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.

Transcription of the ribosomal genes accounts for the majority of transcription in the cell due to the constant high demand for ribosomes. The number of proteins synthesized correlates with an effective ribosomal biogenesis, which is regulated by cell growth and proliferation. In the work presented in this thesis, we have investigated the ribosomal RNA genes 45S and 5S rRNA, which are transcribed by RNA Pol I and RNA Pol III, respectively. The focus of this work is the chromatin remodelling complex B-WICH, which is composed of WSTF, the ATPase SNF2h and NM1. We have studied in particular its role in ribosomal gene transcription. We showed in Study I that B-WICH is required to set the stage at rRNA gene promoters by remodelling the chromatin into an open, transcriptionally active configuration. This results in the binding of histone acetyl transferases to the genes and subsequent histone acetylation, which is needed for ribosomal gene activation. Study II investigated the role of B-WICH in transcription mediated by RNA polymerase III. We showed that B-WICH is essential to create an accessible chromatin atmosphere at 5S rRNA genes, which is compatible with the results obtained in Study 1. In this case, however, B-WICH operates as a licensing factor for c-Myc and the Myc/Max/Mxd network. Study III confirmed the importance and the function of the B-WICH complex as an activator of ribosomal genes. We demonstrated that B-WICH is important for the remodelling of the rDNA chromatin into an active, competent state in response to extracellular stimuli, and that the association of the B-WICH complex to the rRNA gene promoter is regulated by proliferative and metabolic changes in cells. The work presented in this thesis has confirmed that the B-WICH complex is an important regulator and activator of Pol I and Pol III transcription. We conclude that B-WICH is essential for remodelling the rDNA chromatin into a transcriptionally active state, as required for efficient ribosomal gene transcription. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p><p> </p>

Le spermatozoïde des animaux à reproduction sexuée est une cellule extrêmement spécialisée, dont la chromatine très particulière est le siège de nombreux remodelages tant lors de la gamétogenèse que lors de la formation du zygote. Chez D. melanogaster comme chez les mammifères, lors de la spermiogenèse, les histones qui condensent l'ADN sont remplacées par des petites protéines basiques spécifiques du noyau spermatique : les protamines. Cette architecture est stabilisée par des liaisons disulfures. Lors de la fécondation, ces protéines sont éliminées du noyau paternel, qui réincorporent des histones pour former une chromatine fonctionnelle. Toutefois, les mécanismes régissant la mise en place et l'enlèvement des ponts disulfures et des protamines sont inconnus chez la Drosophile.Au cours de ma thèse, j'ai démontré l'importance de deux thiorédoxines sexuelles pour la reproduction.D'une part, j'ai pu montrer que DHD, qui est une thiorédoxine strictement maternelle, est essentielle à l'éviction des protamines de la chromatine paternelle lors de la fécondation. Sans cette protéine essentielle, la décondensation du noyau mâle n'a pas lieu, les protamines ne sont pas enlevées et le développement zygotique ne peut pas avoir lieu. Cette thiorédoxine est directement responsable de la réduction des liaisons disulfures qui stabilisent la chromatine spermatique.D'autre part, j'ai démontré que TrxT, une thiorédoxine exclusivement testiculaire, est nécessaire au bon déroulement de la spermiogenèse. Sans cette protéine, les spermatides subissent des dommages à l'ADN et sont éliminées.Ce travail met en évidence les rôles essentiels des thiorédoxines sexuelles pour la reproduction / In animal sexual reproduction, spermatozoon is a very specialized cell. Its very peculiar chromatin is remodeled both during spermiogenesis and fertilization. During mammalian and drosophilian spermiogenesis, histones involved in DNA condensation are replaced with sperm specific small nuclear basic proteins : the protamines. This sperm specific architecture is stabilized by disulfide bonds. At fertilization,protamines are removed from the male nucleus and maternally-provided histones are incorporated to form a functional paternal chromatin. However, the mecanisms involved in the incorporation and the removal of protamines of their disulfide bonds are unknown in Drosophila.During my PhD, I demonstrated that two sexual thioredoxins are important for spermiogenesis and fertilization in D. melanogaster. In one hand, I showed that DHD, a female specific thioredoxin, is essential for protamine eviction at fertilization. Without this major protein, male nucleus does not decondense, protamines are not removed from sperm chromatin and zygotic development does not occur. Besides, I demonstrated that DHD is directly responsible for the reduction of the disufide bonds which stabilize sperm chromatin.On the other hand, I showed that TrxT, a testis-specific thioredoxin, is needed for spermiogenesis. Without this protein, DNA damages appear on spermatid nuclei, and those spermatozoon are then eliminated during spermatogenesis.This work highlights that drosophilian sex-specific thioredoxins are essential for sexual reproduction success

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.

Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized. In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network. Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Manuscript; Paper 3: Manuscript</p>

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.

DNA organization is an intricate and dynamic process. The approximately two meters of DNA in a single cell is wrapped around small proteins called histones. Histones can be compacted into dense coils or loosely distributed along DNA, allowing for cells to control gene expression. This combination of DNA and histones forms chromatin. This work has focused on understanding the role of Polybromo1 (PBRM1), which is a member of a chromatin remodeling complex. PBRM1 is mutated in 3% of all human cancers and is mutated in 40% of renal clear cell carcinomas (ccRCC), the most common type of kidney cancer. Through my work characterizing PBRM1 as a tumor suppressor, we have found PBRM1 acts as a stress sensor. PBRM1 is a member of the Polybromo1 BRG1 associating factors (PBAF) complex which is a subtype of the larger BAF family of chromatin remodelers. Although BAF is essential for cell viability, knockdown of PBRM1 shows minor phenotypic changes in many cell types under standard cell culturing conditions. However, when cells without PBRM1 experience external stress, the reactive oxygen species levels in the cells are elevated and remain high compared to cells with wild type PBRM1. Depending on the cellular environment of the cell, increase in ROS can be growth promoting or growth inhibiting. PBRM1 is a structurally unique protein, containing two bromo-adjacent homologs, a high mobility group and six tandem bromodomains. Due to the multiple reader domains, it is likely PBRM1 acts to target the complex. Taking advantage of a RCCC cell line not expressing PBRM1, we re-expressed full length PBRM1 containing an asparagine to alanine mutation in each bromodomain, disrupting the acetyl-lysine binding. We have found that the bromodomains are cooperative and are facilitating binding of PBAF to chromatin. We found defects in PBRM1’s ability to suppress growth, bind to chromatin, and regulate gene expression when any of the bromodomains were mutated besides the third bromodomain. These results correlated with patient data. Using acetylated histone peptides, we have identified potential combinations of marks that PBRM1 prefers over single marks. Further work needs to be done to characterize how these histone modifications are altered under stress and they contribute to the role of PBRM1 in stress response.

No description available.

Le complexe de remodelage de la chromatine NuRD, composé des sous-unités catalytiques CHD3 et CHD4, est un régulateur épigénétique de l’expression génique. Nos résultats montrent que NuRD s’associe avec les facteurs de transcription essentiels du mélanome que sont MITF et SOX10. Cependant, malgré une association physique et une co-localisation génomique, CHD4/NuRD ne semble pas agir comme un cofacteur important pour MITF ou SOX10. Néanmoins, la répression de CHD4 conduit à un ralentissement de la prolifération et déréprime l’expression des enzymes PADI1 et PADI3 dans les cellules de mélanome ainsi que dans de nombreux types de cellules cancéreuses. Ainsi, l’induction de ces enzymes, responsables de la conversion des arginines en citrullines, entraîne la citrullination spécifique de PKM2, une enzyme glycolytique essentielle, diminuant ainsi sa sensibilité aux inhibiteurs allostériques, et donc altérant l’équilibre physiologique entre activateurs et inhibiteurs de l’enzyme. L’ensemble de ce travail de thèse a permis de mettre en évidence une nouvelle voie reliant, d’une part la régulation épigénétique de l’expression de PADI1 et PADI3 par CHD4/NuRD ainsi que la reprogrammation de la régulation allostérique de PKM2 via la citrullination d’arginines, au flux glycolytique et au contrôle de la prolifération des cellules cancéreuses d’autre part. / The Nucleosome Remodelling and Deacetylation (NuRD) complex is an epigenetic regulator of gene expression that includes two mutually exclusive ATPase subunits CHD3 and CHD4. Our results show that NuRD associates with essential melanoma cell transcription factors namely MITF and SOX10. However, despite their physical association and genomic co-localization, CHD4-NuRD does not appear to act as a cofactor for MITF or SOX10 regulated gene expression. Nevertheless, CHD4 silencing leads to a slow growth phenotype and de-represses the expression of PADI1 (Protein Arginine DeIminase 1) and PADI3, two enzymes involved in converting arginines to citrullines in melanoma and multiple types of cancer cells. Increased expression of PADI1 and PADI3 enhances citrullination of arginines within the key glycolytic regulatory enzyme PKM2 then promoting excessive glycolysis, lowering ATP levels and slowing down proliferation. PKM2 citrullination lowers its sensitivity to allosteric inhibitors thus shifting equilibrium towards allosteric activators thereby bypassing the normal physiological regulation of glycolysis. Overall, our results lead to describe a novel pathway linking, epigenetic regulation of PADI1 and PADI3 expression by CHD4/NuRD and reprogramming of PKM2 allosteric regulation through arginines citrullination, to glycolytic flux and cancer cell proliferation.

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.