Growth arrest specific (GAS) genes are highly inducible at quiescence (G0) and repressed rapidly in response to mitogens. Aberrant disruption of quiescence can lead to abnormal development and diseases such as cancer, thus, it is important to study the signals and mechanisms responsible for expressions of quiescent specific genes. p20K, a GAS gene whose expression is highly induced in conditions of contact inhibition & hypoxia in chicken embryo fibroblasts (CEF), is studied in this thesis. Preliminary studies demonstrate that p20K activation is dependent on its Quiescence Responsive Unit (QRU), a 48bp promoter region. In addition, the binding sites of a CCAAT/enhancer binding protein (C/EBPβ) and ERK2 on the QRU of p20K promoter overlap with each other regulating the competition between activating (C/EBPβ) and inhibiting (ERK2) of the p20K gene. After culturing CEF with media rich in growth factors (10%FBS), p20K induction is delayed in hypoxia. Moreover, it is the decrease of Phospho-ERK not CHOP level that correlates with p20K inhibition in hypoxia in both 5%CCS and 10%FBS. Western blotting analysis of Hypoxia Inducible Factor 1α (HIF1α) expression indicated that this hypoxia-response factor is induced rapidly and with the same kinetics in CEF subjected to hypoxia cultured in 5%CCS or 10%FBS, indicating that they sense and respond similarly to low oxygen concentrations. These results suggest that p20K induction in hypoxia is caused by growth arrest induced by hypoxia. To further document this process, hypoxia mimicking reagent DMOG, a prolyl-hydroxylase inhibitor that can stabilize HIF in normoxia, was used. Interestingly, p20K expression was highly induced after DMOG treatment in CEF, even if CHOP, an inhibitor of C/EBPβ, was induced in these conditions. Co-Immunoprecipitation results showed that the accumulation of CHOP-C/EBPβ heterodimers was induced during DMOG treatment. Additionally, Proliferation Assay suggested that DMOG treatment significantly inhibited CEF proliferation. Finally, Chromatin Immunoprecipitation Analysis indicated that ERK-2 did not bind to the QRU after DMOG treatment, indicating that ERK-2 dissociation correlates with p20K induction in response to DMOG in CEF. Collectively, these results demonstrate that growth arrest induced by hypoxia or DMOG treatment plays a determinant role in p20K induction. In contrast, CHOP level or CHOP-C/EBPβ heterodimer reduction did not correlate with the induction of p20K. / Thesis / Master of Science (MSc)

Vitamin A and its derivatives, collectively termed retinoids, are essential for proper growth and development as well as maintenance of homeostasis in the adult. Retinoic acid (RA), the major biologically active vitamin A metabolite, is well characterized for its crucial roles in gene activation during embryogenesis. Our lab had previously performed a microarray analysis to identify genes induced by exogenous RA in the tailbud of early mouse embryos. Three genes were chosen from the microarray results for further investigation; Germ Cell Nuclear Factor (GCNF/NR6A1), Prickle1 (Pk1) and Ror2, the latter of which are known members of the planar cell polarity (PCP) pathway. These genes were further examined for RA regulation by embryo culture and RT-PCR, which strongly supported a direct regulatory mechanism of NR6A1 by RA. Further analysis aiming to identify a functional response element in the promoter of the targets was attempted, including chromatin immunoprecipitation (ChIP), made possible by the generation and characterization of a highly specific antibody against RARγ. This antibody was used in a ChIP promoter walk, which identified regions on target gene promoters that are occupied by RARγ in vivo, and therefore likely harbor RA response elements.

Vitamin A and its derivatives, collectively termed retinoids, are essential for proper growth and development as well as maintenance of homeostasis in the adult. Retinoic acid (RA), the major biologically active vitamin A metabolite, is well characterized for its crucial roles in gene activation during embryogenesis. Our lab had previously performed a microarray analysis to identify genes induced by exogenous RA in the tailbud of early mouse embryos. Three genes were chosen from the microarray results for further investigation; Germ Cell Nuclear Factor (GCNF/NR6A1), Prickle1 (Pk1) and Ror2, the latter of which are known members of the planar cell polarity (PCP) pathway. These genes were further examined for RA regulation by embryo culture and RT-PCR, which strongly supported a direct regulatory mechanism of NR6A1 by RA. Further analysis aiming to identify a functional response element in the promoter of the targets was attempted, including chromatin immunoprecipitation (ChIP), made possible by the generation and characterization of a highly specific antibody against RARγ. This antibody was used in a ChIP promoter walk, which identified regions on target gene promoters that are occupied by RARγ in vivo, and therefore likely harbor RA response elements.

Somatic cell nuclear reprogramming (SCNR) by eggs is a way to forcibly transform the nuclei of terminally differentiated somatic cells to an embryonic state and gain totipotency (Gurdon et al., 1958). Additionally, induced pluripotency is applied to transform identities of somatic cells to induced pluripotent stem cells by overexpression of combinatorial Yamanaka factors (iPS, Takahashi et al., 2006). Although both approaches aim to derive cells with highest plasticity, the mechanisms and differences between these procedures are not yet clear. In my thesis, I used quantitative polymerase chain reaction (QPCR) and RNAseq plus 5-bromouridine 5'-triphosphate (BrUTP) pulldown to evaluate the transcriptional reprogramming by maternal factors and overexpressed transcription factors during SCNR by Xenopus oocytes, which are inactive in DNA replication and cell division. QPCR measures changes in the steady-state levels of transcripts within 2 days of nuclear transfer to Xenopus oocytes (Oocyte-NT). Three pairs of Yamanaka factor homologs were tested by QPCR and Yamanaka factor homologues regulated similar sets of pluripotency genes in mouse embryonic fibroblasts (MEFs). Pioneer factor mFoxA1 could not up-regulate most pluripotency genes and their binding targets of neurogenic genes in MEFs while pioneer factors are proposed to bind to their targets even if they may reside in inaccessible chromatin. This shows that the existence of other factors is needed at specified developmental stages. Hence, gene activation by transcription factors in the Oocyte-NT system requires not only corresponding binding on regulatory elements of linked genes but transcription cooperators to exert effective gene activation. Additionally, RNA-seq plus BrUTP pulldown measures the extent to which oocytes change the transcriptional activity of nuclei transplanted to oocytes. Through RNA-seq plus BrUTP pulldown, I compared the reprogrammed transcriptomes of embryonic and somatic cells, including mouse embryonic stem cells, mouse embryonic fibroblasts and mouse myoblasts, to demonstrate the effects of maternal factors and overexpression of transcription factors on gene activities during SCNR by oocytes. Importantly, I find that maternal factors of oocytes and the overexpression of transcription factors exert different strategies to reprogram somatic cells. Oocyte factors reprogram the donor cell nuclei to an oocyte-steady state except for the SCNR resistance genes and xklf2-HA overexpression enhances expression of reprogrammable genes and activates SCNR resistance genes.

Lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) are known to cooperate with the glucocorticoid receptor (GR) to regulate transcription. The current model of GR-mediated transcription classifies KATs as coactivators as they acetylate histones to form an open chromatin conformation and casts KDACs as corepressors that deacetylate histones and condense chromatin. Our recent studies have challenged this long-standing model. In the current study, we show that KDACs act as versatile coregulators, facilitating both the onset and maintenance of GC-induced transcriptional activation and repression. Through siRNA depletion studies, we define KDAC1 as the predominant Class I KDAC for efficient transactivation of a majority of the GR-target genes tested. KDACs 1 and 2 co-operate with each other to activate and repress a few target genes, however KDAC2 alone is not sufficient for activation or repression of the genes, thus questioning the functional redundancy of KDACs 1 and 2. Additionally, we found that there is a unique population of KDAC2 that does not associate with KDAC1 in our cell line. Through a series of siRNA depletion studies, steroid receptor coactivator proteins (SRCs) were shown to be dispensable for GC-induced gene activation and SRC2 was not required for Dex-induced transcriptional repression. We performed ChIP assays to address the mechanism by which Class I KDACs facilitate transactivation and transrepression. At GC-activated genes we found that KDACs are constitutively present at the gene enhancers and that KDAC inhibition does not affect the binding of GR or SRC proteins to chromatin. However, KDACs do influence the histone methylation status of H3K4 at GREs of activated genes and TSSs of repressed genes. To explain the change in the methylation status of this marker, we depleted LSD1, the specific demethylase for mono- and demethylation of H3K4, and found that LSD1 action is required for GC-mediated transrepression. However it is unlikely that KDAC inhibition impairs GR transactivation through effects on LSD1. Glucocorticoid signaling regulates multiple vital biological processes. Glucocorticoids play a major role in regulating carbohydrate, protein and lipid metabolism. They increase hepatic gluconeogenesis to maintain blood glucose concentration in the fasting state. GCs also act as potent anti-inflammatory molecules, stimulate lung maturation in the developing fetus, and affect bone metabolism. Additionally, excess or deficiency of GCs can lead to a variety of psychological abnormalities, indicating their role in CNS functions. Our results indicate that pharmaceutical modulation of KDACs may impair proper glucocorticoid signaling and disrupt vital biological processes. Other steroid hormone receptors function similarly to GR in regulating gene expression and could also be impacted by KDAC inhibition, thus suggesting serious physiological implications in patients. Therefore, the possibility of endocrine modulation should be taken into account when using KDAC inhibitors in the clinic.

No / Skin development, postnatal growth and regeneration are governed by complex and well-balanced programs of gene activation and silencing. The crosstalk between small non-coding microRNAs (miRNAs) and mRNAs is highly important for steadiness of signal transduction and transcriptional activities as well as for maintenance of homeostasis in many organs, including the skin. Recent data demonstrated that the expression of many genes, including cell type-specific master transcription regulators implicated in the control of skin development and homeostasis, is regulated by miRNAs. In addition, individual miRNAs could mediate the effects of these signaling pathways through being their downstream components. In turn, the expression of a major constituent of the miRNA processing machinery, Dicer, can be controlled by cell type-specific transcription factors, which form negative feedback loop mechanisms essential for the proper execution of cell differentiation- associated gene expression programs and cell-cell communications during normal skin development and regeneration. This review summarizes the available data on how miRNA/mRNA regulatory networks are involved in the control of skin development, epidermal homeostasis, hair cycle-associated tissue remodeling and pigmentation. Understanding of the fundamental mechanisms that govern skin development and regeneration will contribute to the development of new therapeutic approaches for many pathological skin conditions by using miRNA-based interventions.

Dans la première partie de cette thèse, les ultrasons focalisés (HIFU) guidés par Imagerie par Résonance Magnétique (IRM) sont utilisés pour l’activation locale transgénique in vivo. Une souris transgénique avec un gène luciferase sous contrôle d’un promoteur thermosensible est utilisée comme modèle biologique. L’hyperthermie locale est induite par une sonde HIFU et contrôlée via un IRM. Dans la seconde partie de ce travail de thèse, les ultrasons sont utilisés pour améliorer le dépôt local de médicaments dans les cellules et les tissus. Le suivi du médicament est effectué à l’aide de différentes techniques d’imageries telles que l’IRM ou l’imagerie par fluorescence. / In the first part of the thesis magnetic resonance imaging (MRI) guided high intensity focused ultrasound (HIFU) is used for the local activation of a transgene in vivo. A transgenic mouse with a luciferase gene under control of a heat sensitive promoter is used as biological model. Local hyperthermia is induced by HIFU and monitored and controlled via MRI. In the second part of the thesis (focused) ultrasound is used for improving local drug delivery in cells and tissue. The fate of the drug is followed by different imaging techniques such as MRI and fluorescence imaging.

It has been frequently postulated that genome or domain-wide histone post-translational modifications induce structural changes to chromatin. Until recently, however, experimental evidence for this hypothesis was lacking. H4 K16 acetylation is the first, and only, of all of the possible post-translational modifications to be directly linked to changes in chromatin conformation. I was interested in clarifying the mechanism by which H4 K16Ac exerts its modulatory effect. To characterize the role of this modification I reconstituted mononucleosomes with H4 K16Ac isolated from chicken erythrocytes by weak cation-exchange HPLC. Analytical ultracentrifuge (AUC) and MgCI2 solubility data suggested that H4 K16Ac structurally relaxes the association of nucleosomal DNA with the histone octamer by weakening intra-nucleosomal DNA-histone electrostatic interaction. Similar to early studies on H4 K16 acetylation, evidence suggests that the phosphorylation of linker histones promotes chromatin decondensation and increases DNA accessibility. In an effort to initiate a study characterizing the structural effects of chromatin fibers containing phosphorylated linker histories, I have optimized a method of purification for nonphosphorylated and monophosphorylated linker histone H5 from erythrocytes of anemic chickens. Preliminary AUC data of oligonucleosomes containing the nonphosphorylated control linker histones (manipulated to the same degree as the experimental monophosphorylated linker histones) showed a salt-dependent sedimentation trend that was consistent with expected values at low ionic concentrations. The purification method described is valuable as it results in a high yield of pure post¬translationally modified H5 product suitable for oligonucleosome reconstitutions. In the third component of this thesis, I describe the creation of a DNA template, Pbsn-208(10), which contains a Rattus norvegicus probasin promoter nucleosome positioning sequence flanked on both sides by 5 tandem repeats of the Lytechinus variegatus nucleosome positioning sequence. Unlike other DNA templates used in the study of chromatin compaction in vitro, the Pbsn-208(10) allows not only the reconstitution of homogenous chromatin fibers but also the differentiation of the middle of the fiber from its flanking ends. Thus, the effects of PTMs on histone-DNA interactions and nucleosome conformation in the center of a chromatin fiber can now be easily investigated. In addition, the Pbsn-208(10) template can help to identify the precise location of H1/H5 within the nucleosome.

Le facteur ULTRAPETALA1 (ULT1) est impliqué dans plusieurs processus développementaux chez Arabidopsis thaliana, dont le maintien de l'homéostasie des méristèmes aériens et la morphogénèse florale. ULT1 est en particulier essentiel à la restriction du territoire d'expression de WUSCHEL (WUS), acteur central du maintien de l'identité des cellules souches. ULT1 est également déterminant dans l'activation spatio-temporelle d'AGAMOUS (AG), gène clé du développement floral, nécessaire à la croissance déterminée de la fleur. Néanmoins les mécanismes moléculaires impliqués dans le fonctionnement d'ULT1 n'ont pas tous été élucidés, notamment la nature de ses partenaires protéiques lui assurant sa spécificité de liaison à l'ADN. Les objectifs du travail de thèse ont été (i) d'identifier de nouveaux interacteurs d'ULT1 et (ii) de caractériser la fonction moléculaire et développementale de l'un d'entre-eux. Par des approches génétique, moléculaire et biochimique, nous avons identifié le répresseur transcriptionnel ULT1 INTERACTING FACTOR 1 (UIF1) et caractérisé sa fonction dans le contrôle de l'activité du méristème floral chez Arabidopsis thaliana. UIF1 est en particulier capable de lier spécifiquement une séquence promotrice du gène WUS. Par cette étude nous apportons un mécanisme pour la reconnaissance spécifique de ses cibles par ULT1. Par une approche gènes candidats, nous avons identifié de nouveaux interacteurs d'ULT1, pouvant expliquer (i) son effet sur le retrait de marques chromatiniennes maintenant un locus inactif (interaction avec la déméthylase RELATIVE OF EARLY FLOWERING 6); (ii) sa fonction trithorax activatrice (interaction avec ARABIDOPSIS TRITHORAX LIKE I); et enfin (III) son rôle dans l'initiation de la transcription de gènes cibles (interaction avec le domaine C-terminal de l'ARN Polymérase II). Ces données positionnent ULT1 à l'interface entre dé-répression chromatinienne et initiation transcriptionnelle. / The ULTRAPETALA1 (ULT1) factor is involved in several developmental processes during Arabidopsis thaliana life cycle such as the homeostasis maintenance at aerial meristems and floral morphogenesis. In particular, ULT1 is critical to the restriction of the expression territory of WUSCHEL (WUS), a central player in stem cell maintenance. ULT1 is also essential for the spatio-temporal activation of AGAMOUS (AG), a key floral developmental gene necessary to flower determinate growth. Nevertheless, the molecular mechanisms through which ULT1 functions haven't all been solved yet, including the nature of its protein partners assuring its binding specificity to DNA targets. The objectives of this thesis were (i) to identify new ULT1 interactors and (ii) to characterize the molecular and developmental function of one of them. By genetic, molecular and biochemical approaches, we identified the ULT1 INTERACTING FACTOR 1 (UIF1) transcriptional repressor and characterized its function in the control of floral meristem activity in Arabidopsis thaliana. In particular, UIF1 is able to specifically bind a promoter sequence in the WUS gene. With this study we provide a mechanism for specific recognition of target genes by ULT1. By a candidate gene approach, we identified novel ULT1 partners, which may explain (i) ULT1 effect on removal of chromatin repressive marks that maintain a locus in an inactive state (interaction with the demethylase RELATIVE OF EARLY FLOWERING 6); (ii) the ULT1 activating trithorax function (interaction with ARABIDOPSIS TRITHORAX LIKE I); and finally (iii) ULT1 role in the transcriptional initiation of target genes (interaction with the C-terminal domain of RNA Polymerase II). This dataset reveals a function for ULT1 at the interplay between chromatin de-repression and transcriptional initiation.

No description available.