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Ubiquitin C-terminal hydrolase L1 (UCHL1) is a multifunctional protein primarily expressed in neuronal cells and involved in numerous cellular processes. UCHL1 has been linked with neurodegenerative diseases and a wide range of cancers but its specific role remains unknown. Previous UCHL1 knockdown studies have shown that UCHL1 controls the expression of pro- and anti-apoptotic genes as well as genes involved in cell cycle regulation but it is unknown how UCHL1 regulates these genes. We have shown that UCHL1 is cross-linked to DNA in DU145 but not in LNCaP or PC3 prostate cancer cells. Therefore, we hypothesized that UCHL1 regulates the expression of pro- or anti-apoptotic genes as well as the genes involved in the cell cycle through its interaction with DNA. By utilizing ChIP and ChIP-seq analyses it is possible to determine the UCHL1 target sequences on the genomic DNA. It was shown that UCHL1 is only expressed in DU145 but not in LNCaP, PC3 or C4-2 prostate cancer cell lines. Additionally, UCHL1 is expressed and cross-linked to DNA in HEK293T cells. It is believed that UCHL1 is silenced by upstream promoter methylation when it is not expressed. However, treatment with the epigenetic drugs 5-aza-2′-deoxycytidine and trichostatin A (TSA) did not result in induction of UCHL1 expression in LNCaP, PC3 or C4-2 prostate cancer cell lines. UCHL1 is also associated with p53. However, ChIP assay results have shown that UCHL1 and p53 do not bind to genomic DNA of upstream promoter regions CDKN1A and BAX genes. Additionally, through UCHL1 ChIP-seq analyses in DU145 and HEK293T cells, we discovered that UCHL1 co-localizes to the DNA with the shelterin complex shedding light on a new role of UCHL1 that has never been described before. / October 2014

The degree of DNA supercoiling in the cell is carefully controlled by DNA topoisomerases. These enzymes catalyze the passage of individual DNA strands (Type I DNA topoisomerases), or double helices (Type II DNA topoisomerases) through one another. The purpose of the present study is to conduct a detailed analysis of the topo llα and β mRNAs expressed in several vertebrate cell lines. The final aim of this project is to analyze the relative roles of topo llα in chromatin condensation and chromosome segregation during mitosis, by performing topo llα gene targeting experiments in the DT 40 chicken lymphoblastoid cell line. The knock-out strategy was based on the observation of a high rate of homologous recombination versus random integration in the DT40 cell line. The topo llα gene was shown to be located on the chicken chromosome 2 (APM unpublished), for which the DT40 cell line is trisomic. The targeting vector completely replaced the 32 kb topo IIα genomic locus, generating a topo llα (-/+/+)cell line, which showed an increased resistance to topo II inhibitors. Paradoxically, 150 uM etoposide or 100 uM mitoxanthrone induced apoptosis within 5 hours in the topo llα (-1+1+) cell line, more rapidly as compared to the normal DT 40 cells. A topo IIα (-I-I+) cell line has also been generated. This study revealed the presence of evolutionarily conserved alternatively spliced forms of both topo llα and β isoforms between birds and humans. Hybridization screening of two chicken cDNA libraries, MSB-1 and DU249, revealed the presence of two distinct forms of both topo llα and β cDNAs. One form of topo llα, designated topo llα-1, encodes the chicken topo llα amino acid sequence previously reported (dbjiAB007445) in the database (unpublished). The second form, designated topo llα-2, encodes a protein containing an additional 35 amino acids inserted after Lysine-322 of chicken topo IIα-1 protein sequence. In the case of topo 11(3 mANA, one form, designated topo IIβ-1, encodes the protein already described (dbjiAB007446). The second form, tapa IIβ-2, would encode a protein missing the next 86 amino acids after Valine-25 in tapa II β-1 protein sequence. The tapa 11β variant is positioned similarly to one previously described in human (Hela) cells. The 5 amino acid insertion in the human tapa 11β-2 variant follows v23. In chicken cells, a longer insertion of 86 amino acids sequence follows v25, the homologous position in the tapa 11β protein. In human cells, the situation with tapa llα is more complex, as revealed by RT-PCR experiments (APM, unpublished) which generated several bands. One of these amplified species was found to contain a 36 amino acids insertion, positioned after residue K321 in the human tapa IIα cDNA, similarly to chicken tapa IIα-2 variant. The second human tapa llα spliced form cDNA was shown to contain a 26 amino acids insertion after residue A401 in the canonical human tapa llα protein sequence. The third cDNA variant isolated from human cells was described to encode a 81 amino acids insertion after residue Q355 positioned within the known human tapa IIα protein. It appears possible that the posttranslational modifications of the a-2 and β-2 isoforms may differ substantially from those of the canonical a-1 and β-1 isoforms. Such variant proteins could fulfil specialized functions, which might be tissue or cell-type specific. In summary, two novel forms of tapa llα and β cDNAs have been identified in three chicken cell lines. These spliced versions of both tapa llα and 13 isoforms seem to be evolutionary conserved, with similar forms occurring in their human counterparts. Future functional analysis of vertebrate tapa IIα and β will have to account for the existence of these novel isoforms, which might encode proteins that may exhibit different regulation of their subcellular localization, interaction with other proteins, or catalytic activity.

Plant embryogenesis is an intriguing developmental process that is controlled by many genes. AGAMOUS Like 15 (AGL15) is a MADS-domain transcriptional regulator that accumulates preferentially during this stage. However, at the onset of this work it was unknown which genes are regulated by AGL15 or how AGL15 is regulated. This dissertation is part of the ongoing effort to understand the biological roles of AGL15. To decipher how AGL15 functions during plant development, a chromatin immunoprecipitation (ChIP) approach was adapted to obtain DNA fragments that are directly bound by AGL15 in vivo. Putative AGL15 targets were isolated, and binding and regulation was confirmed for one such target gene, ABF3. In addition, microarray experiments were performed to globally assess genes that are differentially expressed between wild type and agl15 young seeds. Among them, a gene, At5g23405, encoding an HMGB domain protein was identified and its response to AGL15 was confirmed. Preliminary results suggest that the loss-of-function of At5g23405 might have an effect on somatic embryogenesis, consistent with AGL15 repression of the expression of this gene. Lastly, to address the question about how the regulator is regulated, the cis elements controlling the expression of AGL15 must be identified. Deletion analysis of the AGL15 promoter indicated the presence of putative positive and negative cis elements contributing to the expression of AGL15. Further analysis suggested that AGL15 regulates the expression of its own gene and this regulation may partially be explained by the direct binding of the protein to the AGL15 promoter. The data presented in this dissertation demonstrate that ChIP can be used to identify previously unsuspected targets of AGL15. Based on ChIP, a ChIP-chip technique is being developed in the lab to allow a more global analysis of in vivo binding sites. The identification of target genes and cis elements in AGL15 promoter is a step towards characterization of the biological roles of AGL15.

Each year over 260,000 new cases of breast cancer will be diagnosed in the U.S. and approximately 40,000 women will die of metastatic breast cancer. The etiology of breast cancer is poorly understood and only 5 -10% of cases can be attributed to genetic factors. This suggests that the development of breast cancer may involve environmental factors including diet, lifestyle, and exposure to chemicals. Several lines of experimental and epidemiological evidence have highlighted COX-2 as a potential target for breast cancer prevention. The central hypothesis of this proposal is that activation of COX-2 transcription by epigenetic effectors can be prevented by dietary agents that target the activator protein-1 (AP-1) transcription factor and the aromatic hydrocarbon receptor (AhR). The first specific aim was to determine the mechanism through which conjugated linoleic acid (CLA) and rosmarinic acid (RA) inhibit TPA-induced COX-2 trancription. These studies documented that CLA and RA repressed COX-2 transcription by antagonizing the AP-1 transcription factor. The second specific aim was to investigate whether or not the AhR plays a role in TCDD-induced COX-2 transcription and effects of chemopreventive agents. Results indicated that AhR agonists induced the binding of the AhR to COX-2 and was prevented by CLA and the AhR antagonist, resveratrol (RES) and 3-methoxy-4-nitroflavone (3M4NF). The third specific aim was to examine the effects of AhR agonists and dietary selective AhR modulators on chromatin modifications associated with the COX-2 promoter. Chromatin immunoprecipitation (ChIP) assays revealed that the AhR is recruited to the region of the COX-2 promoter containing a xenobiotic response element and accompanied by recruitment of p300 and acetylation of histone H4. Transcriptional regulation of COX-2 by AhR agonists and dietary antagonists may also involve other post-transcriptional modifications of histones, which along with chromatin remodeling factors modulate the structure of chromatin and recruitment of RNA polymerase II. Overall, the results demonstrated that COX-2 transcription can be targeted by a variety of dietary agents that act through different mechanisms. Therefore, inhibition of transcriptional regulation of COX-2 by selected dietary factors may be a breast cancer preventive strategy that bypasses the side effects of drugs that target COX-2.

Over the past five years the role of epigenetic modifiers in brain development has become increasingly evident. In this regard, Snf2l, a homolog of the chromatin remodeling protein ISWI, was shown to have enriched expression in the brain and be important for neuronal differentiation. Mice lacking functional Snf2l have hypercellularity of the cerebral cortex due to increased cell cycle re-entry. In this thesis I demonstrate the effects of Snf2l-ablation on cortical progenitor cells including increased proliferation and cell cycle deregulation, the consequence of which is a delay in neuronal migration and altered numbers of mature cortical neurons. This phenotype arises from increased expression of Foxg1, a winged-helix repressor expressed in the forebrain and anterior optic vesicle. Moreover, genetically reducing its overexpression rescues the Snf2l-ablated phenotype. Snf2l is bound directly to a promoter region of Foxg1 suggesting that it acts as a repressive regulator in vivo and is an important factor in forebrain differentiation.

In S. cerevisiae initiation of replication occurs from discrete sites in the genome, known as origins and these display a characteristic temporal profile of activation during S phase of the cell cycle. The genomic context of origins has been demonstrated to be important to determine the time of firing, more specifically histone acetylation levels surrounding origins can influence their activation time. How increased acetylation is translated into earlier firing of specific origins is currently unknown. Bromodomains are known to bind acetylated histones in vivo. The bromodomain-containing Yta7p has been identified in a complex with various remodelers of chromatin and subunits of DNA polymerase ǫ. It is also a target of cell cycle and checkpoint kinases. Therefore, Yta7p makes an excellent candidate to bind acetylated histones surrounding replication origins and affect an alteration in the chromatin structure that could influence time of firing. Deletion of the histone deacetylase RPD3 results in a rapid S phase phenotype due to increased histone acetylation at “late-firing” origins. Increased acetylation at “late” origins leads to an advance in the time of firing of those specific origins. The aim of this study was to investigate the hypothesis that the bromodomain-containing protein Yta7p binds to histones with increased acetylation near to replication origins and subsequently influences origin firing. Hence, deletion of YTA7 would abolish the rapid S phase of a ∆rpd3 strain. Indeed the S phase of the ∆rpd3∆yta7 strain was reverted to WT duration. A role for Yta7p in DNA replication is also inferred by two additional lines of evidence presented in this thesis. Synthetic growth defects are evident when YTA7 and RPD3 deletion is combined with mutation of a third replication protein. In addition, ∆rpd3∆yta7 mutants are sensitive to HU, which is a phenotype shared by many strains with deletions in genes that encode proteins involved in DNA replication. Evidence to support a direct role of Yta7p in DNA replication events is provided by identification of an S phase specific binding of Yta7p to replication origins. Moreover, levels of Yta7p bound to early-firing origins are increased compared with their later-firing counterparts. Levels of Yta7p that are bound to “late-firing” origins are only increased in conditions of RPD3 deletion, where the resulting increase in histone acetylation at the “late-firing” origins is associated with advanced time of firing. Time of Yta7p binding at these “late” origins is also advanced concomitantly. This data supports the hypothesis that Yta7p provides a functional link between histone acetylation and time of origin activation. In searching for a specific replication linked function of Yta7p it was observed that recruitment of the FACT subunit Spt16p to replication origins was increased in conditions of YTA7 deletion. A second function for Yta7p in the S phase checkpoint was also demonstrated and the two roles of Yta7p, in DNA replication and S phase checkpoint, were separated depending upon their requirement for the bromodomain. The data produced in this thesis adds to our knowledge of DNA replication events and highlights the importance of histone modifications and chromatin remodeling to the replication field. This thesis describes the direct involvement of a protein, which was previously unassociated, with DNA replication and S phase checkpoint function and provides good ground work for future investigation.

Les protéines des groupes Polycomb et trithorax sont des régulateurs épigénétiques très conservés qui permettent le maintient de l'identité cellulaire en régulant le niveau d'expression des gènes. Ils agissent sur leurs gènes cibles à travers des éléments régulateurs en cis, appelés éléments de réponse aux Polycombs (PRE). Dans des tests transgéniques, il a été montré que deux copies du même PRE sont fréquemment regroupés dans la même région nucléaire. Dans le cas particulier du PRE Fab-7, ce regroupement corrèle avec sa fonction répressive. Durant ma thèse, j'ai tenté de cloner un outil bicolore qui permet la visualisation en 4D de deux PRE Fab-7 stablement intégrés dans le génome de Drosophila melanogaster. De plus, j'ai amélioré le protocole de DNA-FISH du labo. Ceci m'a permis d'identifier vestigial et apterous comme étant des loci qui forment des associations nucléaires, de façon dépendante de la transcription, dans Drosophila melanogaster. / Polycomb- and trithorax-Group proteins are highly conserved epigenetic regulators which maintain cell identities by maintaining states of gene expression. They act on their target genes through /cis/ regulatory elements, named Polycomb Response Elements (PREs). In transgene assays it has been shown that two copies of the same PRE are frequently found clustered in nuclear space and for one particular PRE named Fab-7 clustering is correlated with its repressive function. In the course of this thesis I tried to clone a two colour real-time tool which allows distinguishing in 4D two /Fab-7/s stably integrated into the genome of Drosophila melanogaster. Additionally, I improved the DNA-FISH protocol of the lab and identified vestigial and apterous as potential gene loci forming nuclear associations dependent on transcription in Drosophila melanogaster.

The three-dimensional organization of the genome is important for various processes such as transcription, replication, and repair. Several studies have shown that the genome is organized into long-range and short-range chromatin loops. Gene loops represent a short-range chromatin loop, synonymous with the juxtaposition of promoter and terminator regions of a gene. In Chapter III, I investigate the mechanism of gene-loop formation in a constitutively expressed gene, mouse serum albumin (Alb). The Alb locus appears to exist in a clover-leaf structure, with the promoter in close physical proximity with an upstream enhancer and downstream genic sequences. Furthermore, Alb forms a promoter-terminator gene loop that is dependent on serine 2 phosphorylation of RNA polymerase C-terminal domain. I also investigate the presence of gene loop conformation at the human Nuclear factor NF-kappa-B (NFκB1) gene. In response to cytokine stimulation, NFκB1 transcription proceeds as a wave, with nascent RNA appearing as RNA polymerase traverses along the gene length. This coincides with formation of transient contacts between NFκB1 promoter and genic regions. The cohesin complex is a key mediator of chromatin loops and sister chromatid cohesion. The association of cohesin with chromatin is dependent on the loading complex, Mis4/Ssl3. In Chapter IV, I provide direct evidence for two functionally different populations of cohesin is Schizosaccharomyces pombe. Cohesin that co-localizes with Mis4 represents "cohesive" cohesin. In contrast, cohesin alone is unable to maintain stable sister chromatid cohesion, therefore, "less-cohesive" cohesin. Cohesive cohesin ensures stable cohesion because it is acetylated by the Eso1 acetyltransferase, which preferentially interacts with Mis4. In contrast, less-cohesive cohesin may function in recombination/repair. In Chapter V, I have identified a novel interplay between cohesin loading and transcription by RNA polymerase II. Inhibition of transcription initiation results in loss of Mis4 and consequently, cohesin binding on chromosomal arms regions. Furthermore, cohesin and Mis4 physically interact with RNA polymerase II. In Chapter VI, I summarize the above findings and propose a model that describes the stepwise loading of cohesin onto chromosomal arms during the fission yeast cell cycle. To conclude, I discuss the importance of understanding cohesin and its functions in health and diseases.