Chromatin remodeling complexes modulate DNA accessibility permitting neuronal progenitor cells to proliferate and differentiate to form the mammalian neocortex. In the case of BPTF (Bromodomain PHD transcription Factor), the major subunit of a chromatin remodelling complex called NURF (Nucleosome Remodelling Factor), mutations leading to its haploinsufficiency have been linked to cause a recently annotated human neurodevelopmental disorder called NEDDFL (Neurodevelopmental disorder with dysmorphic facies and distal limb anomalies). Patients with this syndrome are mainly characterized with microcephaly and intellectual disability. We conditionally knockout (cKO) the Bptf gene during neocortical neurogenesis to analyze its role during embryonic and postnatal brain development. The Bptf cKO animals reveal significant forebrain hypoplasia. During cortical neurogenesis, the Bptf cKO mice show a reduction in intermediate neuronal progenitor (INP) cells, an increase in apoptosis as well as a prolonged cell cycle within proliferating progenitors. Similarly, the postmitotic pyramidal neurons of the Bptf cKO mice contained lower levels of Ctip2 and Foxp1. Lastly, our RNA-seq analysis delineated gene pathways deregulated by Bptf removal, which are involved in neurogenesis and neuronal differentiation. Our results indicate that Bptf is critical for murine telencephalon neurogenesis. The hypoplasia demonstrated in the mouse model can resemble the microcephaly displayed by the human NEDDFL patients, highlighting the relevance of chromatin remodelling complexes during intricate neural developmental processes.

L’ADN est constamment exposé à des insultes génotoxiques endogènes et exogènes. Plusieurs mécanismes de réparations de l’ADN sont mis en œuvre pour préserver la stabilité du génome et de l’épigénome. La recombinaison homologue (RH) joue un rôle central dans la réparation des cassures double brin de l’ADN (DSBs) et le redémarrage des fourches de réplication en réponse à un stress réplicatif. Ces deux processus sont tous deux couplés à l’assemblage de la chromatine. Le facteur d’assemblage de la chromatine 1 (CAF-1) est un chaperon d’histone conservé au cours de l’évolution qui fonctionne dans le processus d’assemblage des nucléosomes couplé à la réparation de l’ADN et à la réplication, en déposant sur l’ADN les tétramères d’histones (H3-H4)2 nouvellement synthétisés. Chez la levure Schizosaccharomyces pombe, le complexe CAF-1 est constitué de trois sous-unités, Pcf1, Pcf2 et Pcf3. Il a été montré que CAF-1 agit dans l’étape de synthèse de l’ADN durant le processus de réplication dépendante de la recombinaison (RDR) et protège le désassemblage des D-loop par l’hélicase Rqh1, membre de la famille des hélicases RecQ. Dans cette étude, nous avons adressé le rôle de CAF-1 pendant la réparation de l’ADN par recombinaison homologue chez la levure Schizosaccharomyces pombe. Par l’utilisation d’approches in vivo et in vitro, nous avons validé des interactions protéines-protéines au sein d’un complexe contenant Rqh1, CAF-1, PCNA, et l’Histone H3. Nous avons montré que Rqh1 interagit avec Pcf1 et avec Pcf2 indépendamment l’un de l’autre, et que l’interaction Rqh1-Pcf1 est stimulée par des dommages à l’ADN. Nous avons mis en place une méthode d’analyse de liaison à la chromatine pour suivre l’association de CAF-1 à la chromatine en réponse aux dommages à l’ADN. Nous avons observé qu’un stress réplicatif, mais pas l’induction de cassures double brin de l’ADN, favorise l’association de CAF-1 à la chromatine. Nous avons identifié plusieurs facteurs de la RH nécessaire pour l’association de CAF-1 à la chromatine en réponse à un stress réplicatif. De plus, nous avons mis en évidence des interactions physiques entre Pcf1 et des facteurs de la recombinaison homologue, parmi lesquels RPA et Rad51. Nos données suggèrent que CAF-1 pourrait s’associer aux sites de synthèse d’ADN dépendent de la recombinaison via son interaction avec des facteurs de la RH. L’ensemble des données de cette étude contribuent à renforcer le role de CAF-1 couplé à réparation de l’ADN, et révèlent une interconnexion entre les facteurs de la RH et l’assemblage de la chromatine. / DNA is constantly exposed to both endogenous and exogenous genotoxic insults. Multiple DNA repair mechanisms are exploited to guard the genome and epigenome stability. Homologous recombination (HR) plays a major role in repairing DNA double strand breaks (DSBs) and restarting stalled replication forks under replicative stress. These two processes are both coupled to chromatin assembly. Chromatin assembly factor 1 (CAF-1) is a highly conserved histone chaperone known to function in a network of nucleosome assembly coupled to DNA repair and replication, by depositing newly synthesized histone (H3-H4)2 tetramers onto the DNA. The fission yeast CAF-1 complex consists of three subunits Pcf1, Pcf2 and Pcf3. CAF-1 has been previously reported to act at the DNA synthesis step during the process of recombination-dependent replication (RDR) and protects the D-loop from disassembly by the RecQ helicase family member, Rqh1. In this study, we addressed the role of CAF-1 during homologous-recombination-mediated DNA repair in fission yeast.Using in vivo and in vitro approaches, we validated interactions within a complex containing Rqh1, CAF-1, PCNA, and Histone H3. We showed that Rqh1 interacts with both Pcf1 and Pcf2 independently of each other, and the Pcf1-Rqh1 interaction is stimulated by DNA damage. We developed an in vivo chromatin binding assay to monitor the association of CAF-1 to the chromatin upon DNA damage. We observed that replication stress but not double strand break favors CAF-1 association to the chromatin. We identified that several HR factors are required for CAF-1 association to the chromatin upon replication stress. In support of this, we have identified physical interactions between Pcf1 and HR factors, including RPA and Rad51. Our data suggest that CAF-1 would associate with the site of recombination-dependent DNA synthesis through physical interactions with HR factors. Put together, this work contributes to strengthening the role of CAF-1 coupled to DNA repair, and reveals the crosstalk between HR factors and chromatin assembly.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第22719号 / 理博第4628号 / 新制||理||1665(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 深井 周也, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22829号 / 医博第4668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 遊佐 宏介, 教授 斎藤 通紀, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Chromatin associated RNAs play a key role in mediating epigenetic mechanism. To improve our comprehension of how chromatin-associated RNAs influence gene expression, it is crucial to identify the genomic locus that RNA interacts with on a genome-wide scale. Emerging technologies were developed to capture binding sites of lncRNAs globally. Such techniques rely on the concept of Proximity ligation via a biotinylated adapter to ligate DNA and RNA on each end, such as MARGI, GRID-seq, ChAR-seq and the most recent technology, devised in our lab RNA And DNA Complexes Ligated & Sequenced (RADICL-seq). RADICL-seq has several advantages over the other methods. However, while this method produced a fairly global picture of the chromatin associated “RNA-ome”, the specific association with specific regulatory factors is missing, in addition all emerging technologies lack the sensitivity to capture low-expressed RNAs. Thus, we set out a strategy to address this issue by enriching global RNA-chromatin interactions mediated by a specific factor which will be achieved by combining RADICL with Chromatin Immunoprecipitation (ChIP) method. Since our lab interest is investigating the role of lncRNAs in muscle differentiation, RADICL-ChIP has been performed using the C2C12 mouse skeletal muscle. In particular, the role of identification of RNA moieties interacting with PRC2 PcG proteins in stress response and their genome wide distribution in chromatin.

OBJECTIVE: Despite advancement in targeted therapeutics, women’s cancers remain particularly deadly. The response to Poly (ADP-ribose) Polymerase (PARP) inhibitors, a prominent targeted therapy indicated for use in these cancers, is largely dictated by the cellular status of homologous recombination (HR) and varies among patients possibly due to intrinsic and acquired mechanisms of resistance. A recent effort to identify targetable pathways to enhance toxicity of PARPi identified the Snf2-like chromatin remodeling enzyme, Amplified in Liver Cancer 1 (ALC1), as a key determinant of PARPi sensitivity. While this discovery proposes a link between chromatin re-modeling and PARPi sensitivity, the mechanism underlying the relationship remains unclear. This study aims to validate ALC1 as a determinant of PARPi sensitivity, characterize the phenotype of ALC1 loss, investigate mechanisms of synthetic lethality and identify possibilities for future studies and clinical implementation. METHODS: UWB1.289, SUM149PT, DLD1, U-2 OS, and hTERT-RPE1 cells were cultured and used to validate ALC1-mediated PARPi sensitivity in a variety of viability assays. ALC1 depleted cells were complemented with various functional mutants to assess protein domains that were essential to PARPi-resistance. A Dual CRISPR-Cas9 system was implemented to screen BRCA1 complemented UWB1.289 cells with a sgRNA library targeting a multitude of DNA-repair associated genes in order to assess synthetic lethal/resistant relationships across different DNA-repair pathways. A MNase-sensitivity assay was developed and optimized to assess the global condensation of chromatin with combinational loss of ALC1 and PARP1. The 265-FokI system was employed to quantify the DNA damage response (DDR) to singular induced double strand breaks. Clonogenic assays were used to determine synergy with ionizing radiation. RESULTS: The loss of ALC1 significantly and selectively hypersensitized both BRCA1 and BRCA2-deficient cells to PARPi. In ALC1 depleted cells, the addition of ALC1 cDNA was able to rescue cells from PARPi hypersensitivity while cDNA with mutations in either the macro-domain or ATPase active domain remained sensitive. Screening DNA-repair pathways to assess synergy with PARPi and ALC1 loss in HR-proficient settings revealed that the loss of several HR and Alt-EJ genes selectively re-sensitized cells to PARPi. Interestingly, the loss of BER genes, including several glycosylases, were epistatic or resulted in a protective effect to PARPi. The reduction of NHEJ, NER, MMR or RER did not significantly alter cellular response to PARPi. Further, nucleosome relaxation was significantly inhibited in cells treated with PARPi, ALC1 loss or in combination via MNase assay. The recruitment of repair proteins to DSBs was significantly inhibited by PARPi as assessed by 265-FokI immunofluorescence. The addition of PARPi in the setting of ALC1 loss significantly increased the cytotoxicity of ionizing radiation. Analyzing TCGA data collected from patients’ tumors, ALC1 may be overexpressed in many cancers and alterations in ALC1 may predict patient responses to traditional and targeted cancer therapies. CONCLUSION: Using various models of BRCA1/2 deficiency we were able to validate the ability of ALC1 depletion to hypersensitize HR-deficient cells to PARPi. We provided insight into the mechanisms by which this phenomenon may be taking place, including chromatin compaction and the inhibition of DNA-damage repair. In addition, we provided therapeutic rationale that ALC1 may be targeted with PARP1 in synergy with other DNA-damaging agents. Overall, we believe that ALC1 is a prominent, viable and novel target of inducing PARP inhibitor sensitivity, that may help improve outcomes for patients with PARP inhibitor-resistant HR-deficient cancers. / 2022-06-04T00:00:00Z

In my thesis, I focused on several underexplored areas of RNA splicing regulation. In the first part, I analyzed how chromatin and transcription regulatory elements change pre-mRNA splicing. In the second part, I studied why long non-coding RNAs (lncRNAs) are spliced less efficiently than protein-coding mRNAs. Finally, I was testing the importance of intron for the activating function of lncRNAs. It has been shown that chromatin and promoter identity modulate alternative splicing decisions. Here, I tested whether local chromatin and distant genomic elements that influence transcription can also modulate splicing. Using the chromatin modifying enzymes directly targeted to FOSL1 gene by TALE technology, I showed that changes in histone H3K9 methylation affect constitutive splicing. Furthermore, I provide evidence that deletion of transcription enhancer located several kilobases upstream of an alternative exons changes splicing pattern of the alternative exon. Many nascent lncRNAs undergo the same maturation steps as pre-mRNAs of protein- coding genes (PCGs), but they are often poorly spliced. To identify the underlying mechanisms for this phenomenon, we searched for putative splicing inhibitory sequences. Genome-wide analysis of intergenic lncRNAs (lincRNAs) revealed that, in general, they do not...

Hematopoiesis is coordinated by a complex regulatory network of transcription factors among them PU.1 (Spi1, Sfpi1) and GATA-1 represent key molecules. GATA-1 and PU.1 bind each other on DNA to block each others transcriptional programs to prevent development of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells, transformed erythroid precursors that are blocked from completing the late stages of erythroid differentiation, co-express GATA-1 and PU.1 and as my and others data document, are able to respond to molecular removal (down-regulation) of PU.1 or addition (up-regulation) of GATA-1 by inducing terminal erythroid differentiation. We provide novel evidence that downregulation of GATA-1 or upregulation of PU.1 induces incompletely differentiation into cell cycle arrested monocytic-like cells. Furthermore, PU.1- dependent transcriptome is negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode additional key hematopoietic transcription factors. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Furthermore, transcriptional regulation of these loci by...

Eukaryotes organize their genomes by wrapping DNA around positively charged proteins called histones to form a structure known as chromatin. This structure is ideal for keeping the genome safe from damage, but also becomes an obstacle for the transcriptional machinery to access information stored in the DNA. To facilitate a balance between storage and accessibility, eukaryotes utilize a family of enzymes known as ATP-dependent chromatin remodelers to directly manipulate chromatin structure. The diverse activities of these chromatin remodeling enzymes range from simply sliding nucleosomes to reveal transcription start sites, to editing the composition of a nucleosome by exchanging canonical histones for histone variants. Chromatin remodeling enzymes recognize features of the nucleosome that activate their ATPase domains and enable proper remodeling function. One nuclear epitope that has been extensively studied is the nucleosomal acidic patch. This negatively charged region on the face of the nucleosome has been shown to be essential for remodeling enzymes like Chd1, ISWI, and INO80C. The chromatin remodeler SWR1C edits nucleosomes by removing the canonical histone H2A from nucleosomes and exchanges it for the histone variant H2A.Z, but the role of the acidic patch in this process has not been investigated. In this work, I showed that SWR1C has normal binding affinity to acidic patch mutant nucleosomes and retains ATPase stimulation but can no longer exchange dimers on this substrate. This work also identified a novel arginine anchor on the essential SWR1C subunit, Swc5, that binds specifically to the nucleosomal acidic patch. The data in this work suggest a mechanism where SWR1C engages nucleosomes and uses the Swc5 subunit to recognize the nucleosomal acidic patch to couple ATPase activity to histone dimer exchange.