Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

abstract: Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than deoxyribose nucleic acid (DNA) sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. In 2011, Haynes et al. showed that a synthetic regulator called the Polycomb chromatin Transcription Factor (PcTF), a fusion protein that binds methylated histones, reactivated an artificially-silenced luciferase reporter gene. These synthetic transcription activators are derived from the polycomb repressive complex (PRC) and associate with the epigenetic silencing mark H3K27me3 to reactivate the expression of silenced genes. It is demonstrated here that the duration of epigenetic silencing does not perturb reactivation via PcTF fusion proteins. After 96 hours PcTF shows the strongest reactivation activity. A variant called Pc2TF, which has roughly double the affinity for H3K27me3 in vitro, reactivated the silenced luciferase gene by at least 2-fold in living cells. / Dissertation/Thesis / Masters Thesis Biological Design 2019

Eukaryotic DNA is packaged into highly ordered structures knows as chromatin that further packs the DNA into higher-order structures, limiting the accessibility of the underlying genetic information for the processes like transcription, replication, and repair. However, eukaryotic cells have evolved proteins called chromatin regulators that regulate the accessibility of the genetic information when needed. This dissertation aims to characterize the role of two such proteins, JARID2 and the polycomb repressive complex-2 (PRC2), during skeletal muscle proliferation and differentiation.JARID2 is an inactive yet evolutionarily conserved histone demethylase that is shown to be a sub-stoichiometric component of the PRC2 complex. The PRC2 complex represses gene expression through the trimethylation of lysine 27 of histone 3 (H3K27me) tails. H3K27 methylation leads to chromatin compaction. JARID2 helps in targeting of the PRC2 complex to its target loci. JARID2 is shown to be required for the normal development of mice, as loss of Jarid2 leads to lethality in utero. We, for the first time, show that JARID2 is required for the normal skeletal muscle differentiation. We show that the JARID2 regulates the expression of myogenic regulatory factor, Myod1, both through direct repression and activation through the modulation of canonical Wnt signaling pathway. JARID2, in association with the PRC2 complex, represses Wnt antagonist Sfrp1 to modulate the activity of the canonical Wnt signaling pathway. The translocation of Wnt effector protein, b-catenin, from the cytoplasm to the nucleus modulates the activity of the canonical Wnt signaling pathway during activation. We also show that b-catenin directly regulates the expression of Myod1 gene through its direct binding in the distal regulatory region.We further extend the role of JARID2 during skeletal muscle proliferation. We show that JARID2 also plays an essential role in restraining the skeletal muscle proliferation through its direct repression of positive cell cycle regulators cyclin D1 (Ccnd1) and cyclin E1 (Ccne1). Furthermore, we show that retinoblastoma protein 1 (Rb1), a negative regulator of cell proliferation that promotes cell cycle exit and differentiation, is also directly regulated by JARID2 in PRC2 dependent manner. Together, we show that JARID2 precisely controls cell proliferation and differentiation during skeletal muscle differentiation.Further, we show that the regulation of cell proliferation by JARID2 is PRC2 complex dependent. When the PRC2 complex was depleted or inhibited to a modest level, the cells have an increased cell proliferation ability compared to severe loss or inhibition of EZH2, the catalytic subunit of the PRC2 complex, that leads to the apoptosis of the cells. It is also marked by increased expression of known PRC2 targets genes. We show that the increased proliferation upon modest inhibition or depletion of EZH2 is through direct de-repression of positive cell cycle genes, Ccnd1, and Ccne1. It is the first work that shows a context-dependent role of the PRC2 complex during skeletal muscle proliferation and differentiation.My dissertation also makes an extraordinary discovery as to why myogenin is required for the proper function of MyoD during skeletal muscle differentiation, even though both proteins share a large set of overlapping target genes. We show that myogenin is required for the nucleosome disassembly and reassembly at the target genes through recruitment of the FACT complex, a histone chaperone. We also show that myogenin is required for the assembly of the basic transcription machinery and RNA polymerase II to the target muscle genes during differentiation. Surprisingly, we also show that myogenin reinforces its own expression through the activation of Myod1 expression during skeletal muscle differentiation. Myogenin is a known target of MyoD.Taken together, this dissertation provides a molecular mechanism for the crosstalk between a signaling pathway with chromatin regulatory proteins, JARID2, and the PRC2 complex in regulating skeletal muscle differentiation. It also extends the role of JARID2 and the PRC2 complex - known oncogenes, in precise, context-dependent control of cell proliferation and differentiation in skeletal muscle.

No description available.

no / Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.

During tissues development multipotent progenitor cells establish tissue-specific gene expression programmes, leading to differentiation into specialized cell types. It has been previously shown that the transcription factor p63, a master regulator of skin development, controls the expression of adhesion molecules and essential cytoskeleton components. It has also been shown that p63 plays an important role in establishing distinct three-dimensional conformations in the Epidermal Differentiation Complex (EDC) locus (Fessing et al., 2011). Here we show that in p63-null mice about 32% of keratinocytes showed altered nuclear morphology. Alterations in the nuclear shape were accompanied by decreased expression of nuclear lamins (Lamin A/C and Lamin B1), proteins of the LINC complex (Sun-1, nesprin-2/3) and Plectin. Plectin links components of the nuclear envelope (nesprin-3) with cytoskeleton and ChIP-qPCR assay with adult epidermal keratinocytes showed p63 binding to the consensus binding sequences on Plectin 1c, Sun-1 and Nesprin-3 promoters. As a possible consequence of the altered expression of nuclear lamins and nuclear envelope-associated proteins, changes in heterochromatin distribution as well as decrease of the expression of several polycomb proteins (Ezh2, Ring1B, Cbx4) has been observed in p63-null keratinocytes. Moreover, recent data in our lab have showed that p63 directly regulates Cbx4, a component of the polycomb PRC1 complex. Here we show that mice lacking Cbx4 displayed a skin phenotype, which partially resembles the one observed in p63-null mice with reduced epidermal thickness and keratinocyte proliferation. All together these data demonstrate that p63-regulated gene expression program in epidermal keratinocytes includes not only genes encoding adhesion molecules, cytoskeleton proteins (cytokeratins) and chromatin remodelling factors (Satb1, Brg1), but also polycomb proteins and components of the nuclear envelope, suggesting the existence of a functional link between cytoskeleton, nuclear architecture and three dimensional nuclear organization. Other proteins important for proper epidermal development and stratification, are cytokeratins. Here, we show that keratin genes play an essential role in spatial organization of other lineage-specific genes in keratinocytes during epidermal development. In fact, ablation of keratin type II locus from chromosome 15 in epidermal keratinocytes led to changes in the genomic organization with increased distance between the Loricrin gene located on chromosome 3 as well as between Satb1 gene located on chromosome 17 and keratin type II locus, resulting in a more peripheral localization of these genes in the nucleus. As a possible consequence of their peripheral localization, reduced expression of Loricrin and Satb1 has also been observed in keratins type II-deficient mice. These findings together with recent circularized chromosome conformation capture (4C) data, strongly suggest that keratin 5, Loricrin and Satb1 are part of the same interactome, which is required for the proper expression of these genes and proper epidermal development and epidermal barrier formation. Taken together these data suggest that higher order chromatin remodelling and spatial organization of genes in the nucleus are important for the establishment of lineage-specific differentiation programs in epidermal progenitor cells. These data provide an important background for further analyses of nuclear architecture in the alterations of epidermal differentiation, seen in pathological conditions, such as psoriasis and epithelial skin cancers.

The majority of our physiological and metabolic processes are coordinated by an internal clock, which has evolved as an adaptive response to the daily light-dark cycles. Thus, several physiological and behavioral activities display an oscillatory rhythmic period of 24 hours. This highly conserved molecular mechanism is achieved through a specific program of gene expression, characterized by a complex interaction between clock-core proteins, chromatin remodelers and epigenetic events associated with the oscillatory nature of circadian transcriptional activity in the genome. Clock disruption leads to a wide spectrum of severe health problems including chronic metabolic disorders, muscle waste and cardiopathies. Previous studies revealed that each cell and organ possess an intrinsic clock and that coordination between central versus peripheral clocks is key for health. Furthermore, it has been found that under nutritional challenge such as High Fat Diet (HFD), the circadian transcriptome and metabolome are rapidly remodeled in the mouse model. Surprisingly, metabolome and gene expression analysis on various tissues revealed that skeletal muscle is the most affected under HFD. Mechanisms that regulate circadian cycle and stress induced rapid adaptation and in particular metabolic stress at the chromatin level are largely unknown. In this study, we investigated the role of Polycomb proteins group (PcG) mediate cell memory system by maintaining transcriptional gene silencing, in particular the PRC2-Ezh1. We hypothesized that Ezh1 could play an important role in circadian clock regulation in post-mitotic skeletal muscle, and this pathway has never been explored in this context. We explored the circadian role of PRC2-Ezh1 in the mouse skeletal muscle. Intriguingly, we found that the oscillatory profile of a novel isoform of Ezh1 (Ezh1beta), localized specifically in the cytoplasm and controlling stress induced nuclear PRC2 activity, was completely disrupted under HFD. More interestingly, the circadian pattern of core clock components was impaired in Ezh1 depleted cells. Our data unveils an interesting physiological role of the PcG memory system, from cytoplasm to chromatin, which could indicate a new link between the chromatin remodeler Polycomb proteins and the endogenous clock in adaptation mechanism in skeletal muscle.

No description available.

No description available.

Des études présentées dans cette thèse ont permis de démontrer que le gène du groupe Polycomb (PcG) Bmi1 est essentiel à l’auto-renouvellement des progéniteurs rétiniens immatures et pour le développement rétinien après la naissance. Ce travail illustre chez l’embryon que Bmi1 est hautement enrichie dans une sous-population de progéniteurs rétiniens exprimant le marqueur de surface SSEA-1 et différents marqueurs de cellules souches. À tous les stades de développement analysés, l’absence de Bmi1 résulte en une diminution de la prolifération et de l’auto-renouvellement des progéniteurs immatures. Pour mieux comprendre la cascade moléculaire en absence de Bmi1, nous avons inactivé p53 dans les colonies Bmi1-/-. Cette inactivation a permis une restauration partielle du potentiel d’auto-renouvellement. De plus, en absence de Bmi1, la prolifération et la maintenance de la population de progéniteurs rétiniens immatures localisés dans le corps ciliaire sont aussi affectées après la naissance. Bmi1 permet donc de distinguer les progéniteurs immatures de la population principale de progéniteurs, et est requis pour le développement normal de la rétine. Nous avons également démontré que l’oncogène Bmi1 est requis dans les neurones pour empêcher l’apoptose et l’induction d’un programme de vieillissement prématuré, causé par une baisse des défenses anti-oxydantes. Nous avons observé dans les neurones Bmi1-/- une augmentation des niveaux de p53, de la concentration des ROS et de la sensibilité aux agents neurotoxiques. Nous avons démontré ainsi que Bmi1 contrôle les défenses anti-oxydantes dans les neurones en réprimant l’activité pro-oxydante de p53. Dans les neurones Bmi1-/-, p53 provoque la répression des gènes anti-oxydants, induisant une augmentation des niveaux de ROS. Ces résultats démontrent pour la première fois que Bmi1 joue un rôle critique dans la survie et le processus de vieillissement neuronal. / The studies presented in this thesis establish that the Polycomb Group (PcG) gene Bmi1 is required for the self-renewal of immature retinal progenitor cells (RPCs) and for postnatal retinal development. Work performed in mouse embryos reveals that Bmi1 is highly enriched in a RPC subpopulation expressing the cell surface antigen SSEA-1 and different stem cell markers. Furthermore, at all developmental stages analysed, Bmi1 deficiency resulted in reduced proliferation and self-renewal of immature RPCs. To better understand the molecular cascade leading to this phenotype, we inactivated p53 in Bmi1-deficient colonies. p53 inactivation partially restored RPCs self-renewal potential. Moreover, the proliferation and the postnatal maintenance of an immature RPC population located in the ciliary body was also impaired in absence of Bmi1. Thus, Bmi1 distinguishes immature RPCs from the main RPC population and is required for normal retinal development. We have also shown that the oncogene Bmi1 is required in neurons to prevent apoptosis and the induction of a premature aging-like program characterized by reduced antioxidant defenses. We observed in Bmi1-deficient neurons an increased p53 and ROS levels, and a hypersensitivity to neurotoxic agents. We demonstrated that Bmi1 regulate antioxidant defenses in neurons by suppressing p53 pro-oxidant activity. In Bmi1-/- neurons, p53 induces antioxidant genes repression, resulting in increased ROS levels. These findings reveal for the first time the major role of Bmi1 on neuronal survival and aging.