Os genes de choque térmico groES e groEL de Caulobacter crescentus foram isolados utilizando-se os genes homólogos de E.coli como sonda e por sequenciamento demonstrou-se que estes genes estão organizados na forma de um operon em um fragmento de DNA de aproximadamente 2,5 kb, contendo também sua região regulatória. \"Northern blots\" de RNA total de células crescidas a 300C ou submetidas a choque térmico mostraram a presença de um único RNA de tamanho aproximado de 2,3kb, altamente induzido por choque térmico, permanecendo em altos níveis mesmo após longos períodos de choque térmico. Amostras de RNA total de células sincronizadas, de diferentes estágios do ciclo celular de Caulobacter, foram também analisadas mostrando que os níveis do mRNA groESL variam durante o ciclo, apresentando um máximo na célula prédivisional. Análises através de \"Western blot\" mostraram uma pequena variação nos níveis da proteína GroEL ao longo do ciclo celular, sendo os tempos 60 e 120 minutos, respectivamente, os pontos de mínimo e máximo acúmulo da proteína concordando com os resultados obtidos em \"N orthern blots\". O mesmo tipo de análise foi feito com extratos totais obtidos a partir uma população mista de células crescidas a 300C e submetidas a choque térmico, observando-se o acúmulo da proteína até 60 minutos depois do choque térmico, com aumento da ordem de 5 vezes nos níveis de GroEL, níveis estes que diminuem lentamente a partir deste ponto. Os inícios de transcrição foram determinados em experimentos de \"primer extension\" utilizando-se RNA total de células incubadas 300C e de células submetidas a diferentes condições de choque térmico. Dois possíveis sítios de início de transcrição foram determinados nas posições -119 e -88 do ATG da metionina iniciadora de groES, sendo as regiões -10 e -35 dos promotores correspondentes (P 1 e P2) identificadas. Somente a transcrição iniciando a partir de P2, que apresenta características de um promotor transcrito pelo σ32, aumenta durante o choque térmico. Fusões de transcrição com o vetor repórte placZ/290 e a região 5\' regulatória do operon groESL foram construídas para identificar as sequências responsáveis pelo controle por choque térmico e pelo controle temporal. Fusões de transcrição contendo deleções na região 5\' do operon mostraram que sequências a montante do promotor P2 não são necessárias para a indução por choque térmico ou para o controle temporal. Fusões de transcrição contendo mutações sítio-dirigidas na repetição invertida, encontrada a 3\' do promotor P2, antes do gene groES, revelaram que este elemento, conhecido como CIRCE, regula negativamente a expressão de groESL a 300C e mutações neste elemento levam à perda do controle temporal deste operon. / The heat shock genes groES and groEL of Caulobacter crescentus were isolated using the homologous genes of E.colí as a probe. DNA sequence analysis has shown that these genes are organized as an operan in a fragment of about 2.5kb, which includes the 5\' regulatory region. Northern blot analysis of total RNA from cells grown at 300C or heat shocked treated has shown the presence of a single mRNA species for groESL, of approximately 2.3kb in size, which presented increased leveis even after long periods of heat shock. Samples of total RNA from synchronized cells, corresponding to different stages of the Caulobaaer cell cycle, were also analysed, showing that the amount of groESL mRNA varies during the cycle, with maximum leveis in predivisional cells. Western blot analysis of GroEL leveis in Caulobaaer has shown that the amount of the protein decreases during the first 60 minutes of C.crescentus cell cycle and then starts to increase again. These results corroborate the data obtained with Northern blot analysis. A similar experiment was performed after exposing a mixed population of C.crescentus cells to different times of heat shock at 400C. Western blot of extracts of these cells showed a fivefold increase in the leveis of GroEL after 60 minutes of heat shock, which then begins to decrease. Primer extension experiments were performed using total RNA from cells incubated at normal growth temperature or after heat shock treatment. Two possible transcription start sites were determined at positions -119 and -88 from the ATG of the groES initiator methionine and the -10 and -35 regions of the corresponding promoters (P 1 and P2) were identified. Only trancription initiating from the P2 promoter, which has caracteristics of a σ32 promoter, I ncreases during heat shock .Transcription fusions with the reporter vector placZ/290 and the 5\' regulatory region of the groESL operan were contructed in order to identify the sequences responsible for heat shock and cell cycle contral. Deletion analysis in the 5\' region of the operon showed that no sequences upstream of the P2 promoter are necessary for heat shock induction or for temporal contral. Site-directed mutagenesis in the inverted repeat found 3\' of the P2 promoter, in front of the groES gene, revealed that this element, also known as CIRCE, negatively regulates groESL expression at 300C and mutations in it lead to loss of temporal control of this heat shock operon.

Candida albicans is the most common fungal pathogen that infects humans. The research described in this thesis focuses on an in-depth characterization of the regulatory pathways controlling white-opaque switching, mating and biofilm formation, and the relationships among these programs in this pathogen. It was demonstrated in 2006 that minority opaque cells of C. albicans release pheromone to signal majority white cells of the opposite mating type to form enhanced biofilms. The white cell biofilms in turn facilitate opaque cell chemotropism, an essential step in mating. The white cell response is a general characteristic of C. albicans, occurring in all tested strains and in all common lab media. By generation of deletion mutants of select genes in the opaque cell mating pathway, it was demonstrated that the pathway regulating the white cell response shares all of the components of the opaque mating pathway, from the pheromone receptor through the MAP kinase cascade with the exception of the downstream transcription factor. In addition, it was demonstrated that a C. albicans-specific region in the first intracellular loop, IC1, of the α-pheromone receptor is required for the white, but not the opaque, pheromone response. Furthermore, the cis-acting element in the promoters of genes induced by pheromone in white cells was identified. The white-specific pheromone response element, WPRE, is important for the regulation of the white pheromone response and induction of white-specific genes by pheromone. Finally, based on a misexpression library screening of transcription factors previously implicated in adhesion, cell wall biogenesis, filamentation or biofilm formation, the transcription factor Tec1 was identified to be the key regulator in the white pheromone response pathway. Tec1 binds to the WPRE in the promoters of genes induced by pheromone in white cells to mediate the white cell response. The white pheromone response pathway appears to be a relatively young pathway that borrowed the upstream components from the opaque mating pathway, the transcription factor from the ancestral filamentation pathway, and the downstream genes from the pathway regulating biofilm formation in a/α cells of C. albicans. Therefore, the configuration of the white response pathway provides a unique glimpse and possibly a paradigm for the evolution of signal transduction pathways in eukaryotes.

Cells must employ a diversity of strategies to regulate the quantity and functionality of different proteins during development and adult homeostasis. Post-transcriptional regulation of gene transcripts by microRNAs (miRNAs) is recognized as an important mechanism by which the dosage of proteins is regulated. Despite this, the physiological relevance of direct regulation of an endogenous gene transcript by miRNAs in vivo is rarely investigated. PAX6 is a useful model gene for studying miRNA regulation directly. PAX6 is highly dosage-sensitive transcription factor that is dynamically expressed during development of the eye, nose, central nervous system, gut and endocrine pancreas, and is mutated in the haploinsufficiency disease aniridia. Several miRNAs have been implicated in regulating PAX6 in different developmental contexts. Notably, miR-7 appears to regulate Pax6 during specification of olfactory bulb interneurons in the ventricular-subventricular zone (V-SVZ) of the brain and during development of the endocrine pancreas. Here, we produced a bioinformatics tool to enable selective mutation of candidate microRNA recognition elements (MREs) for specific miRNAs while ensuring that new MREs are not inadvertently generated in the process. We then performed the first comprehensive analysis of the mouse Pax6 3’ untranslated region (3’UTR) to identify MREs that may mediate miRNA regulation of Pax6 and to identify miRNAs capable of interacting with the 3’UTR of Pax6. Using Pax6 3’UTR genetic reporter assay, we confirmed that two MREs for miR-7-5 located at 3’UTR positions 517 and 655 function together to regulate PAX6. We generated mice harbouring mutations in the Pax6 3’UTR that disrupt these miR-7-5p MREs, individually or in combination, to explore the biological relevance of miRNA regulation directly. PAX6 protein abundance was elevated in double miR-7-5p MRE mutants relative to wild type and single mutants in the ventral V-SVZ. However, this increase in PAX6 was not associated with an altered dopaminergic periglomerular neuron phenotype in the olfactory bulb. Our findings suggest that, in vivo, microRNA regulation can be mediated through redundant MRE interactions. This work also reveals that directly mutating predicted MREs at the genomic level is necessary to fully characterize the specific phenotypic consequences of miRNA-target regulation. / Graduate

<p>After finishing a human genome reference sequence in 2002, the genomics community has</p><p>turned to the task of interpreting it. A primary focus is to identify and characterize not only</p><p>protein-coding genes, but all functional elements in the genome. The effort has identified</p><p>millions of regulatory elements across species and in hundreds of human cell-types. Nearly</p><p>all identified regulatory elements are found in non-coding DNA, hypothesizing a function</p><p>for previously unannotated sequence. The ability to identify regulatory DNA genome-wide</p><p>provides a new opportunity to understand gene regulation and to ask fundamental questions</p><p>in diverse areas of biology.</p><p>One such area is the aim to understand the molecular basis for phenotypic differences</p><p>between humans and other primates. These phenotypic differences are partially driven</p><p>by mutations in non-coding regulatory DNA that alter gene expression. This hypothesis</p><p>has been supported by differential gene expression analyses in general, but we have not</p><p>yet identified specific regulatory variants responsible for differences in transcription and</p><p>phenotype. I have worked to identify regulatory differences in the same cell-type isolated</p><p>from human, chimpanzee, and macaque. Most regulatory elements were conserved among</p><p>all three species, as expected based on their central role in regulating transcription. How-</p><p>ever, several hundred regulatory elements were gained or lost on the lineages leading to</p><p>modern human and chimpanzee. Species-specific regulatory elements are enriched near</p><p>differentially expressed genes, are positively correlated with increased transcription, show</p><p>evidence of branch-specific positive selection, and overlap with active chromatin marks.</p><p>ivSpecies-specific sequence differences in transcription factor motifs found within this regu-</p><p>latory DNA are linked with species-specific changes in chromatin accessibility. Together,</p><p>these indicate that species-specific regulatory elements contribute to transcriptional and</p><p>phenotypic differences among primate species.</p><p>Another fundamental function of regulatory elements is to define different cell-types in</p><p>multicellular organisms. Regulatory elements recruit transcription factors that modulate</p><p>gene expression distinctly across cell-types. In a study of 112 human cell-types, I classified</p><p>regulatory elements into clusters based on regulatory signal tissue specificity. I then used</p><p>these to uncover distinct associations between regulatory elements and promoters, CpG-</p><p>islands, conserved elements, and transcription factor motif enrichment. Motif analysis</p><p>identified known and novel transcription factor binding motifs in cell-type-specific and</p><p>ubiquitous regulatory elements. I also developed a classifier that accurately predicts cell-</p><p>type lineage based on only 43 regulatory elements and evaluated the tissue of origin for</p><p>cancer cell-types. By correlating regulatory signal and gene expression, I predicted target</p><p>genes for more than 500k regulatory elements. Finally, I introduced a web resource to</p><p>enable researchers to explore these regulatory patterns and better understand how expression</p><p>is modulated within and across human cell-types.</p><p>Regulation of gene expression is fundamental to life. This dissertation uses identified</p><p>regulatory DNA to better understand regulatory systems. In the context of either evolution-</p><p>ary or developmental biology, understanding how differences in regulatory DNA contribute</p><p>to phenotype will be central to completely understanding human biology.</p> / Dissertation

Pluripotent stem cells have the capacity to develop into different cell lineages and can be manipulated into certain cell types through the use of small molecule inducers. Retinoic acid (RA) signaling through retinoic acid receptors (RAR) and retinoid X receptors (RXR) has the ability to direct lineage determination but has yielded disappointing results in promoting skeletal myogenesis in embryonic stem (ES) cells. RXR is crucial in embryonic development although it is generally considered to act as a silent partner for other nuclear receptors such as RAR. Our findings demonstrate that rexinoid specific signaling enhances skeletal myogenesis and requires β-catenin but not RAR. Moreover, RXR signalling in mouse ES cells can efficiently enhance skeletal myogenesis and closely recapitulates sequential events observed in vivo. Since ES cells closely represent the properties of the developing embryo, efficiently generating skeletal muscle provides a means to further scrutinize signaling pathways in myogenic development in view of developing therapies for muscle related diseases.

Pluripotent stem cells have the capacity to develop into different cell lineages and can be manipulated into certain cell types through the use of small molecule inducers. Retinoic acid (RA) signaling through retinoic acid receptors (RAR) and retinoid X receptors (RXR) has the ability to direct lineage determination but has yielded disappointing results in promoting skeletal myogenesis in embryonic stem (ES) cells. RXR is crucial in embryonic development although it is generally considered to act as a silent partner for other nuclear receptors such as RAR. Our findings demonstrate that rexinoid specific signaling enhances skeletal myogenesis and requires β-catenin but not RAR. Moreover, RXR signalling in mouse ES cells can efficiently enhance skeletal myogenesis and closely recapitulates sequential events observed in vivo. Since ES cells closely represent the properties of the developing embryo, efficiently generating skeletal muscle provides a means to further scrutinize signaling pathways in myogenic development in view of developing therapies for muscle related diseases.

<p>Copper (Cu) is a co-factor that is essential for oxidative phosphorylation, protection from oxidative stress, angiogenesis, signaling, iron acquisition, peptide hormone maturation, and a number of other cellular processes. However, excess copper can lead to membrane damage, protein oxidation, and DNA cleavage. To balance the need for copper with the necessity to prevent accumulation to toxic levels, cells have evolved sophisticated mechanisms to regulate copper acquisition, distribution, and storage. The basic components of these regulatory systems are remarkably conserved in most eukaryotes, and this has allowed the use of a variety of model organisms to further our understanding of how Cu is taken into the cell and utilized.</p><p>While the components involved in Cu uptake, distribution, and storage are similar in many eukaryotes, evolution has led to differences in how these processes are regulated. For instance, fungi regulate the components involved in Cu uptake and detoxification primarily at the level of transcription while mammals employ a host of post-translational homeostatic mechanisms. In <italic>Saccharomyces cerevisiae</italic>, transcriptional responses to copper deficiency are mediated by the copper-responsive transcription factor Mac1. Although Mac1 activates the transcription of genes involved in high affinity copper uptake during periods of deficiency, little is known about the mechanisms by which Mac1 senses or responds to reduced copper availability. In the first part of this work, we show that the copper-dependent enzyme Sod1 (Cu,Zn superoxide dismutase) and its intracellular copper chaperone Ccs1 function in the activation of Mac1 in response to an external copper deficiency. Genetic ablation of either <italic>CCS1</italic> or <italic>SOD1</italic> results in a severe defect in the ability of yeast cells to activate the transcription of Mac1 target genes. The catalytic activity of Sod1 is essential for Mac1 activation and promotes a regulated increase in binding of Mac1 to copper response elements in the promoter regions of genomic Mac1 target genes. Although there is precedent for additional roles of Sod1 beyond protection of the cell from oxygen radicals, the involvement of this protein in copper-responsive transcriptional regulation has not previously been observed. </p><p>Higher eukaryotes including mice and humans regulate Cu uptake predominately by means of post-translational control of the localization and stability of the Cu transport proteins. One of these proteins, Ctr1, is the primary means of Cu uptake into the cell, and members of the highly conserved Ctr family of Cu ion channels have been shown to mediate high affinity Cu(I) uptake into cells. In yeast and cultured human cells, Ctr1 functions as a homo-trimer with each monomer harboring an amino-terminal extracellular domain, three membrane spanning domains, a cytoplasmic loop, and a cytoplasmic tail. In addition to the highly conserved Ctr1 Cu ion importer, the baker's yeast <italic>S. cerevisiae</italic> expresses a related protein called Ctr2. Experimental evidence demonstrates that unlike yeast and mammalian Ctr1, yeast Ctr2 is localized to the vacuolar membrane where it mobilizes Cu stores to the cytoplasm under conditions of Cu limitation. </p><p>In mice and humans a gene encoding a protein with significant similarity to the <italic>Ctr</italic> family has been identified, denoted <italic>Ctr2</italic>. Publications from others suggest that mammalian Ctr2 may either be a low affinity Cu importer at the plasma membrane or, similar to yeast Ctr2, may mobilize Cu from intracellular organelles such as the lysosome to the cytosol. In agreement with a previous report we found that a fraction of mouse Ctr2 is localized to the plasma membrane and that its membrane topology is the same as Ctr1. Interestingly, over-expression of Ctr2 by stable transfection results in decreased intracellular bioavailable Cu. To begin to understand the physiological role of Ctr2, mice bearing a systemic deletion of the <italic>Ctr2</italic> gene were generated. The <italic>Ctr2-/-</italic> mice are viable but hyper-accumulate Cu in all tissues analyzed. Moreover, protein levels of the Ctr1 Cu importer are dramatically altered in tissues from the <italic>Ctr2</italic> knock out mice, and over-expression of Ctr2 in cultured mammalian cells enhances processing of the Ctr1 protein into a less active form. Taken together these results suggest that mammalian Ctr2 functions in the cell as a negative regulator of Cu import via Ctr1.</p> / Dissertation

Gene expression is critical for animal development as cells divide and differentiate into multiple cell types. Cell-type specific gene expression is controlled by enhancers, DNA sequences that can direct expression of a target gene from hundreds of kilobases away. Gene promoters contact at least two enhancers on average, and enhancers may also contact each other. A key question is therefore how enhancers operate in this complex regulatory DNA context. It has long been assumed that enhancers act as independent modules based on their ability to drive gene expression when isolated in reporter constructs. To test assumptions of enhancer modularity, I probed interactions between two developmental enhancers from the even-skipped locus in Drosophila melanogaster blastoderm embryos. My results contradict the classic definition of enhancers; I found that the arrangement of enhancers relative to one another and the promoter influences levels of gene expression while not affecting its spatial pattern within the embryo. These results are described in Chapter 2. However, these enhancers are modular in one aspect: when fused directly together, they still direct their distinct spatial expression patterns. In Chapter 3 I describe a collaboration with Md Abul Hassan Samee in Saurabh Sinha's group at the University of Illinois Urbana-Champaign to apply computational sequence-to-expression models to my data. We found that a mechanistic model describing interactions between transcription factors was unable to fit our data well; in contrast, a phenomenological model that finds active sequences fits the data much better. These results indicate that to predict gene expression from sequence we will need to learn how enhancer boundaries are defined. In summary, I present evidence that the organization of enhancers within a locus impacts expression of the target gene. This finding overturns assumptions about enhancer modularity and emphasizes the importance of considering higher level interactions across a locus. Structural variation is common in natural populations, and our results highlight a novel way in which these sequence variants may alter gene expression. To realize the long-standing goal to predict gene expression directly from sequence we must investigate how enhancers interact within a complex locus.

In the AcMNPV genome, ~28% of the genes are arranged divergently on opposite strands with an intergenic region of <1 kbp. In this configuration, a bidirectional promoter generally drives expression of both genes. However, no baculovirus bidirectional promoters have been characterized in any detail. We chose the AcMNPV chiA/v-cath intergenic region to serve as a model to characterize transcriptional regulation of bidirectional gene pairs during AcMNPV infection. We sequentially truncated putative upstream regulatory regions of chiA and v-cath to identify sequences essential for transcriptional initiation. Forty bp of the chiA gene 5’-flanking region was sufficient to support chiA transcription at half the level of the AcΔCC+CC repair virus. Interestingly, v-cath transcription from viruses containing only 40 bp of their upstream 5’-flanking region was found to be higher by 4-fold relative to the level of native expression. Linker-scanning mutagenesis that inserted 5 bp linkers spanning the chiA/v-cath intergenic region identified nucleotides critical for the transcriptional activation of both genes. From this, nucleotides -36 to -45, of the v-cath gene were found to negatively regulate v-cath mRNA expression. Quantitative RT-PCR studies revealed a 2-4 fold higher chiA mRNA expression relative to v-cath possibly explaining why translation of CHIA can be detected 6 hours earlier than V-CATH. This study identifies upstream regions of viral chiA and v-cath required for initiation of transcription and provides the first insight into baculovirus mechanisms for transcriptional regulation of interdependent gene pairs.

INTRODUCTION Estrogen is implicated as an important factor in stimulating breast cancer cell proliferation, and presence of estrogen receptor (ER) is an indication of a good prognosis in breast cancer patients. Mcl-1 is an anti-apoptotic Bcl-2 family member that is often overexpressed in breast tumors, correlating with poor survival. Estrogen has been previously shown to regulate Bcl-2 family members, leading to an evasion of apoptosis, however the role of estrogen in regulating Mcl-1 expression is unclear. I hypothesize that estrogen increases the expression of anti-apoptotic gene Mcl-1 through binding of ERα to a half estrogen response element (ERE) site within the promoter of Mcl-1 gene. This leads to increased Mcl-1 expression in breast cancer cells, ultimately contributing to an evasion of apoptosis. METHODS Four distinct breast cancer cell lines: MCF-7 and ZR-75, which both express ERα, and SKB-BR-3 and MDA-MB-231, which do not express ERα, to investigate the role of estrogen plays in regulating Mcl-1 expression. Cells were grown in serum-starved white media with charcoal-stripped FBS for five days prior to treatment with estrogen. Cells were treated with ERα antagonists Tamoxifen and Fulvestrant in combination with estrogen. Also, siRNA knockdown of ERα was performed and mRNA expression was evaluated. Chromatin immunoprecipitation (ChIP) was used to investigate if ERα binds to a specific ERE halfsite within the Mcl-1 promoter. To further validate this data, a streptavidin pull-down assay was performed using a biotinlabeled probe specific to this region. RESULTS In ERα positive cell lines, estrogen treatment increased Mcl-1 expression at both the protein and mRNA level. In two ERα negative cell lines, SK-BR-3 and MDA-MB-231, estrogen failed to increase in Mcl-1 protein expression. ERα antagonists decreased estrogen mediated Mcl-1 expression at both the protein and message level. Upon knockdown of ERα, Mcl-1 mRNA expression after estrogen treatment was also decreased. ChIP showed an enrichment of ERα to the Mcl-1 promoter at a region 3683 bp upstream of the translation start site containing a half ERE site. Streptavidin-pull down assay showed both ERα and transcription factor Sp1 bind to this region and mutation of the half ERE site eliminated this binding. CONCLUSIONS These results suggest that estrogen is involved in regulating Mcl-1 expression specifically through a mechanism involving ERα. Ultimately, a better understanding of the role of estrogen in regulating Mcl-1 expression will determine whether Mcl-1 is a valid molecular target for breast cancer therapy.