Nitrogen is an essential building block of proteins and nucleic acids and, therefore, crucial for the biosphere. Nearly 79 % of the air consists of nitrogen, but in the form of nitrogen gas (N2), which cannot be utilized by most organisms. Nitrogen-fixing microorganisms such as cyanobacteria have a central role in supplying biologically useful nitrogen to the biosphere. Therefore, it is important to achieve further understanding of control mechanisms involved in nitrogen fixation and related processes. This thesis concerns different molecular aspects of the transcription factor NtcA from the heterocystous cyanobacterium Anabaena PCC 7120. Apart from performing oxygenic photosynthesis, Anabaena PCC 7120 is also capable of fixing nitrogen. NtcA is a protein regulating transcription of a wide range of genes and in particular genes involved in cyanobacterial global nitrogen control. NtcA binds as a dimer to the promoter regions of target genes such as those involved in nitrogen fixation and heterocyst differentiation. NtcA from Anabaena PCC 7120 was heterologously expressed in E. coli and a high yield of recombinant protein was achieved through purification by Ni-IMAC chromatography. The purified NtcA was used to examine DNA binding motifs preferred by NtcA in vitro using a semi-random library of DNA sequences. The preferred binding sequence for NtcA is TGTA – N8 – TACA and at least five of the bases in the palindromic binding site are necessary for binding. Differences in the consensus sequence in vivo may reflect variations in the structural conformation of NtcA under various physiological conditions. Since an earlier study suggested redox-regulated NtcA-DNA binding the role of the two cysteine residues of NtcA were investigated. Binding studies using three mutants, Cys157Ala, Cys164Ala, and Cys157Ala / Cys164Ala, demonstrated that all these NtcA variants bind to DNA with a slightly higher affinity in the presence of the reducing agent DTT. The studies indicate that the binding mechanism is not dependent on a conformational change of NtcA caused by breaking of intra-molecular disulfide bonds. Crystallization followed by structural studies rendered a partial crystal structure of NtcA. The structure verifies that NtcA is a dimeric protein. Each subunit has three domains: the N-terminal domain, a dimerization helix connecting the N-terminal domain with the C-terminal domain, as well as making up the dimer interface, and a C-terminal domain including the DNA binding helix-turn-helix motif. Furthermore, an NtcA binding site was found in the promoter region of the hupSL gene, encoding an uptake hydrogenase in Nostoc punctiforme (ATCC 29133), indicating that yet another gene is transcriptionally controlled by NtcA, thereby further emphasizing the multifaceted role of NtcA in cyanobacteria.

Adenovirus E1A is needed to activate early viral genes and induce cell cycle progression to optimise the conditions for viral replication. This is mostly achieved through interactions between the first exon of E1A and cellular transcriptional regulatory proteins. The carboxy terminus of E1A binds the cellular corepressor of transcription C-terminal Binding Protein (CtBP), resulting in derepression of CtBP target genes. Inducible stable U2OS cell lines were established, expressing wild type E1A (E1Awt) and a mutant unable to bind CtBP (E1A∆CID). Low inducible levels and loss of protein expression after prolonged induction together with induction of apoptosis were consistent with the fact that wild type E1A is a cytotoxic protein and correlated with the ability of CtBP to repress proapoptotic genes. E1A∆CID did not induce apoptosis and could be expressed at high levels for prolonged time periods. Moreover, the binding of CtBP contributed to E1A-induced activation of viral E1B and E4 genes, through possible targeting of Sp1 and ATF transcription factors. In a micorarray study on mRNA levels in E1A-expressing cells, several genes consistent with the tumour suppressive and apoptotic properties of E1Awt were identified as differentially expressed. Furthermore, the differences between the two cell lines correlated with the presence of binding sites for CtBP-interacting transcription factors in the promoters of regulated genes, enabling the possible identification of new CtBP target genes. Finally, a molecular characterisation of the CtBP mechanism of repression revealed that positioning proximal to the basal promoter element was required for efficient repression, suggesting that CtBP interferes with the basal transcriptional machinery. Two separate domains were identified in CtBP, conferring transcriptional repression and activation when expressed alone, achieved through their interaction with HDACs and HATs, respectively. However, together they cooperate to ensure maximal repression through recruitment of histone deacetylase and inhibition of histone acetyl transferase activity. Together, these data shows important modulation of E1A activities by the binding of CtBP and suggests the involvement of acetylation/deacetylation complexes for the regulation of E1A function.

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Genetics is among the most rewarding fields of biology for the theoretically inclined, offering both room and need for modeling approaches in the light of an abundance of experimental data of different kinds. Many aspects of the field are today understood in terms of physical and chemical models, joined by information theoretical descriptions. This thesis discusses different mechanisms and phenomena related to genetics, employing tools from statistical physics along with experimental biomolecular methods. Five articles support this work. Two articles deal with interactions between proteins and DNA. The first one reports on the properties of non-specific binding of transcription factors proteins in the yeast Saccharomyces cerevisiae, due to an effective background free energy which describes the affinity of a single protein for random locations on DNA. We argue that a background pool of non-specific binding sites is filled up before specific binding sites can be occupied with high probability, thus presenting a natural filter for genetic responses to spurious transcription factor productions. The second article describes an algorithm for the inference of transcription factor binding sites for proteins using a realistic physical model. The functionality of the method is verified on a set of known binding sequences for Escherichia coli transcription factors. The third article describes a possible genetic feedback mechanism between human cells and the ubiquitous Epstein-Barr virus (EBV). 40 binding regions for the major EBV transcription factor EBNA1 are identified in human DNA. Several of these are located nearby genes of particular relevance in the context of EBV infection and the most interesting ones are discussed. The fourth article describes results obtained from a positional autocorrelation analysis of the human genome, a simple technique to visualize and classify sequence repeats, constituting large parts of eukaryotic genomes. Applying this analysis to genome sequences in which previously known repeats have been removed gives rise to signals corroborating the existence of yet unclassified repeats of surprisingly long periods. The fifth article combines computational predictions with a novel molecular biological method based on the rapid amplification of cDNA ends (RACE), coined 5’tagRACE. The first search for non-coding RNAs encoded in the genome of the opportunistic bacterium Enterococcus faecalis is performed here. Applying 5’tagRACE allows us to discover and map 29 novel ncRNAs, 10 putative novelm RNAs and 16 antisense transcriptional organizations. Further studies, which are not included as articles, on the monitoring of secondary structure formation of nucleic acids during thermal renaturation and the inference of genetic couplings of various kinds from massive gene expression data and computational predictions, are outlined in the central chapters. / QC 20110316

[Truncated abstract] Human complement receptor 2 (CR2/CD21) is a cell surface glycoprotein detected on specific cells involved in immunity, which binds complement C3 cleavage fragments, cellular ligands IFN-? and CD23 as well as the EBV coat protein, gp350/220. During the early stages of B-cell development CR2/CD21 is silenced. Expression is initiated on immature B-cells escaping negative selection. During peripheral maturation CR2/CD21 is up-regulated with B-cell sub-populations showing distinctive surface levels (comparatively low, intermediate or high). CR2/CD21 is silenced upon terminal plasmacytic differentiation. Appropriate timing and expression level of CR2/CD21 is important for the development of a healthy B-cell repertoire. Previous studies have identified sequences within the proximal promoter and first intron of CR2/CD21 that cooperate within native chromatin to control cell-specific silencing. Further, analysis of cultured human cells has revealed chromatin structural variation causing DNase I hypersensitivity at these regulatory sites in a CR2/CD21-expressing mature B-cell line (Raji) which are absent in a non-lymphoid cell type (K562). The primary focus of the present study involved characterising chromatin structural variation over previously recognized DNase I hypersensitive regions at the CR2/CD21 locus in human cells to understand how chromatin structure might regulate developmental expression of CR2/CD21. ... These studies provide evidence that notch signaling influences CR2/CD21 expression in human cell lines. First, in vivo binding of CBF1 to CR2/CD21 sequences in the proximal promoter and CRS implies that CR2/CD21 is a direct target of notch activation. Second, the effect of exogenous notch signalling molecules on CR2/CD21 proximal promoter activity was modulated by factors binding tandem E-boxes near the transcriptional start site suggesting that the notch pathway may also influence CR2/CD21 expression via control of HLH molecules. Third, initiation of CR2/CD21 expression was observed in a nonexpressing pre-B cell line (Reh) by co-culture with stromal cells expressing a notch ligand (OP9-DL) but not control stroma (OP9-GFP). Together, these findings support a role for notch regulation of B-cell maturation and invite speculation that initiation of CR2/CD21 expression following negative selection of immature B-cells involves crosstalk between HLH transcriptional regulators and the notch pathway. Furthermore, the Reh/OP9-DL co-culture system may provide a model to directly study the relationship between cell signalling molecules, transcription factor regulation, chromatin structural variation and differentiation of B-cells.

Philosophiae Doctor - PhD / Tsetse flies (Glossina spp) are the biological vectors for Trypanosomes, the causative magents of Human African Trypanosomiasis (HAT). HAT is a debilitating disease that continues to present a major public health problem and a key factor limiting rural development in vast regions of tropical Africa. To augment vector control efforts, the International Glossina Genome Initiative (IGGI) was established in 2004 with the ultimate goal of generating a fully annotated whole genome sequence for Glossina morsitans. A working draft genome of Glossina morsitans was availed in 2011. In this thesis, transcriptional regulatory features in Glossina morsitans were analysed using the draft genome. A method for TSS identification in the newly sequenced Glossina morsitans genome was developed using TSS-seq tags sampled from two developmental stages of Glossina morsitans. High throughput next generation sequencing reads obtained from Glossina morsitans larvae and pupae were used to locate transcription start sites (TSS) in the Glossina morsitans genome. TSS-seq tag clusters, defined as a minimum number of reads at the 5’ predicted UTR or first coding exon, were used to define transcription start sites. A total of 3134 tag clusters were identified on the Glossina genome. Approximately 45.4% (1424) of the tag clusters mapped to the first coding exons or their proximal predicted 5’UTR regions and include 31 tag clusters that mapped to transposons. A total of 1101 (35.1%) tag clusters mapped outside the genic region and/or scaffolds without gene predictions and may correspond to previously un-annotated transcripts or noncoding RNA TSS. The core promoter regions were classified as narrow or broad based on the number of TSS positions within a TSS-seq cluster. Majority (95%) of the core promoters analysed in this study were of the broad type while only 5% were of the narrow type. Comparison of canonical core promoter motif occurences between random and bona fide core promoters showed that, generally, the number of motifs in biologically functional genomic windows in the true dataset exceeded those in the random dataset (p <= 0.00164, 0.00135, 0.00185 for the narrow, broad with peak and broad without peak categories respectively). Frequency of motif co-occurrence in core promoter was found to be fundamentally different across various initiation patterns. Narrow core promoters recorded higher frequency of the TATA-box and INR motifs and two-way motif co-occurrence showed that the TATA-box-INR pair is over-represented in the narrow category. Broad core promoters showed higher frequency of the BREd and MTE motifs and two-way motif co-occurrence showed that the MTE-DPE pair is over-represented in broad core promoters. TATA-less promoters account for 77% of the core promoters in this analysis. TATA-less core promoters showed a higher frequency of the MTE and INR motifs in contrast to observations in Drosophila where the DPE motif has been reported to occur frequently in TATA-less promoters. These motif combinations suggest their equal importance to transcription in their corresponding promoter classes in Glossina morsitans.

La polymérase d'A.R.N. la protéine obligatoire (le RbpA) est l'activateur transcriptional global (mondial) de l'espèce actinomycetes qui est essentielle pour la croissance et qu'augmente la tolérance de bactéries aux antibiotiques. RbpA de la tuberculose Mycobacterium (Mtb) stimule spécifiquement la transcription par la polymérase d'A.R.N. (RNAP) contenant σA ou les sous-unités σB, mais aucune de la 11 autre alternative σ des facteurs. Il a été rapporté que le fonctionnement de RbpA est dépendant de promoteur et c'est indispensable pour le déroulement de promoteur du promoteur sigAP constitutif.Pour déchiffrer la nature de spécificité de promoteur de RbpA, nous avons utilisé des essais biochimiques, mutagensis et des approches de génomique. Nous avons identifié ce remplacement 'TG' le motif entre-14 à-17 positions (postes) dans le promoteur sauvage-type sigAP fait la transcription indépendante de RbpA. Aussi, nous avons montré que la capacité d'augmentations de RbpA de RNAP pour fondre le promoteur sigAP aux températures sous-optimales et stabilise des complexes de promoteur. Mutational l'analyse de résidus d'acide aminé H166 et E169 dans la région σB 3.0 (σR3.0) a démontré une implication de σR3.0 dans la stabilisation RbpA-servie-d'intermédiaire de complexes de promoteur RNAP. Plus loin, la substitution à RbpA au résidu d'acide aminé R79 a affecté la stabilité complexe de promoteur, tandis que les substitutions à RbpA resdiues K73, K74 a affecté l'initiation de transcription. Cependant, aucun des mutants RbpA n'a étudié l'ouverture ici affectée de l'ADN de promoteur par RNAP. Les rôles différentiels joués par ces résidus RbpA dans la stabilisation de complexe de promoteur et l'initiation de transcription ensemble avec l'effet produit par les mutations σB suggèrent l'implication de R3.0 σB dans l'action de RbpA. Ensuite, nous avons exécuté la large de génome cartographie des gènes RbpA-dépendants du σB regulon en utilisant une Analyse de Puce à ADN de Finale(d'Écoulement) in vitro (des ROMS). L'analyse ROM a montré la preuve claire de 15 augmentation de pli du nombre de gènes activés par σB-RNAP en présence de RbpA. L'analyse de bio-informatique de 140 gènes contrôlés par la paire de RbpA-σB nous a permis d'identifier la signature caractéristique dans le-10 consensus ('TANNNT') spécifique à la sous-unité σB.Notre étude sur l'impact d'échelle de génome de RbpA, ensemble avec le déchiffrement du mécanisme moléculaire d'action de RbpA, souligne une importance de l'interaction entre σR3.0 et RbpA dans la transcription Mtb. Basé sur nos résultats nous proposons que RbpA puisse jouer un rôle du remplacement(remplaçant) fonctionnel pour-10 motifs prolongés(étendus) dans l'espèce mycobacterium. / RNA polymerase binding protein A (RbpA) is global transcriptional activator from actinomycetes species which is essential for growth and which increases tolerance of bacteria to antibiotics. RbpA from Mycobacterium tuberculosis (Mtb) specifically stimulates transcription by RNA polymerase (RNAP) containing either the σA or σB subunits but none of the other 11 alternative σ factors. It has been reported that the functioning of RbpA is promoter-dependent and it is indispensible for promoter unwinding of the constitutive sigAP promoter. To decipher the nature of promoter specificity of RbpA, we used biochemical assays, mutagensis and genomics approaches. We found that placing ‘TG-motif' between -14 to -17 positions in sigAP wild-type promoter makes transcription independent of RbpA. Also, we have shown that RbpA increases ability of RNAP to melt sigAP promoter at sub-optimal temperatures and stabilises promoter complexes. Mutational analysis of amino acid residues H166 and E169 in the σB region 3.0 (σR3.0), interacting with TG-motif, demonstrated an implication of σR3.0 in RbpA-mediated stabilisation of RNAP promoter complexes. Substitution in RbpA at amino acid residue R79 affected the promoter-complex stability, while the substitutions at RbpA resdiues K73, K74 affected the transcription initiation. However, none of the RbpA mutants studied here affected opening of the promoter DNA. The differential roles played by these RbpA residues in promoter complex stabilization and transcription initiation together with the effect produced by the σB mutations suggest the implication of σR3.0 in RbpA action. Next, we performed genome-wide cartography of the RbpA-dependent genes from the σB regulon by using an in vitro RunOff Microarray Analysis (ROMA). ROMA analysis has shown clear evidence of 15 fold increase in the number of genes activated by σB-RNAP in the presence of RbpA. Bioinformatics analysis of 140 genes controlled by RbpA-σB pair allowed us to identify characteristic signature in the -10 consensus (‘TANNNT’) specific to σB subunit. Our study underlines an importance of the interplay between σR3.0 and RbpA in Mtb transcription. Based on our results we propose that RbpA may play a role of functional replacement for TG-motif of the extended -10 elements in mycobacterium species.

DNA damage is a constant threat to our genomes which drives genome instability and contributes to cancer progression. DNA damage interferes with important DNA transactions such as transcription and replication. DNA lesions are removed by repair pathways that ensure genome stability during transcription and replication. Here, we identify and characterize distinct roles for the ultra violet stimulated scaffold protein A (UVSSA) in the maintenance of genome stability during transcription in human cells. First, we unravel a novel function for UVSSA in transcription-coupled repair of DNA interstrand crosslinks (ICLs), genotoxic adducts that covalently bind opposing strands of the DNA and block transcription and replication. UVSSA knockout cells are sensitive to ICL inducing drugs, and UVSSA is specifically required for transcription-coupled repair of ICLs in a fluorescence-based reporter assay. Based on analysis of the UVSSA protein interactome in crosslinker treated cells we propose a model for transcription-coupled ICL repair (TC-ICR) that is initiated by stalling of transcribing RNA polymerase II (Pol II) at an ICL. Stalled Pol II is first bound by CSA and CSB, followed by UVSSA which recruits TFIIH to initiate downstream lesion removal steps. Second, we establish that UVSSA counteracts MYC dependent transcription stress to promote genome stability in cells aberrantly expressing the cMYC oncogene. UVSSA knockdown sensitizes cells to MYC expression, resulting in synthetic sickness and increased doubling time. UVSSA knockdown impacts Pol II dynamics in MYC activated cells. We conclude that UVSSA is required for regulation of Pol II during MYC induced transcription to prevent transcription stress. Together, these studies expand our understanding of UVSSA’s role in genome stability during transcription and elucidates the poorly understood transcription-coupled ICL repair pathway.

No description available.

The combinatorial code of cPcdh isoforms creates a diversified cell-surface molecular signature for cell-cell recognition in neural networks. This genetic architecture, combined with a regulated expression pattern and trans-homophilic binding properties, provides insights into cell specialization and signaling. Anomalies in cPcdhs, which include genetic mutations, epigenetic modifications, structural variations, and altered gene expression profiles, are associated with several neurological, neuropsychiatric, and systemic conditions, highlighting the importance of cPcdh investigations. This study focuses on the transcriptional regulation of the Pcdhα gene cluster. Each neuron displays a specific Pcdhα alternate exon repertoire, necessitating an understanding of the transcriptional dynamics. Using the SK-N-SH human neuroblastoma cell line and methodologies such as cRNA-seq and Start-Seq, these dynamics are examined. The application of CRISPR-Cas9 gene editing and a dCas9-VPR gain-of-function assay in the HEK293T cell line reveals the role of as-lncRNA and its interaction with DNA methylation within the Pcdhα gene cluster. This study identifies the role of noncoding as-lncRNA in RNA transcription and provides information on CTCF binding and Pcdhα promoter activation. The research also examines the gastrointestinal domain, as cPcdhs are linked to various diseases. Shifting focus from the canonical realm of the CNS, the research embarks on a preliminary yet pivotal exploration of the gastrointestinal domain. As cPcdhs intersect with a plethora of diseases, an incisive understanding of their expression could yield revelations into tissue susceptibilities with potential disease ramifications. Employing a novel single-domain antibody technique coupled with immunohistochemistry, the endeavor casts a precise lens into the gastrointestinal expression dynamics of Pcdhα and Pcdhγ. These insights not only fortify the understanding of cPcdh within neural structures but also beckon a deeper inquiry into their multifaceted biological roles.