The nucleosome remodeling factor (NURF) is a mutli-protein complex that plays a role in the regulation of gene expression through its ability to remodel nucleosomes. The largest subunit of this complex, Bptf (Bromodomain PHD Finger Transcription Factor) is important for many cellular processes as a transcriptional regulator and improper function results in disease or malignancy. To further understand the genome-wide recruitment of the NURF complex, the interaction partner for the N-terminal PHD finger domain of Bptf was investigated through pull down assays followed by mass spectrometry. It was determined that this domain does not recognize histones; instead it recognizes a nonhistone protein, Thoc4 or Hmgb1. The expression of a cDNA corresponding to Bptf was also tested for expression in mouse ES cells after the addition of two exons found to be missing in the original cDNA. Addition of this sequence did not allow for exogenous Bptf expression in ES cells.

Tumor immunoediting is a dynamic process in which the immune response attacks tumor cells by detecting danger signals and tumor antigens. In order to survive, tumor cells develop mechanisms to avoid detection or destruction by the immune system. To counteract this, several strategies are being developed to enhance the antitumor immune response, including the depletion of immunosuppressive cells, enhancing the activation of antitumor immune cells and increasing tumor cell immunogenicity. These therapies have seen limited success individually, however, and it is likely that combination therapy with novel targets will be necessary to see reproducible beneficial responses. Epigenetic modifications are attractive therapeutic targets because they are reversible and affect gene expression in cancer cells. Within this framework, this study aimed to elucidate the role of the chromatin remodeling complex nucleosome remodeling factor (NURF) in cancer immunoediting by silencing of bromodomain PHD-finger containing transcription factor (BPTF), the largest and essential subunit of NURF. Using two syngeneic mouse models of cancer, BPTF was found to suppress T cell antitumor activity in the tumor microenvironment. In vitro, enhanced cytolytic activity was observed for individual CD8 T cell clones only from mice bearing BPTF-silenced tumors, implicating the involvement of novel antigens. Mechanistic investigations revealed that NURF directly suppresses the expression of genes encoding immunoproteasome subunits Psmb8 and Psmb9 and the antigen transporter genes Tap1 and Tap2. PSMB8 inhibition reversed the effects of BPTF ablation, consistent with a critical role for the immunoproteasome in improving tumor immunogenicity. Thus, NURF normally suppresses tumor cell antigenicity and its depletion improves CD8 T cell antitumor immunity. In a concurrent study using different tumor lines, BPTF was also found to suppress natural killer (NK) cell antitumor immunity in vivo. Enhanced NK cell cytolytic activity toward BPTF-depleted targets in vitro was dependent on the natural cytotoxicity receptors (NCR). Molecular studies revealed that BPTF directly activates heparanase (Hpse) expression, resulting in reduced cell surface abundance of the NCR co-ligands: heparan sulfate proteoglycans. Thus, NURF represses NCR co-ligand abundance and its depletion enhances NK cell cytotoxicity. Therefore, NURF emerges as a candidate therapeutic target to enhance CD8 T or NK cell antitumor immunity.

The nucleosome remodeling factor (NURF) is an evolutionary conserved ATP-dependent chromatin remodeling factor. It was first isolated from Drosophila as a complex with enzymatic activity that once recruited to nucleosome, it slides the nucleosome to provide accessibility for transcription factors. Since then, numerous works from animal models and cell lines show the role of NURF as a regulator of gene expression. NURF interacts with H3K4me3 and sequence specific transcription factors that recruit the complex to promoter regions. Whether this is the only mechanism by which NURF regulates gene expression is not known. However, other ATP-dependent chromatin remodeling complexes are known to regulate gene expression independent from transcription initiation. In order to explore the role of NURF in regulating gene expression, we utilized two genome wide approaches to map NURF binding and NURF dependent changes in chromatin structure using ChIP-Seq and FAIRE-Seq, respectively. From these analyses, we discovered that NURF broadly localizes in the genome with preferences to gene bodies and 3’ends of genes. Also, we found that NURF maintains open chromatin regions at upstream, intron and downstream of genes. These novel findings shed light on new roles for NURF complex within genes, in addition to its classical role at promoter regions. Furthermore, we discovered the function of a previously uncharacterized domain in the NURF specific subunit BPTF. We show that the N-terminal the plant homeodomain (PHD) of BPTF directly interacts with THOC4, a protein associated with RNA-pol 2. Also, we show using ChIP analyses that this interaction recruits BPTF to gene bodies. Next, we investigated functional consequences for NURF recruitment to gene bodies using Cyclin D1 (Ccnd1) gene as a model. These analyses revealed that NURF is required for normal mRNA processing and loss of NURF induces intron retention, which results in unstable transcripts. Finally, we show that the defect in mRNA processing is not specific to the Ccnd1 gene, as we observe similar defects in four other BPTF dependent genes. Together, our work uncovered new role of mammalian NURF complex in regulating gene expression through mRNA processing.

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.

Le mélanome est un cancer de la peau très agressif. Microphthalmia-associated transcription factor (MITF) est un facteur de transcription clé contrôlant le développement de la lignée mélanocytaire, ainsi que la prolifération et l’invasion des cellules de mélanome. Pour mieux comprendre les fonctions de MITF, nous avons identifié ses cofacteurs impliques dans la régulation de la transcription. Nous avons montré que le complexe de remodelage de la chromatine NURF interagit avec MITF. Ma thèse a consisté à élucider le rôle de NURF dans le mélanome et les mélanocytes. La perte de BPTF, la principale sous-unité de ce complexe, induit un arrêt de la prolifération et une entrée en senescence des cellules de mélanome. Nous avons montré que BPTF et MITF coopèrent pour réguler l’expression de gènes impliqués dans la prolifération and invasion suggérant que BPTF et un cofacteur de MITF. De façon inattendue, l’inactivation de BPTF spécifiquement dans les mélanocytes entraine la perte progressive et totale de la pigmentation du pelage en raison de l’incapacité des cellules souches mélanocytaire à produire une descendance fonctionnelle. C’est la première fois que l’interaction fonctionnelle entre NURF et MITF est démontrée in vitro, complétée par des observations phénotypique uniques in vivo, contribuant à la compréhension de la biologie des mélanocytes et du mélanome. / Melanoma is a highly aggressive form of skin cancer. Microphthalmia-associated transcription factor (MITF) isa key regulator of development of the melanocyte lineage and proliferation and invasion of melanoma cells.To further elucidate the functions of MITF, we identified factors co-regulating transcription with MITF. We identified the NURF chromatin-remodeling complex as MITF interactor. My thesis aims to elucidate the role of NURF in melanoma and melanocytes. Loss of BPTF, the principal subunit of the complex, led to arrest of proliferation and entry into senescence of melanoma lines. We showed BPTF and MITF co-regulate genes involved in proliferation and invasion suggesting that BPTF acts as cofactor for MITF. Remarkably, the mouse model of melanocyte-specific BPTF ablation led to progressive and complete loss of coat pigmentation due to the inability of the melanocyte stem cells to produce functional progeny. This is the first report of NURF-MITF functional interaction in vitro, complemented with a unique in vivo phenotype, both adding to a general understanding of melanocyte and melanoma biology.

Le mélanome est un cancer très agressif en raison de sa capacité rapide à former des métastases et de développer une résistance aux traitements existants.
 MITF (Micropthalmia-associated Transcription Factor) est un facteur de transcription clé à toutes les étapes de développement du lignage mélanocytaire et dans la physiopathologie du mélanome. Afin de comprendre les mécanismes impliqués dans la régulation de l’activité et de la stabilité de MITF, nous avons identifié ses partenaires protéiques parmi lesquels figurent de nombreuses sous-unités des complexes de remodelage de la chromatine ATP-dépendant PBAF et NURF. Ce travail caractérise le rôle et l’étendue de la coopération entre BRG1/PBAF et plusieurs facteurs de transcription clés tels que MITF et SOX10 dans le fonctionnement des cellules de mélanome, qui recrutent activement de BRG1 à la chromatine et contribuent ainsi à la mise en place de la signature épigénétique caractéristique des cellules de mélanome prolifératives. Par ailleurs, l’utilisation de différents modèles murins a permis de révéler in vivo la contribution fonctionnelle distincte mais complémentaire de ces deux complexes de remodelage associé à MITF aux cours de trois stades majeurs du lignage mélanocytaire : le développement embryonnaire des mélanocytes, leur différentiation ainsi que lors de l’initiation et la progression du mélanome. Ce travail contribue ainsi à une meilleure compréhension du fonctionnement biologique des mélanocytes, du mélanome et du remodelage de la chromatine chez les eucaryotes. / Malignant melanoma is the most deadly form of skin cancer due to its quick metastatic spread and the development of resistance to available treatments.
MITF (Micropthalmia-associated Transcription Factor) is a transcription factor and master regulator of melanocyte lineage development and melanoma physiopathology. In order to investigate the mechanisms involved in the regulation of MITF activity and stability, we identified its numerous partners by tandem affinity purification coupled to mass spectrometry, which include several subunits of the PBAF and NURF ATP-dependant chromatin remodelling complexes. The present work characterizes the role and extent of cooperation between BRG1/PBAF and several key transcription factors including MITF and SOX10 in melanoma cell function, that actively recruit BRG1 to chromatin to establish the epigenetic landscape of proliferative melanoma cells. Furthermore, using different mouse models we revealed the distinct but complementary functional contribution of these two MITF-associated chromatin remodelers in vivo at three majors stages of melanocyte lineage development: embryonic development of melanocytes, their differentiation and during melanomagenesis. Thus, this work contributes to a better understanding of processes regulating the biological function of melanocytes, melanoma and more widely chromatin remodelling events in eukaryotes.

Understanding the impact of epigenetic mechanisms on tumorigenesis is essential, as epigenetic alterations are associated with tumor initiation and progression. Because epigenetic changes are reversible, they are potential targets for cancer therapy. Nucleosome Remodeling Factor (NURF) is a chromatin-remodeling complex that regulates gene expression by changing nucleosome positioning along the DNA sequence. Previous studies have shown a role for NURF in embryonic development as well as regulating genes involved in tumor progression. In this work we investigated the impact of eliminating NURF function in tumorigenesis in vivo. BALB/c mice challenged with syngeneic 67NR breast cancer cell lines, injected into the mammary fat pad, lacking NURF, due to knockdown of its essential subunits Bptf, showed reduction in tumor growth comparing to control tumors. The observed reduction in tumor growth was abrogated in immunodeficient mice lacking a functional immune system. Bptf KD and control 67NR cells grew at similar rates in vitro. Similar findings were observed in our lab using 66cl4 breast cancer cell lines. Using immunofluorescence staining, no significant difference in CD8+, CD4+, NK and MDSC cells infiltrations into the tumor microenvironment was observed in 66cl4 tumors. Preliminary results from 67NR tumors suggested more CD4+ and CD8+ cells. Gene expression profile of tumor tissues from BALB/c mice injected with 67NR and 66cl4 cell lines showed enrichment of genes associated with immune response. Our findings suggested a role of the immune system in targeting tumor cells lacking Bptf in vivo.

Increasingly the role of epigenetic machinery as a bridge between underlying DNA sequence and cellular phenotype is being discovered. The establishment of a myriad of unique cellular types sharing identical gene sequences in a multicellular organism gives a broad sense for the inherent role of epigenetic influence on cell differentiation. Importantly, the epigenetic mechanisms involved in establishing cell identity unsurprisingly contribute to diseased states, including cancer. Recent research continues to elucidate contributory roles of epigenetic mechanisms, such as DNA methylation, histone modification, and microRNA regulation, in human cancers. Additionally, chromatin remodelers, such as the Nucleosome Remodeling Factor (NURF), have been identified as important regulators for normal cell biology. While much has been done to identify and characterize the role of NURF chromatin remodeling complex as a key regulator of development in a number of model organisms, little has been published on the implications of NURF in diseases such as cancer. Our preliminary data shows dysregulation of E-cadherins, N-cadherins, and MHC-I genes in Bptf (an essential subunit of NURF) knocked down murine breast cancer cell lines. These proteins have well documented roles in the development and metastatic progression of cancers. To study the effect of Bptf knockdown on the development and progression of cancer we injected Bptf knocked down mouse breast cancer cell lines, 4T1, 66cl4, and 67NR, into syngenic BALB/c mice. Our findings reveal decreased tumor growth in 66cl4 and 67NR as measured by tumor weight at 3-4 weeks post injection. Tumor growth did not appear to be significantly affected in 4T1 challenged mice. However, mice inoculated with Bptf knockdown 4T1 cell lines have decreased metastasis to lungs as compared to control while metastasis of 66cl4 tumors to the lungs appear unaffected. To assess the role of the immune system in decreasing tumor growth in BALB/c mice, we injected 66cl4 tumors into NOD-SCID-Gamma (NSG) immune deficient mice. The tumors from these mice show no difference in tumor growth between Bptf knockdown and control tumors, implicating a role for the immune system regulating the decreased tumor weight in BALB/c mice. To delineate which immune cell effector may impede breast cancer carcinogenesis, we performed an in vitro natural killer (NK) cell cytotoxicity assay against 66cl4 tumors and found greater susceptibility to NK killing in Bptf knockdown tumors.