory. Telencephalon-specific conditional heterozygotes exhibited slight changes in white matter, increased repetitive movements and altered social behaviours. Telencephalon-specific conditional knockouts presented with decreased brain size, brain weight, and cortex thickness due to decreased upper layer neurons, and anxiety phenotypes. These data reveal an unexpected complexity in the impacts of Chd4 mutations on neurodevelopmental processes and behaviour.

Indiana University-Purdue University Indianapolis (IUPUI) / Understanding β-cell development and function is essential for generating more effective treatment options for individuals with diabetes. A key player in pancreatogenesis, islet development, and mature β-cell function is the Pdx1 transcription factor (TF). Pdx1 activity is modulated through interactions with various coregulators, including the Swi/Snf chromatin remodeling and Nucleosome Remodeling and Deacetylase (NuRD) complexes. Loss of one Swi/Snf ATPase subunit, Brg1, in early pancreatogenesis reduces final pancreas mass, and β-cell-specific deletion of both subunits, Brg1 and Brm, leads to glucose intolerance and loss of insulin production in the β-cell. Here, we hypothesized Swi/Snf governs endocrine progenitor cell development and postnatal islet function. To test this, we generated conditional murine knockouts of Brg1 (Brg1Δendo;Brm+/-), Brm (Brg1Δendo/+;Brm-/-), or both subunits (DKOΔendo) during endocrine cell development. No DKOΔendo mice were recovered at weaning, and loss of Brg1 but not Brm led to severe glucose intolerance, ad-lib fed hyperglycemia, and reduced insulin levels by four weeks of age. Brg1Δendo;Brm+/- mice had fewer islets and compromised insulin secretion. Together, these data suggest that loss of Brg1 during endocrine cell development has negative impacts on postnatal islet function, with loss of both Brg1 and Brm being early postnatal lethal. Pdx1 has been shown to also interact with the Chd4 helicase subunit of the NuRD complex. Here, we demonstrate Pdx1:Chd4 interactions are increased under stimulatory conditions and hypothesize that Chd4 modulates expression of Pdx1-bound genes critical for β-cell function. To test this, we generated a tamoxifen inducible, β-cell-specific Chd4 knockout mouse model (Chd4Δβ). Four weeks following Chd4 removal, Chd4Δβ mutants were glucose intolerant with severe insulin secretion defects. Additionally, Chd4Δβ islets contained fewer mature insulin granules and secreted more proinsulin. RNA-sequencing from Chd4Δβ β-cells identified numerous upregulated (eg Hk2, Mycl) and downregulated genes (eg MafA, Chga, Chgb, Slc2a2). Through ATAC-sequencing, we discovered several differentially accessible genomic regions, including Chd4-bound and Pdx1-controlled MafA Region 3, which had reduced accessibility in Chd4Δβ β-cells. Lastly, we demonstrate that CHD4 impacts human β-cell function and PDX1:CHD4 interactions were reduced in human donor β-cells with type 2 diabetes, demonstrating loss of these interactions is a significant feature of diabetes pathogenesis.

Chromodomain Helicase DNA-Binding Protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double stranded break (DSB) repair. Here, we show that depletion of CHD4 in Acute Myeloid Leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ATM pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic-agent induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34+ hematopoetic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor formatting behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21922号 / 生博第407号 / 新制||生||53(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 垣塚 彰, 教授 豊島 文子, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Le rôle fonctionnel des transcrits de l’hétérochromatine péricentromérique reste à ce jour largement incompris chez les eucaryotes supérieurs. Néanmoins, il a été montré que ces transcrits sont soumis à un contrôle très précis, fonction du cycle cellulaire. La régulation de la transcription est fortement contrôlée par la structure de la chromatine qui peut être modifiée localement en changeant la composition biochimique du nucléosome, notamment par l’utilisation des variantes d’histones. L’objectif de ma thèse a été de mieux comprendre le rôle de la protéine chaperonne d’histone DAXX et de sa variante d’histone H3.3 dans la régulation de la transcription des séquences répétées péricentromériques. Par la méthode de purification TAP-TAG, les partenaires spécifiques de DAXX ont été identifiés à partir d’extraits solubles nucléaires de fibroblastes embryonnaires murins. Ces analyses ont mis en évidence que CAF-1, classiquement associé à H3.1, et les facteurs de remodelage de la chromatine ATRX et CHD4 interagissent spécifiquement avec DAXX. Le rôle de ces protéines dans le contrôle de la transcription de l’hétérochromatine péricentromérique a ensuite été mis en évidence par une approche combinant l’interférence ARN et la Q-PCR. Enfin, les résultats suggèrent fortement que ces mécanismes de régulation ont lieu au niveau des corps nucléaires PML. L’ensemble de ces données montre qu’il existe une régulation spatio-temporel très fine de la structure de la chromatine régulant la transcription de l’hétérochromatine péricentromérique. / The functional role of pericentromeric heterochromatin transcripts remains largely unknown in higher eukaryotes. Nevertheless, it has been shown that these transcripts are subject to very precise control, depending on the cell cycle. Regulation of transcription is tightly controlled by chromatin structure that can be modified locally by changing the biochemical composition of the nucleosome, including the use of histone variants. The aim of my thesis was to better understand the role of the histone chaperone protein DAXX and its histone variant H3.3 in the regulation of transcription of pericentromeric repeats. By the method of TAP-TAG purification, DAXX specific partners were identified from soluble nuclear extracts of murine embryonic fibroblasts. These analyzes revealed that CAF-1, classically associated with H3.1, and the chromatin remodeling factors, ATRX and CHD4, specifically interact with DAXX. The role of these proteins in the control of transcription of pericentromeric heterochromatin was then highlighted by an approach combining RNAi and Q-PCR. Finally, the results strongly suggest that these regulatory mechanisms take place at PML nuclear bodies. Taken together, these data show that there is a spatio-temporal regulation of the fine structure of chromatin regulates transcription of pericentromeric heterochromatin.

An understanding of the human fetal to adult hemoglobin switch offers the potential to ameliorate β-type globin gene disorders such as sickle cell anemia and β-thalassemia through activation of the fetal γ-globin gene. Chromatin modifying complexes, including MBD2-NuRD and GATA-1/FOG-1/NuRD play a role in γ-globin gene silencing, and Mi2β (CHD4) is a critical component of NuRD complexes. In the studies presented in Chapter 2, we observed that the absence of MBD2 in a sickle cell mouse model leads to a decrease in the number of sickled cells observed in the peripheral blood, and significantly increases survival in these mice. Although further studies will be necessary to fully understand the effect of MBD2 knockout in sickle cell disease mice, absence of MBD2 appears to partially ameliorate the sickle cell anemia phenotype in vivo. In the studies presented in Chapter 3, we observed that knockdown of Mi2β relieves γ-globin gene silencing in β-YAC transgenic murine CID hematopoietic cells and in CD34+ progenitor derived human primary adult erythroid cells. We show that independent of MBD2-NuRD and GATA-1/FOG-1/NuRD, Mi2β binds directly to and positively regulates both the KLF1 and BCL11A genes, which encode transcription factors critical for γ-globin gene silencing during β-type globin gene switching. Remarkably, less than 50% knockdown of Mi2β is sufficient to significantly induce γ-globin gene expression without disrupting erythroid differentiation of primary human CD34+ progenitors. These results indicate that Mi2β is a potential target for therapeutic induction of fetal hemoglobin.