L’ARN polymérase (ARNP) est l'enzyme centrale d'expression des gènes. Toutes les formes de vie possèdent de l’ARNP. C’est un complexe protéique formé de plusieurs sous-unités responsables du processus de transcription qui aboutit à la synthèse de l’ARN à partir d’une matrice ADN. Les procaryotes possèdent un seul type d’ARNP responsable de la synthèse de tous les ARNs de la cellule, alors que les eucaryotes possèdent trois types d’ARNPs pour la synthèse des différents types d’ARNs.L’ARNP est la cible d’un grand nombre de protéines et de petites molécules de régulation dont certains antibiotiques utilisés en première ligne pour le traitement de diverses maladies infectieuses. La sous-unité sigma de l’ARN polymérase bactérienne est impliquée dans toutes les étapes de l'initiation de la transcription qui est le point crucial de l'expression des gènes. Les sous-unités sigma activent par exemple les gènes de virulence des bactéries pathogènes et sont impliquées dans la persistance qui est une forme de survie aux traitements antibiotiques.Ce projet a permis de déterminer le rôle de la sous-unité sigma de l'ARN polymérase bactérienne dans la résistance à la lipiarmycine (Fidaxomicin). Nous avons utilisé des approches biochimiques, biophysiques et génétiques pour l’étude de la dynamique des interactions ADN-protéine dans les complexes formés par l’ARN polymérase, l’antibiotique et de l’ADN des promoteurs.Les résultats de cette étude montrent que la sensibilité de l’ARNP dépend fortement de la structure de la région 3.2 de sigma et que les régions 1.2 et 3.2 de la sous-unité sigma sont impliquées dans la formation du complexe d’initiation de la transcription. Les mutations au niveau de ces régions affectent allostériquement l'action de la lipiarmycine en compromettant la formation du complexe ouvert. Ces résultats suggèrent que la conformation et la mobilité de la région 3.2 dépendent fortement de sa séquence. Ces travaux contribueront de manière significative à la compréhension des bases moléculaires de la résistance aux antibiotiques; Les approches méthodologiques développées pendant ce projet pourront être étendues à l'analyse d’autres antibiotiques ciblant l’ARNP bactérienne et à l’analyse des autres facteurs de transcription. / The RNA polymerase (RNAP) is the central enzyme for genes expression. All forms of life own RNAP. It is a multi-protein complex composed of several subunits responsible of the process of the transcription. The prokaryotes have only one type of RNAP responsible of synthesis of all RNAs in the cells, whereas eukaryotes have three types of RNAPs for the synthesis of the various types of RNAs.RNAP is the target of a large number of proteins and small regulatory molecules including antibiotics used the treatment of various infectious diseases. The sigma subunit of the bacterial RNAP is implicated in all steps of transcription initiation which is the crucial point of genes expression.For example some of the sigma subunits activate genes of virulence in pathogenic bacteria and are implied in the persistence which is a form of survival to the antibiotic treatments.This project aimed to explore the role of the sigma subunits of RNAP bacterial polymerase in resistance to the lipiarmycine (Fidaxomicin). We used biochemical approaches, biophysics and genetics for the study of the dynamic of the interactions DNA-protein in the complexes formed by RNA polymerase, the antibiotic and the promoter DNA. The results of our study show that sensitivity of RNAP to the drug strongly depends on the structure of the sigma region 3.2 and that the regions 1.2 and 3.2 of the sigma subunit are implied in the formation of the RNAP-promoter open complex. Mutations in these regions allosterically affected action of lipiarmycin by impairing the formation of the open complex.These results suggest that conformation and mobility of the region 3.2 depend on its sequence. The outcomes of our work could be used for development of new more effective drugs and could help to progress the studies of the fundamental mechanisms of the transcription.

Many genes demonstrate highly dynamic pulsatile expression, with characteristic bursts of activity. Dynamic expression of the human prolactin (hPrl) gene in pituitary cells has previously been investigated identifying key temporal characteristics, influenced by the process of chromatin remodelling. Earlier work on the related pituitary human growth hormone (hGH) proximal promoter (-496/+1bp) indicated that it displayed similar dynamic behaviour. The human GH gene contains an extensive long-distance regulatory sequence, including a locus control region (-14/-32kbp) that has been shown to regulate chromatin remodelling and confer tissue-specificity of hGH expression. In this work I aimed to study dynamic regulation of the hGH gene promoter in detail. Initially I investigated the efficiency of several methods to express the luciferase gene in a 180kb hGH genomic fragment using bacterial artificial chromosome recombineering, to allow the investigation of single cell transcription dynamics. Although a functional recombinant BAC was not finalised during the course of the work, I carried out detailed time course studies using shorter hGH-reporter constructs. Using quantitative microscopy to study live single cells, I compared the dynamic characteristics of a 5kb hPrl promoter fragment with those of -840/+1bp and -3348/+1bp hGH-luciferase promoter-reporter constructs. Whilst previous hPrl analysis utilised a binary mathematical model assuming a simplified two-state (ON/OFF) process of gene transcription, I validated and applied a novel stochastic switch model (SSM), assuming instead that transcription rate can switch between any variable states at any time. Through doing so I observed an asymmetry in transcription rate switching, suggesting an all-or-nothing activation of a single UP-switch, with a greater number of rate decreasing DOWN-switches. The -3348/+1bp construct produced double the number of DOWN-switches, whilst the -840/+1bp construct produced 1.5 DOWN-switches in a 48h period. The cycling of transcriptional activity seen by the shorter construct was modified through the addition of forskolin, activating cAMP signalling. However, significant modification of the transcriptionally inactive refractory period seen with the -3348/+1bp construct (reduced from 3h to 1.9h) required histone modification through application of trichostatin A, a HDAC inhibitor. In conclusion, different promoter elements confer different transcriptional timing and dynamics. A subtler transcriptional modelling, such as used here in the SSM, reveals new insights into the phenomena of transcriptional switching, but the mechanisms involved remain to be determined.

Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.

The Hepatitis Delta Virus (HDV) is an RNA pathogen that uses the host DNA-dependent RNA polymerase II (RNAP II) to replicate. Previous studies identified the right terminal domain of genomic polarity (R199G) of HDV RNA as an RNAP II promoter, but the features required for HDV RNA to be used as an RNA promoter were unknown. In order to identify the structural features of an HDV RNA promoter, I analyzed 473,139 sequences representing 2,351 new R199G variants generated by high-throughput sequencing of a viral population replicating in 293 cells. To complement this analysis, I also analyzed the same region from HDV sequences isolated from various hosts. Base pair covariation analysis indicates a strong selection for the rod-like conformation. Several selected RNA motifs were identified, including a GC-rich stem, a CUC/GAG motif and a uridine at the initiation site of transcription. In addition, a polarization of purine/pyrimidine content was identified, which might represent a motif favourable for the binding of the host Polypyrimidine tract-binding protein-associated-splicing-factor (PSF), p54 and Paraspeckle Protein 1 (PSP1). Previously, it was shown that R199G binds both RNAP II and PSF, that PSF increased the HDV levels during in vitro transcription and that p54 binds R199G. In the present study, I showed that PSP1 also associates with HDV RNA and I investigated whether these proteins are required for HDV replication. My results show that knockdown of PSF, p54 and PSP1 resulted in a decrease of HDV accumulation. These proteins are highly concentrated in paraspeckles, which are nuclear structures involved in storage of transcripts generated by RNAP II. I found that upon viral replication in 293 cells, PSP1 appeared as bigger foci present outside of the nucleus, while PSF and p54 foci remained in the nucleus. NEAT1 is a long non-coding RNA essential for the formation of paraspeckles. Upon HDV replication, I found an increase of the intensity and size of NEAT1 foci that correlates with an increase of NEAT1 transcripts. Altogether, these data suggest that HDV replication results in an alteration of the paraspeckles structures, providing foundation for further investigation of the paraspeckles role in HDV cycle. Overall, the present study addresses the importance of the HDV RNA structure and of the host paraspeckle proteins for HDV replication.

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Duas linhagens filogenéticas principais (LI e L2) foram definidas em Trypanosoma cruzi, com base em sequências de genes de rDNA e mini-exon e análise por RAPD (Souto et al. 1996). Neste trabalho investigamos a estrutura e atividade dos promotores do cistron ribossômico das duas linhagens de T. cruzi. O promotor da cepa Dm28 (L2) foi clonado e sua seqüência foi comparada com seqüências publicadas da região homóloga de CL (L1) e La Cruz (L2). A identidade entre os dois promotores de L2 foi de 98%, e entre estes e o de L1 foi de 82%. O ponto de início de transcrição foi mapeado, apresentando a mesma localização nas duas linhagens. A atividade dos promotores de L1 e L2 foi determinada em construções plasmidiais contendo o gene reporter da cloranfenicol acetil transferase (CAT). Experimentos de expressão transitória mostraram que o promotor de L1 foi funcional apenas em isolados de L1, enquanto que o promotor de L2 foi funcional em ambas as linhagens. A expressão do promotor de L2 em isolados de L1 foi maior do que a do promotor homólogo. Analisamos ainda a atividade dos dois promotores em um grupo de isolados que apresentam dois tipos de cistrons ribossômicos (grupo 1/2). Neste grupo de cepas observamos que ambos os promotores presentes nas construções são funcionais, embora o promotor de L2 induza maior expressão de CAT. Por outro lado, demonstramos que nestes isolados apenas o cistron ribossômico de tipo 2 é expresso in vivo. Neste trabalho analisamos ainda a eficiência de entrada das construções plasmidiais nas cepas de T. cruzi; caracterizamos a atividade de regiões do promotor de L2 e a eficiência do processo de \"trans-splicing\" para gerar mRNA de CAT contendo a seqüência de mini-exon na extremidade 5\'. / Two major phylogenetic lineages (L1 and L2) have been defined in Trypanosoma cruzi based on rDNA and mini-exon sequences and RAPD analysis (Souto et al., 1996). In the present work we have investigated the structure and activity of ribosomal RNA promoters from the two T. cruzi lineages. The promoter region of Dm28 strain (L2) was cloned and its sequence was compared with the homologous regions from the CL (L1) and La Cruz (L2) strains, whose sequences were previously published. The identity found between promoters of the two L2 strains was 98%, while the identity between L2 and L1 promoters was 82%. The transcription start point mapped in the two Lineages showed the same localization. The activity of L1 and L2 promoters was investigated through the use of plasmid constructs bearing bacterial chloramphenicol acetyl transferase (CAT) as reporter gene. Experiments of transient expression showed that the L1 promoter drove high CAT activity in L1 isolates, but essentially no activity in L2 strains. On the other hand, L2 promoter was functional in both Lineages. The expression driven by L2 promoter in L1 isolates was higher than that driven by the homologous promoter. We have also analysed the activity of both L1 and L2 promoters in a particular group of T. cruzi isolates (group 1/2) which contains two types of rRNA cistrons. It was observed that both promoters were functional in this group of strains, although L2 promoter drove higher CAT activity. This is an interesting result since we have shown that in group 1/2 isolates only the type 2 rRNA cistron is expressed in vivo. In the present study, we have also analysed the transfection efficiency of the plasmid constructs in T. cruzi strains; the activity of segments of the L2 promoter; and the efficiency of the \"trans-splicing\" process involved in the generation of mature CAT mRNA containing the mini-exon sequence at the 5\' end.

Neste trabalho, estudamos duas malformações craniofaciais mendelianas, decorrentes de um distúrbio do desenvolvimento dos primeiro e segundo arcos faríngeos: a síndrome de Treacher Collins (STC) e a síndrome Aurículo-condilar (SAC). A identificação de genes e de mecanismos moleculares associados a essas condições, além de contribuir para a compreensão do desenvolvimento das estruturas derivadas desses arcos faríngeos, é fundamental para o desenvolvimento do diagnóstico molecular, uma ferramenta importante para diagnóstico diferencial e aconselhamento genético. Contribuímos para uma melhor caracterização clínica da SAC com a descrição de uma nova família com 11 afetados. Após excluirmos quatro genes/regiões candidatas para síndromes de 1º e 2º arcos faríngeos, realizamos estudos de ligação usando marcadores polimórficos ao longo do genoma. Mapeamos o primeiro lócus associado à SAC, 1p21.1-q23.3 (lod score=3.0), e nossos dados sugerem que há heterogeneidade genética para essa patologia. Com relação ao estudo da STC, realizamos uma extensa revisão da nomenclatura das mutações patogênicas descritas na literatura, além de investigar mecanismos mutacionais atípicos na STC, corroborando a hipótese de que mutações nos exons que sofrem splicing alternativo são capazes de gerar o fenótipo da síndrome. Demos continuidade à caracterização do espectro de mutações no gene TCOF1 e investigamos a correlação genótipo-fenótipo numa amostra de 58 pacientes com STC. A análise dos dados de polimorfismos da região codificadora do gene permitiu que fizéssemos inferências sobre o regime de seleção ao qual o TCOF1 está submetido, e os resultados sugeriram que o gene TCOF1 está sob seleção purificadora, que atua sobre mutações fracamente deletérias. Também inferimos a fase para um conjunto de polimorfismos da região codificadora, verificando se havia associação de algum haplótipo à gravidade do quadro clínico ou à predisposição para a doença. Dada a observação de ausência de correlação haplótipo/genótipo-fenótipo, testamos a hipótese de que variações nos níveis de expressão do alelo normal poderiam ser responsáveis pela variabilidade clínica observada nos pacientes portadores da STC. Para tanto, iniciamos o estudo funcional das regiões regulatórias do gene TCOF1 , inclusive, de regiões distantes do promotor mínimo, preditas como enhancers. Identificamos polimorfismos em sua região promotora, sendo um deles capaz de diminuir os níveis de transcrição e de afetar a ligação do fator de transcrição YY1 ao promotor do TCOF1 . Caracterizamos a ação de YY1 como repressora in vitro. Testamos também a hipótese de haploinsuficiência como mecanismo associado à STC. Quantificamos os níveis de transcritos do TCOF1 em indivíduos afetados e normais e observamos uma diferença significativa. Nosso trabalho corrobora a hipótese de haploinsuficiência e mostra pela primeira vez que pacientes têm degradação de transcritos. Também investigamos a possibilidade de os níveis de transcritos estarem correlacionados à variabilidade fenotípica. Comparamos os dados de expressão de cada paciente à freqüência de nove sinais clínicos principais da STC, mas nenhuma correlação foi observada. Investigamos o padrão de metilação da ilha CpG do gene TCOF1 em pacientes e em controles, com o intuito de verificar se diferentes níveis de metilação inter-individual estariam associados à grande variação na expressão gênica. Demonstramos que a metilação da ilha CpG não é o mecanismo regulatório por trás dessa ampla variação de expressão do TCOF1 / In the present study, we investigate two craniofacial mendelian disorders, resulting from abnormalities in the development of the first and second pharyngeal arches: the Treacher Collins Syndrome (TCS ) and the auriculo condylar syndrome (ACS). The identification of genes and molecular mechanisms associated to these conditions, in addition to contributing to the understanding of the development of structures derived from these pharyngeal arches, is fundamental for the development of molecular diagnostics, an important tool for differential diagnosis and genetic counseling. We contributed to a better clinical characterization of ACS with a description of a new family with 11 affected individuals. After excluding four candidate genes/regions for syndromes of the 1st and 2nd pharyngeal arches, we carried out linkage studies using polymorphic markers throughout the genome. We mapped the first locus associated to ACS, 1p21.1-q23.3 (lod score=3.0), and our data suggest genetic heterogeneity exists for this pathology. With respect to the study of TCS , we carried out an extensive review of the nomenclature for the pathogenic mutations described in the literature, in addition to investigating atypical mutation mechanisms in TCS , corroborating the hypothesis that mutations in the exons that undergo alternative splicing can result in the TCS phenotype. We continued the characterization of the mutation spectrum for TCOF1 and we investigated the genotype-phenotype correlation in a sample of 58 patients with TCS . The analysis of polymorphisms in the coding region allowed us to make inferences about the selective regime experienced by TCOF1 , and our results suggested that TCOF1 is under purifying selection, which acts upon weakly deleterious mutations. We also inferred the phase for a set of polymorphisms in the coding region, testing whether there was association between any haplotype and the severity of the phenotype or the susceptibility to the disease. Given the observation of no correlation between haplotype/genotype and phenotype, we tested the hypothesis that variation in the levels of expression of the normal allele could be responsible for the clinical variability observed in TCS patients. To do this, we started a functional study of the TCOF1 regulatory regions, including regions distant from the minimal promoter, predicted to be enhancers. We identified polymorphisms in the promoter region, one of which reduced the levels of transcription and affected the binding of the YY1 transcription factor to the TCOF1 promoter. Using an in vitro assay we characterized YY1 as a repressor. We also tested the hypothesis of haploinsufficiency as a mechanism associated to TCS . We quantified the levels of TCOF1 transcripts in normal and affected individuals and found a significant difference. Our study corroborates the haploinsufficiency hypothesis and for the first time shows that patients have transcript degradation. We also investigated the possibility that transcripts levels are correlated to phenotypic variability. We compared the expression data for each patient with the frequency of nine clinical signs of TCS , but no correlation was found. We investigated the pattern of methylation on the CpG island of TCOF1 in patients and controls, in order to test whether different levels of methylation among individuals were associated to the great variation in gene expression. We demonstrated that methylation of the CpG island is not the regulatory mechanisms underlying the broad variation in TCOF1 expression.

O desenvolvimento de sistemas de expressão através da modificação de fortes promotores conhecidos vem sendo amplamente utilizado para a produção de proteínas com potencial utilização biotecnológica em diversos hospedeiros como S. cerevisiae. O objetivo do trabalho foi construir um promotor quimérico através de modificações do promotor cbh1 de T. reesei e inserção de elementos de resposta a metais provenientes do promotor ZRT1 de S. cerevisiae. O promotor desenhado foi utilizado na construção de um sistema de expressão, que foi inserido na levedura e teve sua atividade analisada pelo teste de ONPG. Foi possível observar que, conforme a concentração de zinco aumenta, a atividade do promotor diminui. O promotor teve maior atividade no meio sem zinco e menor no meio com 1000 μM de zinco. Esses resultados confirmam que o sistema funciona de forma eficiente em S. cerevisiae. Também foi possível observar que os mutantes crescidos em meio com limitação de zinco e, consequentemente com maior atividade do promotor, tiveram sua taxa de crescimento alterada. O que é esperado devido ao fenômeno conhecido como estresse metabólico, comum durante a produção de proteínas recombinantes em leveduras, na qual a cultura apresenta uma diminuição significativa do crescimento. / The development the expression systems by modifying strong promoters has been widely used for the production of proteins with potential biotechnological use in various hosts such as S. cerevisiae. This study aimed to construct an expression system constituted of the chimeric promoter built with cbh1 promoter modified by inserting metal responsive elements from the promoter of ZRT1 gene of S. cerevisiae. The S. cerevisiae was transformed and the system was induced with different concentrations of zinc and was tested using ONPG as substrate. It was observed that under high zinc concentrations promoter activity is low. At low zinc concentrations the opposite effect is observed, and the promoter reaches its highest activities. These results confirm that the system functions efficiently in S. cerevisiae. It was also observed that the mutant grown in environment with zinc limitation, hence with higher activity of the promoter, showed reduced growth rate. Indeed, this is expected due to the phenomenon known as metabolic burden, characterized by a joint stress during the production of recombinant proteins in yeast, under conditions which the culture has a significant growth reduction.

Cannabinoids and cannabinoid receptors have been shown to play important roles in immune regulation particularly as modulators of anti-inflammatory cytokines and antibody production. The predominant cannabinoid receptor involved in this immune regulation is cannabinoid receptor 2 (CB2), which is robustly expressed in B cells. Utilizing a combination of bioinformatics, 5' RACE, real time RT-qPCR, and reporter assays, we showed that human B cells from peripheral blood mononuclear cells (PBMC) expressed one CB2 transcript while mouse B cells from spleen express three CB2 transcripts. Alignment of the sequenced B cell RACE products to either the mouse or human genome, along with the GenBank mRNA sequences, revealed that the transcripts isolated in this study contained previously unidentified transcriptional start sites (TSSs). In addition, expression construct testing of the genomic region containing the TSSs of the mouse CB2 exon 1 and 2 transcripts showed a significant increase of promoter activity. Bioinformatics analysis for cis-sequences in the promoter regions identified DNA binding sites for NF-kB, STAT6, and Elk1 transcription factors activated by LPS, IL-4 and anti-CD40. Regarding variations in CB2 transcript expression among the immune cell subtypes, RACE analysis showed that the exon 1b transcript is seen in B cells but not in T cells, dendritic cells or macrophages. Furthermore, RT-qPCR showed variations in transcript expression during B cell development as well as in resting versus LPS or IL-4/anti-CD40 stimulated B cells. The exon 1a transcript was predominant in pre-, immature and resting B cells whereas the exon 1b and 2 transcripts were enhanced in mature and activated B cells. These data showed for the first time that human B cells use one TSS for CB2 expression while mouse B cells use multiple TSSs for the expression of three CB2 transcripts, in which the expression of the individual transcript is related to immune cell type and/or cell activation state. Additionally, this is the first report in mouse B cells defining TSSs that are in genomic areas with promoter activity thus suggesting the location of two promoter regions. Defining the CB2 transcript expression during various stages of B cell activation provide clues to therapeutic methods.

The transcriptome represents the entirety of RNA molecules within a cell or tissue at a given time. Recent advances have facilitated the production of large-scale, global interrogations of transcriptomes, finding that genomes are extensively transcribed and contain diverse classes of RNAs (Dinger et al., 2009). Information generated by high-throughput analyses of mRNA transcription start sites (TSSs) such as CAGE (Cap Analysis of Gene Expression) indicate that eukaryotic genomes have complex landscapes of transcription initiation. The TSS is important for the annotation of cis-regulatory sequences, because it provides a link between the mRNA transcript and the promoter. The patterns of TSS distributions observed within mRNA 5' end profiling studies prevent straightforward annotation of putative promoters. To address this challenge, we developed a method to identify- on a genome-wide basis- the putative promoter, which we define by TSS distributions and designate the transcription start region (TSR). We applied a clustering method to identify and annotate TSRs within the budding yeast Saccharomyces cerevisiae using a full-length cDNA dataset (Miura et al., 2006). To validate these TSR annotations, we performed an integrative genomic analysis using multiple datasets. Our method identified TSRs at positions consistent with bona fide promoters in S. cerevisiae. In addition, using 5'RACE, we find overall agreement between computationally-defined TSRs and TSSs identified experimentally. From this analysis, we find that a significant proportion of genes exhibiting alternative promoter usage within sporulation are associated with respiration, suggesting that this is regulated on a condition-specific basis in budding yeast. We further developed our TSS clustering method into a bioinformatics tool called TSRchitect, which identifies and annotates TSRs from large-scale TSS profiling information. TSRchitect is capable of handling both tag and sequence-based TSS information and efficiently computes TSRs from global TSS datasets on a desktop computer. We find support for TSRchitect's annotations in human from a CAGE experiment from the ENCODE (Encyclopedia of DNA Elements) project. Finally, we use TSRchitect to identify TSRs from the transcriptomes of diverse eukaryotes. We investigated the conservation of TSRs among orthologous genes. We frequently identify multiple TSRs for a given gene, suggesting that alternative promoter usage is widespread. Overall, using TSS profiling data derived from separate tissues within mouse and human, we find that the positions of TSRs are relatively stable across tissues surveyed; however, a small fraction of genes exhibit tissue-specific differences in TSR use. As transcriptome profiling information continues to be generated at an rapid pace, computational approaches are increasingly important. It is anticipated that the method and approach we describe within this dissertation will contribute to an improved of gene regulation and promoter architecture in eukaryotes.