The thymus in mice and humans originates from the third pharyngeal pouch endoderm. This process is divided into early Foxn1-independent stages and later Foxn1-dependent stages. Foxn1 is indispensible for the differentiation of thymic epithelial progenitor cells (TEPCs) as the development of thymus in Foxn1 mutant mice is arrested around E12.5. The transcriptional changes associated with the developmental of the thymus are poorly understood. In particular, the transcriptional regulation of Foxn1 in the developing thymic rudiment has not been definitively identified. Recently, Pax1, Pax9, Tbx1, and E2Fs have been implicated in transcriptional regulation of Foxn1. However, with the exception of E2Fs, evidence regarding their direct involvement in regulating Foxn1 expression is missing. Therefore, the aims of this thesis were to study the transcriptional regulation of Foxn1 through identification of its regulatory regions and studying the transcriptional changes associated with the developing thymus. These aims were addressed through the use of chromatin-immunoprecipitation technique combined with next-generation sequencing and gene expression analyses of the developing TEPCs. The data presented in this thesis identified H3K4me3 and H3K27ac marked Foxn1 promoter and five H3K4me1 and H3K27ac marked putative enhancer regions. The combination of gene expression analyses and transcription factor binding sites within the above regions suggested Ets1, Isl1, Foxc1, Nfia, Nfib, Srf, Foxo1, Nfatc2, Ing4, Foxa2, Hes1, E2Fs, and p53 as candidate transcriptional regulators of Foxn1. Nfatc2 appears also to be a target of Foxn1 that could play an important role in thymus development by regulating a large set of genes. Comparison of wild type and Foxn1 null thymus showed that Foxn1 could act as positive regulator of Pax1 and negative regulator of Gata3 and Eya1, genes important for third pharyngeal pouch development. The comparison of transcriptome of E10.5 and E11.5 third pharyngeal pouch cells and E12.5 TEPCs showed that genes involved in tissue development are downregulated while those involved in antigen presentation, a process important for thymus function, are upregulated during development. These results also demonstrated a decrease in the activity of transcription factor network involving Hox genes and an increase in the activity of a network involving Nfkb, Rela, and Irf genes. Analysis of signalling pathways suggested that the NFκB signalling pathway could be important for thymus development after E12.5 while TGFβ signalling pathway appeared to be detrimental to Foxn1 expression in thymic epithelial cells. Together, I identified several transcription factors that could be involved in transcriptional regulation of Foxn1 in TEPCs, several genes that could be a target of FOXN1, changes in transcription factor network and signalling pathways associated with the developing thymic rudiment.

The present study revealed that FOXO1 is present in the nuclei of non-dividing pituitary cells and in a subset of differentiated cells with highest level of expression in somatotrophs, followed by corticotrophs, thyrotrophs and gonadotrophs throughout development and in adulthood stage. A significant difference in Foxo1 transcript between age-matched males and females at 8-9 weeks of age was demonstrated in the anterior pituitary for the first time. IHC data demonstrating (i) FOXO1 co-localization with p27kip1 (ii) an increase in FOXO1 immunopositive cells within anterior pituitary in p27KO embryos compared to WT (iii) absence of FOXO1 in the nucleus of BrdU positive cells suggested that in absence of p27Kip1 FOXO1 might be important for preventing unbridled cell proliferation. Data suggested that FOXO1 might not be important for initiating pituitary cell differentiation but might be involved with p27kip1 in maintaining pituitary cell quiescence. Increase in nuclear localization of FOXO1 in the pituitary of Foxp3 mutant (lacking insulin signaling) suggested that it might be a down-stream target of insulin/PI3K/PKB pathway in the pituitary as it is in several other tissues.

The transcription factor DEAF-1 is the mammalian homologue of a critical Drosophila developmental gene and is essential for neonatal survival in mice. Haploinsufficiency of Deaf-1 in the testis of adult mice was initially thought to cause loss of spermatogenesis and disrupted morphology of the seminiferous tubules, and this heterozygosity was thought to be sufficient to disrupt epigenetic programming in the developing sperm to produce inheritance of testicular defects in both heterozygous and genotypically normal offspring. Although Deaf-1 knockout mice do display disrupted testis structure, infertility at advanced age, hyperproliferation of early germ cells, and abnormal staging of seminiferous tubules, this phenotype was also observed in normal mouse strains that were born in the SIUC vivarium. Mice ordered from a vendor and raised at SIUC did not show testicular defects. This suggests an environmental factor at the SIUC vivarium may act as an endocrine disruptor during embryonic testicular development. Deaf-1-/- mice die soon after birth, often as the result of exencephaly, a gross neural tube defect (NTD). Unlike many mouse models, exencephalic Deaf-1-/- mice do not display a higher incidence of NTDs in females as compared to their male littermates. DEAF-1 promotes Bax-mediated apoptosis; studies using terminal UTP nick-end labeling (TUNEL) suggest a global increase in apoptosis in both exencephalic and normal Deaf-1-/- fetuses during neurulation as compared to their Deaf-1+/+ littermates. This indicates that Deaf-1 is crucial for correct apoptotic patterning during development, which, in turn, is essential for neural tube closure. Finally, cDNA microarray comparison of e14.5 Deaf-1 knockout and wildtype fetuses reveals expression of translation initiation factor 4g3 (Eif4g3) to be downregulated in Deaf-1-/- fetuses. Electrophoretic mobility shift assay using recombinant DEAF-1, and chromatin immunoprecipitation assay of a human cell line confirmed DEAF-1 could bind the eIF4G3 promoter both in vitro and in vivo. Additionally, transcription of the Deaf-1 Antisense Transcript (Das) was found to be significantly downregulated in both e14.5 fetuses and e18.5 fetal brains from Deaf-1-/- mice, suggesting that either lack of Deaf-1 protein or lack of exons 2 through 5 in Deaf-1 knockout mice causes changes in levels of the noncoding RNA that shares Deaf-1's promoter in the mouse.

Orientador: Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T12:54:32Z (GMT). No. of bitstreams: 1 Monte-Mor_BarbaradaCostaReis_D.pdf: 7171978 bytes, checksum: 5c73cb6c7edd6138186b17d972fce769 (MD5) Previous issue date: 2007 / Resumo: Policitemia vera (PV) é uma síndrome mieloproliferativa (SMP) crônica que ocorre por proliferação clonal de progenitores hematopoéticos multipotenciais. Pacientes com PV apresentam expansão das três principais linhagens mielóides na medula óssea, levando à produção aumentada de hemácias, granulócitos e plaquetas. Características importantes de PV incluem elevação de hematócrito em presença de níveis normais ou diminuídos de eritropoetina (EPO) e a formação de colônias eritróides endógenas (CEE). A mutação JAK2 V617F, presente na maioria dos pacientes com PV, causa ativação constitutiva de JAK2 e parece ser responsável pelo fenótipo observado. Apesar disso, as mudanças transcricionais desencadeadas pela mutação ainda não foram completamente caracterizadas. Neste trabalho, utilizou-se Serial Analysis of Gene Expression (SAGE) para realizar um estudo de expressão gênica global em células da medula óssea de um paciente com PV portador da mutação, ao diagnóstico e em células normais de doadores saudáveis. Genes com expressão aumentada em PV estão envolvidos em processos biológicos importantes, como transdução de sinal, diferenciação e proliferação celular, ciclo celular, apoptose, resposta imune e regulação da transcrição. JUNB foi um dos genes identificados e, usando reação em cadeia da polimerase quantitativa em tempo real (qRT-PCR), detectou-se expressão aumentada de JUNB em dois outros pacientes com PV JAK2 V617F-positivos. Por meio de linhagens murinas Ba/F3-REPO e culturas primárias de eritroblastos humanos, observou-se que JUNB é expresso após adição de EPO e que a proteína JunB é constitutivamente induzida por JAK2 V617F. Além disso, interferência na expressão de JUNB diminuiu o potencial clonogênico e proliferativo de progenitores eritróides humanos Ainda, em PV, a eritropoese causada por JAK2 V617F mostrou-se mais sensível à diminuição de expressão de JunB do que a eritropoese normal. Assim, esses resultados sugerem que JunB tenha um papel fundamental no desenvolvimento de SMPs causadas por JAK2 V617F / Abstract: Polycythemia vera (PV) is a chronic myeloproliferative disorder that arises through clonal proliferation of multipotent hematopoietic progenitors. PV patients present bone marrow trilineage expansion, leading to increased production of mature red cells, granulocytes and platelets. Important PV features are elevated red cell mass, despite normal or subnormal erythropoietin (EPO) levels, and endogenous erythroid colony (EEC) formation. The JAK2 V617F mutation, present in the majority of polycythemia vera (PV) patients, causes a JAK2 constitutive activation and seems to be responsible for the PV phenotype. However, the transcriptional changes triggered by the mutation have not yet been totally characterized. Serial analysis of gene expression (SAGE) was used to perform a large scale gene expression study in bone marrow cells of a newly-diagnosed PV patient harboring the JAK2 V617F mutation and in normal bone marrow cells from healthy donors. Genes overexpressed in PV are involved in important biological processes, such as signal transduction, cellular differentiation, cellular proliferation, cell cycle, apoptosis, immune response and transcriptional regulation. JUNB was one of the genes up-regulated in PV and overexpression of JUNB was also detected by quantitative real time polymerase chain reaction (qRT-PCR) in hematopoietic cells of another two JAK2 V617F PV patients. Using Ba/F3-EPOR cell lines and primary human erythroblast cultures, JUNB was found to be expressed after Epo addition and JunB protein was shown to be constitutively induced by JAK2 V617F. In addition, JUNB knock down reduced the clonogenic and proliferative potential of human erythroid progenitors. Furthermore, in PV, the JAK2 V617F driven erythropoisis was more sensitive to JunB downregulation than normal erythropoiesis. Hence, these results demonstrate that JunB may play a major role in the development of JAK2 V617F myeloproliferative disorders / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

Plants defence responses are often associated with the development of the so-called hypersensitive response (HR), a form of PCD that confines the pathogen to the infection site. The sharp boundary of the HR suggests the existence of efficient mechanisms that control cell death and survival. The Arabidopsis transcription factor AtMYB30 positively regulates plant defence and HR responses by enhancing the synthesis of sphingolipid-containing Very Long Chain Fatty Acids (VLCFA) after bacterial infection. The activity of AtMYB30 is tightly controlled inside plant cells through protein-protein interactions and post-translational modifications. During my PhD, we identified a protease of the subtilase family (AtSBT5.2) as a AtMYB30-interacting partner. Interestingly, we have shown that the AtSBT5.2 transcript is alternatively spliced, leading to the production of two distinct gene products that encode either a secreted [AtSBT5.2(a)] or an intracellular [AtSBT5.2(b)] protein. The specific interaction between AtMYB30 and AtSBT5.2(b), but not AtSBT5.2(a), leads to AtMYB30 specific retention outside of the nucleus in small intracellular vesicles. atsbt5.2 Arabidopsis mutant plants, in which both AtSBT5.2(a) and AtSBT5.2(b) expression was abolished, displayed enhanced HR and defence responses. The fact that this phenotype is abolished in an atmyb30 mutant background suggests that AtSBT5.2 is a negative regulator of AtMYB30-mediated disease resistance. Importantly, overexpression of the AtSBT5.2(b), but not the AtSBT5.2(a), isoform in the atsbt5.2 mutant background reverts the phenotypes displayed by atsbt5.2 mutant plants, suggesting that AtSBT5.2(b) specifically represses AtMYB30-mediated defence.

The vertebrate vasculature is formed by populations of endothelial and mural cells that arrange into functionally and molecularly distinct arterial, venous and capillary beds. Although a number of signalling pathways and transcriptional regulators have been implicated in these processes of vascular differentiation, a clear picture of how arterial-specific gene regulation is achieved is yet to emerge. In this study I have investigated the transcriptional regulation of arterial identity from two different directions: characterisation of enhancers to identify the transcription factors that bind and direct arterial specification; and direct study of the function of one particular transcription factor expressed specifically in the arterial vasculature. I have identified a novel gene enhancer that directs arterial-specific expression of Flk1 (Vegfr2) in transgenic mouse and zebrafish models. Dissection of inputs from individual transcription factor binding sites within this enhancer shows a requirement for Gata factors for enhancer function in endothelial cells, whereas arterial-specification is directed by Rbpj-mediated repression of enhancer activity in veins. This work demonstrates that Flk1 expression in arterial endothelial cells is downstream of the Notch/Rbpj pathway, and also describes a novel transcriptional mechanism of arterial differentiation. In parallel, I have uncovered a novel role for the transcription factor Tbx2 in the regulation of arterial mural cell identity. Histological analysis demonstrates the previously unreported expression of Tbx2 exclusively in mural cells of peripheral arteries and microvessels, and genetic deletion experiments in mice suggest a role for Tbx2 in mural cell recruitment, survival, proliferation, and differentiation upstream of Notch3 and Pdgfrβ. Together, these results contribute valuable insights into our understanding of the establishment of vascular identity by identifying novel transcriptional regulators of arterial fate in both endothelial and mural cells.

The NF-κB family of transcription factors controls a number of essential cellular functions. Pirin is a non-heme iron (Fe) redox specific co-regulator of NF-κB (p65) and has been shown to modulate the affinity between the homodimeric p65 and the DNA. The allosteric effect of the active Fe(III) form of Pirin on the DNA is not known and has not been investigated. We carry out multiple microsecond-long molecular dynamics simulations of the free DNA, p65-DNA complex, and the Pirin-p65-DNA supramolecular complexes in explicit water. We show that, unlike the Fe(II) form of Pirin, the Fe(III) form in the Pirin-p65-DNA supramolecular complex enhances the interactions and affinity between p65 and the DNA, in agreement with experiments. The results further provide atomistic details of the effect of the Fe(III) form of Pirin on the DNA upon binding to p65 to form the supramolecular complex.

The healthy development of a living cell requires precise spatial-temporal gene expression. The code that dictates when and where genes are expressed is stored in a pattern of specific sequence motifs, which can be recognised by transcription factors. Understanding the interaction between these DNA sequence motifs and transcription factors will help to elucidate how genomic sequences build transcriptional control networks. However, the DNA-binding specificities of ~1400 human transcription factors are largely unknown. The in vivo DNA-binding events of transcription factors involve great subtlety, because most transcription factors recognise degenerate sequence motifs and related transcription factors often prefer similar or even identical sequences. Forkhead transcription factors exemplify these challenges. To understand how members within the Forkhead transcription factor family gain their binding and functional specificities, we used chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) to interrogate the genome-wide chromatin binding events of three Forkhead transcription factors: FOXM1, FOXO3 and FOXK2. We find that FOXM1 specifically binds to the promoters of a large array of genes whose activities peak at the G2 and M phases of the cell cycle. The canonical Forkhead consensus GTAAACA is not enriched within the FOXM1 cistrome. It gains its own specific binding events and biological functions by interacting and cooperating with the MMB complex. FOXO3 and FOXK2 are recruited to chromatin by the canonical Forkhead consensus GTAAACA, and they bind both shared and specific regions in the genome. FOXO3 mostly binds to the regions which are also bound by FOXK2, but no competitive or assisted binding between FOXO3 and FOXK2 is detected within those regions. Overall, these results help explain how individual members of the Forkhead transcription factor family gain binding specificity within the genome yet raises new questions of how functional specificity is achieved by other family members.

Genome-wide binding and expression studies in Drosophila melanogaster have revealed widespread roles for Dichaete and SoxNeuro, two group B Sox proteins, during fly development. Although they have distinct target genes, these two transcription factors bind in very similar patterns across the genome and can partially compensate for each other's loss, both phenotypically and at the level of DNA binding. However, the inherent noise in genome-wide binding studies as well as the high affinity of transcription factors for DNA and the potential for non-specific binding makes it difficult to identify true functional binding events. Additionally, externalfactors such as chromatin accessibility are known to play a role in determining binding patterns in Drosophila. A comparative approach to transcription factor binding facilitates the use of evolutionary conservation to identify functional features of binding patterns. In order to discover highly conserved features of group B Sox binding, I performed DamID-seq for SoxNeuro and Dichaete in four species of Drosophila, D. melanogaster, D. simulans, D. yakuba and D. pseudoobscura. I also performed FAIRE-seq in D. pseudoobscura embryos to compare the chromatin accessibility landscape between two fly species and to examine the relationship between open chromatin and group B Sox binding. I found that, although the sequences, expression patterns and overall transcriptional regulatory targets of Dichaete and SoxNeuro are highly conserved across the drosophilids, both binding site turnover and rates of quantitative binding divergence between species increase with phylogenetic distance. Elevated rates of binding conservation can be found at bound genomic intervals overlapping functional sites, including known enhancers, direct targets of Dichaete and SoxNeuro, and core binding intervals identified in previous genome-wide studies. Sox motifs identified in intervals that show binding conservation are also more highly conserved than those in intervals that are only bound in one species. Notably, regions that are bound in common by SoxNeuro and Dichaete are more likely to be conserved between species than those bound by one protein alone. However, by examining binding intervals that are uniquely bound by one protein and conserved, I was able to identify distinctive features of the targets of each transcription factor that point to unique aspects of their functions. My comparative analysis of group B Sox binding suggests that sites that are commonly bound by Dichaete and SoxNeuro, primarily at targets in the developing nervous system, are highlyconstrained by natural selection. Uniquely bound targets have different tissue expression profiles, leading me to propose a model whereby the unique functions of Dichaete and SoxNeuro may arise from a combination of differences in their own expression patterns and the broader nuclear environment, including tissue-specific cofactors and patterns of accessible chromatin. These results shed light on the evolutionary forces that have maintained conservation of the complex functional relationships between group B Sox proteins from insects to mammals.

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.