Dissertations / Theses on the topic 'Molecular biology, Hematopoiesis, Gene regulation'

To identify new genes functionally involved in erythropoiesis during development and maturation, we analysed by DNA microarray three cell populations of different maturity during mouse fetal liver development (E11.5 - E13.5). Among genes whose expression increases in parallel with erythroid maturation there is Sox6, a member of the Sry–related transcription factors family. Sox6 is known to play a major role in erythropoiesis: its ablation in mouse causes a strong relative increase of the expression of embryonic (εy) versus other non-alpha globin genes in late gestation, and a high number of circulating nucleated and misshapen erythrocytes. To study the role of Sox6 in human erythropoiesis, we overexpressed it by lentiviral vector transduction both in the K562 erythroleukemic cell line and in human primary CD34+ cord blood cells . Sox6 induces significant differentiation in both models as shown by morphological and FACS analysis. Moreover, several erythroid specific transcripts are greatly increased , i.e. mRNAs for enzymes controlling the heme-biosynthetic pathway, for transcription factors and for all globins (although the ratio between epsilon and gamma-globin is decreased), suggesting that Sox6 is a general positive regulator of erythroid genes expression. Despite their erithroleukemic origin, K562 overexpressing Sox6 grow at a very low rate when compared with control cells, and die in culture within about ten days after transduction. Sox6 overexpression causes in fact a strong increase of SOCS3 (suppressor of cytokine-signaling) transcript, which is known to block Jak2 signalling. Since erythroblasts undergo cell-cycle withdrawal during terminal differentiation, our working hypothesis is that Sox6 may induce the erythroid terminal maturation program through a block in the cell-cycle progression, possibly by acting as a repressor of the Jak signalling pathway. We then used the Sox6 consensus from the εy-globin promoter to perform a bioinformatic genome-wide search for similar evolutionarily conserved motifs, and we found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in-vitro and in-vivo, and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells. Moreover, the binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by a strong downregulation of the endogenous Sox6 transcript. Finally, we demonstrated that Sox6 expression, in human erythroid cultures and in mouse bone marrow cell populations, peaks at the erythroblast stage and decreases along with erythroid differentiation. Together these observations suggest that the negative Sox6 autoregulation mediated by the double Sox6 binding site within its own promoter, might be relevant to control the Sox6 transcriptional downregulation observed in late erythroid maturation

The development of the hematopoietic system occurs in two waves: a first wave of primitive erythropoiesis, which consists in the production of a single lineage, primitive erythrocytes, and a second wave of definitive hematopoiesis, which describes the generation of many specialized blood cell types from common hematopoietic stem cells. Whereas definitive hematopoiesis is fairly well understood, involves signals from the environment and the expression of lineage-specific transcription factors, the molecular mechanisms regulating primitive erythropoiesis remain to be defined. The aim of this thesis was to clarify the roles of the Stem Cell Leukemia (SCL) gene and Vascular Endothelial Growth Factor (VEGF) during primitive and definitive hematopoiesis. Although gene targeting experiments indicate essential roles for VEGF/Flk-1 signaling and SCL at the onset of hematopoiesis, their exact functions remain elusive. This work has revealed that different thresholds of VEGF are required for the migration of hematopoietic precursors from mesoderm to sites of hematopoiesis and for their subsequent expansion. Furthermore, it shows that SCL, a basic helix-loop-helix transcription factor, acts downstream of VEGF signaling to ensure the survival of primitive erythrocytes. During definitive hematopoiesis, conditional knock-out experiments establish a non-redundant role for SCL during erythroid and megakaryocytic differentiation. Yet, it remains unclear whether SCL is essential for commitment to these lineages. Results presented in this thesis suggest that SCL is not involved in commitment to these pathways, but rather acts to consolidate and expand the erythroid and megakaryocytic compartments, following lineage choice. Finally, despite the central role for SCL during hematopoietic development, the mechanisms regulating its tissue specific expression remain unknown. This work provides molecular and functional evidence that demonstrate that the homeodomain-
Taken together, this work has elucidated molecular mechanisms which underlie cell fate decisions. It describes how the activity of a master regulator of erythroid differentiation, SCL, is regulated both by signals from the environment and at the transcriptional level, through combinatorial interactions between lineage-specific transcription factors.

Solute carrier family 11 member 1 protein (SLC11A1), also known as natural resistance-associated macrophage protein 1 (NRAMP1), plays an important role in the host immune defense and inflammatory response. It is a highly conserved transmembrane protein which transports divalent metal cations in a proton-dependent manner. It regulates iron homeostasis in macrophages and exerts pleiotropic effects on macrophage activation. In mice, natural resistance or susceptibility to a range of intracellular pathogens is controlled by the Slc11a1 gene. In human, genetic polymorphisms of the SLC11A1 gene have been shown to be associated with a susceptibility to a variety of infectious and autoimmune diseases. The expression of the SLC11A1 gene is strictly regulated during myeloid differentiation. The human promyelocytic leukemia cell lines such as HL-60 and U937 cells are useful models to study the regulation of SLC11A1 gene expression during experimentally induced granulocytic, monocytic, or macrophage-like differentiation. Herein, we demonstrated that during the PMA-induced differentiation of HL-60 cells and human monocytes toward macrophages, β-actin translocates from the cytoplasm to the nucleus where it is associated with RNA polymerase II and binds to the promoter of the SLC11A1 gene. β-actin knockdown inhibits the SLC11A1 promoter-driven transcription, and neutralization of nuclear actin by in vivo microinjection of antibodies against β-actin into nuclei significantly blocks the expression of SLC11A1 mRNA. Further studies revealed that an AP-1-like element present in the proximal region of the SLC11A1 gene promoter is essential for PMA-induced transcriptional activation of this gene. β-actin, as a subunit of the SWI/SNF complex, and another subunit BRG1 are associated with the transcription factor ATF-3 and are recruited to the AP-1 like element in an ATF-3-dependant manner . ATF-3 cooperates with BRG1 and β-actin to activate the SLC11A1 promoter. Furthermore, a proximal (GT/AC)n repeat (t(gt)5ac(gt)5ac(gt)9g) region adjacent to the AP-1-like element is converted into a Z-DNA structure in response to PMA treatment, and BRG1 is involved in this process. Our results suggest that recruitment of the SWI/SNF complex initiates Z-DNA formation and subsequently helps to transactivate the SLC11A1 gene. Previous studies have shown that SLC11A1 is extensively glycosylated and phosphorylated, and is localized at the membrane of late endosomes/lysosomes in macrophages. The present study revealed that SLC11A1 is tyrosine-phosphorylated during the differentiation of U937 cells into macrophages induced by PMA. Using the kinase inhibitor PP2 and RNA interference experiments, we demonstrated that Src family kinases including c-Src are required for the tyrosine phosphorylation of SLC11A1 protein. In vitro phosphorylation assays showed that SLC11A1 is a direct substrate for active c-Src kinase. Furthermore, tyrosine 15 is identified as the tyrosine phosphorylation site by Src family kinases and phosphorylation of tyrosine 15 modulates SLC11A1-mediated nitric oxide production. We also showed that the Src family kinases including c-Src are also involved in lysosomal targeting of SLC11A1. These results suggest an important role of Src family kinases in subcellular localization and function of SLC11A1 in macrophages. Overall, our studies contributed important information on the regulation of expression of SLC11A1 in macrophages and its role in regulation of macrophage functions.
La protéine « Solute carrier family 11 member 1 » (SLC11A1), aussi connue sous le nom « natural resistance associated macrophage protein 1 » (NRAMP1), jour un rôle important dans la défense immunitaire et réponse inflammatoire de l'hôte. C'est une protéine transmembranaire hautement conservée qui transporte des cations divalents métalliques d'une manière dépendent des protons. Elle régularise l'homéostasie du fer dans les macrophages et a des effets pléiotropiques sur l'activation de ces cellules. Chez les souris, le gène Slc11a1 contrôle la resistance naturelle ou la susceptibilité aux pathogènes intracellulaires. Chez les humains, les polymorphismes génétiques de SLC11A1 sont associés à une susceptibilité à une variété de maladies infectieuses ou auto-immunitaires. L'expression du gène SLC11A1 est strictement régularisée pendant la différenciation myéloïde. Les lignées cellulaires humaines dérivées de la leucémie aiguë promyélocytaire, telles que la HL-60 et l'U937, sont des modèles utiles pour étudier le contrôle de l'expression du gène SLC11A1 pendant la différenciation de type granulocytaire, monocytaire ou de macrophage induite expérimentalement. Ici, nous avons démontré que durant la différenciation induite par PMA des cellules HL-60 et des monocytes humains aux macrophages, β-actine passe du cytoplasme au noyau où il s'associe avec l'ARN polymérase II et se fixe sure le promoteur du gène SLC11A1. Le « knock-down » de la β-actine inhibe la transcription menée par le promoteur de gène SLC11A1. Dans le noyau, l'expression de l'ARN de SLC11A1 est bloqué significativement en neutralisant l'actine par la microinjection in vivo des anticorps contre β-actine. Autres études ont démontré qu'un élément « semblable à AP-1 » est présent dans la région proximale du promoteur de SLC11A1 et celui-ci est essentiel pour l'activation transcriptionnelle de ce gène induite par PMA. β-actine, étant une sous-unité du complexe SWI/SNF, et la sous-unité BRG1 sont associés avec le facteur de transcription ATF-3. Ensemble, elles sont recrutées à l'élément semblable à AP-1 en une manière qui dépendante sur ATF-3. ATF-3 coopère avec BRG1 et β-actine pour activer le promoteur de SLC11A1. De plus, la région du répète proximale (GT/AC)n [t(gt)5ac(gt)5ac(gt)9g] adjacent au élément semblable à AP-1 est convertit en une structure de Z-ADN en réponse au traitement de PMA, un processus dans lequel BRG1 est impliqué. Nos résultats suggèrent que le recrutement du complexe SWI/SNF amorce la formation de Z-ADN et aide à transactiver le gène SLC11A1. Des études précédentes ont démontrés que SLC11A1 est extensivement glycosylée et phosphorylée, et que cette protéine se trouve chez les macrophages dans les membranes des endosomes tardifs ou des lysosomes. L'étude présentée ici a révélé que SLC11A1 est phosphorylée sur les tyrosines pendant la différenciation des cellules U937 aux macrophages par PMA. En utilisant l'inhibiteur de kinase PP2 et des essais d'interférence d'ARN, nous avons démontré que les kinases de la famille Src, incluant c-Src, sont requises pour la phosphorylation de tyrosine de la protéine SLC11A1. Les essais in vitro de phosphorylation ont montré que SCL11A1 est un substrat direct pour la kinase active c-Src. De plus, la tyrosine 15 a été identifiée comme étant le site de phosphorylation de kinases de la famille Src. La phosphorylation de tyrosine 15 fait moduler la production de l'oxyde nitrique de laquelle SLC11A1 fait parti. Nous avons aussi montré que les kinases de la famille Src ont un rôle important dans la localisation subcellulaire et dans le fonctionnement de SLC11A1 dans les macrophages. Globalement, nos études ont contribué d'information importante sur la régularisation de l'expression de SCL11A1 dans les macrophages et son rôle dans le fonctionnement des macrophages.

The gene encoding the glycolytic enzyme phosphoglycerate kinase is transcriptionally regulated at two levels. Expression of enzyme is related to the glycolytic activity of the cell, and is highest in transcriptionally active cells. Expression is also regulated by X chromosome inactivation, as the somatically expressed Pgk-1 gene is X-linked. The role of 5$\sp\prime$-flanking cis-acting DNA elements and trans-acting factors in the regulation of Pgk-1 expression was examined. The murine Pgk-1 gene contains an upstream activator sequence (UAS) in its 5$\sp\prime$-flanking region. This region was found to be responsible for elevating transcription levels at least ten-fold above basal Pgk-1 promoter levels in P19 embryonal carcinoma (EC) cells. Part of this activity was attributed to the R2 protein binding site, first identified by DNase 1 footprinting techniques. Mutation of the middle region of R2 resulted in a 5-fold reduction in expression of a Pgk-1 driven construct in stable transfection experiments into P19 cells. It was also determined that the R2 site was not important for transcription in P19 cells induced to differentiate with retinoic acid (RA). In undifferentiated P19 cells, another UAS protein binding site, R1, was identified by band shift analysis. R1 could not be detected by footprint analysis, suggesting that the affinity of binding at R1 was lower than at R2. The mutation of the R2 site did not abolish protein binding, which led to the hypothesis that multiple factors were binding the DNA at R2. R1 was also hypothesized to interact with multiple factors. However, fractionation of the P19 nuclear extract and use in band shift studies against the R1 DNA resulted in a single fraction with binding activity, suggesting a single R1 DNA binding protein and a non-DNA binding component. This non-DNA binding component at R1 was found to be tissue- or species-specific. Southwestern analysis in conjunction with fractionation experiments suggested that one of the R2 DNA binding proteins was approximately 70 kD and that the R1 DNA binding protein was 120 kD. Treatment of the P19 cells with RA led to a reduction in gene expression. Two days after exposure to the drug, the contribution to expression from the UAS was reduced by 50%, and four days after exposure the UAS no longer contributed to gene expression. Protein binding to the UAS was also altered after RA-treatment. A new site of protein interaction was detected in the distal region of the UAS, at R3, and binding at R1 was altered. There was, therefore, a correlation between protein interaction within the UAS and gene expression during differentiation. The results presented in this thesis demonstrate that the regulation of PGK-1 occurs, at least in part, at the level of gene expression, and that the UAS has an important role to play in regulating expression levels of the gene during differentiation. The results also suggest that the transcriptional stimulatory activity of the UAS depends on higher-order interactions between multiple low affinity DNA binding proteins which change upon differentiation.

The beta3-adrenergic receptor (beta3-AR) represents a very attractive target for the development of anti-obesity drugs. Selective agonists have been developed based on the rodent beta3-AR and successfully used to treat obesity and Type 2 diabetes in these animals. In humans, however, these particular compounds appear to be less effective and not as specific for the beta3-AR as they are in rodents. An additional problem is the low level of expression of these receptors in human adipose, which may ultimately limit the clinical use of stronger more specific agonists. The objective of this research was to examine the regulation of the beta3-AR gene expression. In order to characterize functionally the cis-acting elements necessary for 3-AR proximal promoter activity, a series of luciferase reporter constructs containing various portions of the mouse 5'-flanking region of the gene, spanning from -1.4 kb to -0.126 kb relative to the translation start site (since there is no unique transcription start site), were generated and their transcriptional activity was evaluated by transient transfection experiments. We identified a 13 by element located between -0.208 kb to -0.196 kb that is essential for basal and constitutive proximal promoter activity. The beta3-AR mRNA has a rapid turnover and is readily expressed in the absence of apparent stimuli, in cells cultured in medium with charcoal-stripped serum. Out of several possible regulators we focused on glucocorticoids and thiazolidinediones, the former because it was a potentially important negative regulator, the latter because it could possibly stimulate the expression. In HIB-1B brown adipocytes, glucocorticoids appear to have two opposing effects on beta3-AR gene expression: they rapidly and directly inhibit transcription but also induce a rapidly turned-over protein that stimulates transcription of the gene. The level of beta3-AR expression in response to glucocorticoids may ultimately depend on the relative magnitu

The signaling pathways that regulate the fate of cells located in the anterior lateral (AL) region of the vertebrate embryo are not well understood. Mesodermal cells in this region will assume the heart muscle phenotype while adjacent endoderm gives rise to foregut tissues such as the liver. The AL endoderm supplies key signaling molecules to promote the survival and differentiation of the precardiac mesoderm. These AL endoderm factors are know to up-regulate transcription factors, such as Nkx2--5, that regulate cardiac genes. However, little is known about how the AL endoderm is patterned and the exact mechanism by which the cardiac transcription factors function within the mesoderm. Therefore, two projects were pursued to understand the developmental pathways that promote early heart development. One project looks at defining the mechanism by which the Nkx2 homeobox genes regulate cardiac gene expression in mouse embryonic stem cells. Unfortunately, this project was plagued with difficulties. Mouse ES cells were used as a model system to study cardiac differentiation. However, these cells were found to contain a potent genome protection mechanism that prevented the stable integration of transcription factors. This phenomenon is addressed and discussed within the thesis. The second project defines the role of retinoids in patterning the AL endoderm. In this study, the homeobox gene cHex, a gene required for hepatocyte development, was used as an AL endoderm marker. It was found that retinoids act directly on the cHex gene promoter to reduce its activity and restrict its expression domain to the AL endoderm.

Atrial natriuretic factor (ANF), a 28 amino acid peptide hormone, is the major secretory product of the heart. Because of its diuretic, natriuretic and vasodilating activities, this peptide may be involved in the maintenance of proper fluid and electrolyte balance and blood pressure control. In order to study the transcriptional regulation of ANF, we have isolated the rat ANF gene and we have established a system of cardiocytes in primary cell culture for studies on the hormonal, tissue-specific and developmental regulation of the ANF gene. Using this in vitro system, as have demonstrated that thyroid hormone increases ANF mRNA levels about 2- to 4-fold in atrial and ventricular cells in primary cardiocyte cell cultures, respectively. Similarly, glucocorticoids augment by about 3-fold both atrial and ventricular ANF mRNA levels in cardiac cells in culture. Glucocorticoids exert this effect at the transcriptional level probably via the binding of glucocorticoid receptor to a DNA element in the distal 5$ sp prime$-flanking sequences of the gene as suggested by DNA-mediated transfection studies in cardiocyte cultures. In order to better understand the mechanisms governing the cardiac-specific as well as developmental expression of the ANF gene, we have analyzed ANF promoter sequences by transient transfection studies in primary cardiocyte cultures. Our data show that the ANF promoter is active only in cells of cardiac origin. Moreover, up to $-$1.6 kb of 5$ sp prime$ upstream sequences are necessary for full expression of the ANF gene in cardiac cells. Within these sequences, two particular elements, a proximal and a distal, are necessary for full ANF transcriptional activity. The proximal element can confer cardiac specificity to an otherwise non tissue-specific heterologous promoter. Further upstream sequences, between $-$2.5 and $-$1.6 kb appear to be implicated in the developmental control of ANF gene expression, as assessed by differential activity in 1 and 4 d

USP4 is a deubiquitinating enzyme whose levels have been shown to be elevated in certain human lung tumors. USP4 is thought to possess oncogenic properties due to its ability to promote tumors in nude mice assays. The lack of an overall effect on ubiquitin levels in overexpression studies has led to the hypothesis that USP4 may act on a few select substrates to edit their ubiquitination status. Although the structure/function relationship is more documented, the physiological substrates and role in vivo are not. In order to elucidate the mechanism by which USP4 could potentially exert its tumorigenic effect, an RNA knockdown approach was undertaken. The effect of changes in USP4 levels was investigated to determine if USP4 plays a role in transcription or mRNA stability. The data suggest that USP4 does not affect levels of mRNAs containing an ARE sequence in NIH-3T3 or Cos-7 cells. Although USP4 was not shown to have an mRNA stabilizing effect, USP4 was found to bind CBP in vivo using an immunoprecipitation experiment and to exert an effect on basal transcription levels. This data suggests that USP4 levels may affect global transcription, perhaps through binding with the transcriptional co-activator CBP.

In order to characterize cis-acting sequences and trans-acting factors important in regulating the expression of the mouse mdr1 gene, in vitro DNAsel footprinting experiments were carried out on a mdr1 promoter segment between positions $-$245 and +84, using nuclear protein extracts prepared from cell lines expressing different endogenous amounts of mdr1 mRNAs. Three footprinted sequences were detected on the non-coding strand of the $-$245 to +84 mdr1 promoter fragment (between -77 to -56, between -46 to -24, and between +5 and +15) with nuclear extracts from mdr1 expressing cells (CMT-93, LTA, and Y-1 cells). In addition, a specific footprinted sequence ($-$14 to +5) was detected on both strands only with nuclear extracts from the mdr1 non-expressing cell line (RAG cells) suggesting the presence and binding of a putative negative regulatory factor in these cells. However, replacement of this sequence in the mdr1 basal promoter ($-$93 to +84) by a heterologous, although similar positioned SV40 sequence did not restore promoter activity in RAG cells. The basal mdr1 promoter was further characterized in bidirectional deletion mutants, in order to identify cis-acting elements important for general transcriptional regulation. These studies further localized the mdr1 basal promoter between positions $-$74 and +84, and also suggested the presence of possible positive and negative cis-acting sequence elements modulating the activity of this basal promoter.

The calcium-sensing receptor (CaSR), expressed in parathyroid, thyroid and kidney, is essential for maintenance of calcium homeostasis. Extracellular calcium (Ca2+o) affects several hepatic functions including bile secretion, metabolic activity, regeneration, and the response to xenobiotics. We have demonstrated the presence, in hepatocytes, of a functional CaSR. Western blot analysis using a specific CaSR antibody showed staining in both whole liver and hepatocyte extracts. Immunohistochemistry and in situ hybridization of rat liver sections showed expression of CaSR protein and mRNA by a subset of hepatocytes. CaSR agonists, gadolinium (Gd3+; 0.5-3.0 mM) and spermine (1.25-20 mM), in the absence of Ca2+o, elicited dose-related increases in intracellular calcium (Ca2+i) in isolated rat hepatocytes loaded with Fura 2-AM and this response was abrogated when IP3-sensitive calcium pools had been depleted by pretreatment with either thapsigargin or phenylephrine. Addition of the deschloro-phenylalkylamine compound, NPS-R467, but not the S enantiomer, NPS-S467, increased the sensitivity of the Ca 2+i mobilization response to 1.25 mM spermine. Bile flow ceased after Ca2+o withdrawal and its recovery was enhanced by spermine in isolated perfused liver preparations. Ca2+ and Gd3+ increased bile flow and NPS-R467 but not the S compound enhanced the response to a sub maximal Ca2+ concentration. Thus, the data demonstrate that rat hepatocytes harbor a CaSR capable of mobilizing Ca2+i from IP3-sensitive stores and that activation of the CaSR stimulates bile flow.
The human CASR gene contains at least 7 exons and spans more than 100 kilobases. Little is known about the 5' flanking region and transcriptional regulatory sequences that control expression of the CASR in specific cells. The human CASR gene has two promoters (P1 and P2) yielding alternative transcripts containing either exon 1A or exon 1B 5'-untranslated region sequences that splice to exon 2 some 242 bp before the ATG translation start site. We have cloned the CASR promoter and transcriptional start sites were identified in parathyroid gland and in human thyroid C-cell (TT) cells; that for promoter P1 lies 27-bp downstream of a TATA box, whereas that for promoter P2, which lacks a TATA box, lies in a GC-rich region.

The beta-amyloid precursor protein (APP) is an extensively processed membrane protein with a single membrane spanning domain. One of the natural proteolytic products of APP is a peptide of 39 to 43 amino acids in length, beta-amyloid, that has been identified as a major constituent of senile plaques, a neuropathological hallmark of Alzheimer disease (AD). In Down syndrome (DS) (trisomy 21), overexpression of the APP gene, located on chromosome 21, is thought to be responsible for the early formation of senile plaques in the brain of DS patients. We investigated the transcriptional regulation of the APP gene in order to identify the cis-elements located in its promoter and the trans-acting factors recognizing these elements. Identification of the factors regulating APP gene expression, could help define conditions leading to the development of AD. The APP promoter possesses multiple transcriptional start sites, has a high percentage of GC and lacks a consensus TATA box. We have characterized, using electrophoretic mobility shift analysis (EMSA), DNase I footprinting, site-directed mutagenesis and transient transfection studies, three elements located in close proximity of the major transcriptional start sites of APP. We present evidence that two of the elements are recognized by the ubiquitous transcription factors Sp1 and USF respectively. Both the Sp1 and USF elements are essential for full activity of the APP promoter, regulate the APP promoter in an additive manner and synergize with the factor(s) recognizing a GC-rich element located upstream in transient transfection assays in NG108-15 and HepG2 cell lines. We also show that treatment by cyclic AMP of NG108-15 cells which had been transiently transfected with reporter constructs driven by different portions of the APP promoter caused an increase in reporter gene activity mediated by sequences located between --2991 to --488 and --303 to --204, even though the upstream sequences of APP do not contain a canoni

Prolactinomas account for 40% of all tumors in the pituitary, an important gland of the endocrine system, and secrete excess amount of prolactin hormone. We recently identified a member of the TGF-beta superfamily of growth factors, activin, as an important regulator of prolactin expression and cell growth. Activin inhibits prolactin expression through inhibition of expression of a pituitary-specific transcription factor, named Pit-1. Pit-1 plays major roles in the development and maintenance of the anterior pituitary gland integrity. In this study, I investigated the intracellular signaling pathways and transcriptional mechanisms by which activin controls Pit-1 activity. I demonstrate that in addition to the down-regulation of mRNA and proteins levels of Pit-1, a functional p38 MAPK signaling pathway is required for activin to down-regulate human Pit-1 gene promoter activity in a Smad-independent manner. This study allows a better understanding of the regulatory effects of activin in the pituitary gland.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Chromatin regulation is a key mechanism for controlling gene expression patterns during development and differentiation. The histone H2A variant H2A.Z is highly conserved among eukaryotes and is of particular interest because it has an essential, yet unknown role in early development. H2A.Z is enriched at the promoter regions of most genes that harbor H3K4me3 in mouse embryonic stem cells (mESCs) including both active and silent, poised genes, marked additionally by polycomb-mediated H3K27me3 and compromising a large cohort of developmental regulators. How H2A.Z mediates these contrasting gene expression states is not known. H2A.Z displays homology to canonical H2A throughout the histone fold domain, however considerable divergence exists outside of this domain, suggesting specialized functions. Here we developed a quantitative chromatin immunoprecipitation followed by mass spectrometry approach to identify downstream effectors of H2A.Z. We identified BET (bromodomain and extraterminal) transcriptional regulator proteins including Brd2 as highly enriched in H2A.Z chromatin. We demonstrate by ChIP-seq that Brd2 significantly overlap H2A.Z at the promoter region of active genes. Conversely, PRC1 -dependent H2A.Z ubiquitination prevents Brd2 occupancy at poised, bivalent genes. Loss of H2A.Z ubiquitination of by mutation of Cterminal lysines results in a Brd2 recruitment and de-repression of bivalent genes. Moreover, inhibition of Brd2 by small molecule inhibition or siRNA-mediated depletion restores repression and leads to a recruitment of PRC2. In contrast, siRNA inhibition of another BET family member Brd4, does not restore repression suggesting that Brd2 and Brd4 play distinct roles in ESCs. This thesis provides novel insights into how H2A.Z acts as a molecular rheostat to regulate the balance between active and silent genes in ESCs, and more broadly a model for its role in responsive systems including development and cancer.
by Paul A Fields.
Ph. D.

In this thesis I have studied the RNA molecule and its function and characteristics in the regulation of gene expression. I have focused on two events that are important for the regulation of the transcriptome: Translational regulation through micro RNAs; and RNA editing through adenosine deaminations.

Micro RNAs (miRNAs) are ~22 nucleotides long RNA molecules that by semi complementarity bind to untranslated regions of a target messenger RNA (mRNA). The interaction manifests through an RNA/protein complex and act mainly by repressing translation of the target mRNA. I have shown that a pre-cursor miRNA molecule have significantly different information content of sequential composition of the two arms of the pre-cursor hairpin. I have also shown that sequential composition differs between species.

Selective adenosine to inosine (A-to-I) RNA editing is a post-transcriptional process whereby highly specific adenosines in a (pre-)messenger transcript are deaminated to inosines. The deamination is carried out by the ADAR family of proteins and require a specific sequential and structural landscape for target recognition. Only a handful of messenger substrates have been found to be site selectively edited in mammals. Still, most of these editing events have an impact on neurotransmission in the brain.

In order to find novel substrates for A-to-I editing, an experimental setup was made to extract RNA targets of the ADAR2 enzyme. In concert with this experimental approach, I have constructed a computational screen to predict specific positions prone to A-to-I editing.

Further, I have analyzed editing in the mouse brain at four different developmental stages by 454 amplicon sequencing. With high resolution, I present data supporting a general developmental regulation of A-to-I editing. I also present data of coupled editing events on single RNA transcripts suggesting an A-to-I editing mechanism that involve ADAR dimers to act in concert. A different editing pattern is seen for the serotonin receptor 5-ht2c.

A hallmark of DNA methylation is the fact that 60 to 80% of CpG dinucleotide sequences in the vertebrate genome are methylated at the 5th-position of cytosine while the remaining unmethylated sequences are nonrandomly distributed throughout the genome generating a pattern of methylation that is both tissue and gene specific. Several lines of evidence suggest that methylation patterns correlate with the expression level of eukaryotic genes and that DNA methylation plays an important role in regulating the state of differentiation of mammalian cells. The methylation of DNA is catalyzed by the DNA methyltransferase (DNA MeTase) enzyme, which transfers a methyl group from S-adenosylmethionine to DNA. Cells that exhibit different DNA methylation patterns express similar mRNA levels and DNA MeTase activities. It has therefore been suggested that patterns of methylations are the result of an interplay between the level of the nonspecific DNA MeTase enzyme and other site- or tissue-specific nuclear factors; changing either one of these parameters will result in a change in DNA methylation patterns. In accordance with this hypothesis it has been proposed that regulation of DNA methyltransferase gene expression plays a role in the maintenance and generation of DNA methylation patterns, as is the case in prokaryotic systems. Bestor et al., cloned the cDNA encoding the mouse DNA MeTase gene but nothing was known about the elements regulating its expression. To further understand regulation of the mouse DNA MeTase gene expression I cloned and sequenced the 5$ sp prime$-upstream region of the gene and demonstrated that: (A) The mouse DNA MeTase promoter is a unique housekeeping promoter lacking the classical binding sites for known transcription factors. (B) The mouse DNA MeTase is induced by the Ras-AP-1 pathway. (C) Induction of the mouse DNA MeTase by the Ras-AP-1 leads to profound changes in cell morphology, methylation patterns and tumorigenicity all of which can be inhib

The promoter region of the rat gene encoding the enzyme carbamyl phosphate synthetase I (CPS) has been cloned and characterized. The start of transcription was positioned to 138-140 nucleotides upstream of the translation initiation codon. From transient transfection analyses, the functional promoter region of the CPS gene extends 5$ sp prime$ to about 525 nucleotides of the start site of transcription. This promoter region contains six footprinted regions when analyzed in liver nuclear extracts. All sites can be recognized by endogenous C/EBP from liver nuclear extracts while the endogenous DBP binds to all sites except site II. Either C/EBP or DBP can trans-activate the CPS promoter in Hep G2 cells. Sites I, III and IV seem to be significantly protected only in the presence of liver nuclear extracts as opposed to brain, kidney or spleen extracts. However, sites II, V and VI interact with factors in all tissues, while their patterns of DNA-protein complexes in gel retardation analyses show apparent differences with all four tissues.

Wilms' tumor (WT) is an embryonic renal neoplasm which affects 1 in 10,000 children. It has long been considered an excellent model for studying the relationship of cancer to development. A tumor suppressor gene wt1 , implicated in predisposition to WT, is known to play an essential role in regulating development of the kidney and of the genitourinary system. A number of genes involved in growth regulation and cellular differentiation are modulated by WT1 and a number of proteins are known to associate with WT1. Some of these proteins are able to alter the transcriptional properties of WT1 influencing whether WT1 acts as a repressor or activator.
We demonstrate that WT1 represses different classes of activation domains previously shown to stimulate the initiation and elongation steps of transcription in vivo. We also showed that WT1 can repress transcription over a significant distance. Nuclear run-on assays revealed that the mechanism of repression by WT1 occurs at the level of transcription initiation.
We identified a novel WT1-interacting protein named Bone Marrow Zinc Finger 2 (BMZF2). The BMZF2·gene encodes a potential transcription factor harboring 18 zinc fingers and mainly expressed in fetal tissues. In vivo and in vitro pull-down experiments showed that WT1 and BMZF2 associate. This interaction inhibits WT1-mediated transcriptional activation. Additionally, BMZF2 harbors a transcriptional repression domain. These results suggest that BMZF2 interferes with the transactivation properties of WT1.
We performed an expression array screen utilizing WT1 inducible cell lines and found the human vitamin D receptor (VDR) as a major target. Nuclear run-on experiments, transient transfection assays, deletion mutagenesis, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays suggested that the WT1(-KTS) isoforms bind directly to the VDR promoter and activate VDR gene expression. Our results suggest that the human VDR gene represents a downstream target of WT1. We discuss the potential regulation of VDR by WT1 in normal and malignant tissues.

The proximal promoter of the rat carbamyl phosphate synthetase I gene contains 4 cis-acting regulatory regions, designated sites GAC, I, II, and III. Transient transfection assays revealed that the GAG element functions as the main activator element, whereas sites I and III bind repressor proteins and site II binds an anti-repressor. We propose that sites I and III repress the CPS I promoter either by quenching the sole activator(s) bound to the GAG element, or by directly repressing the basal transcription apparatus. Footprint analyses of the promoter with and without a mutation within site II suggested that the site II-anti-repressor may interfere with the site I and site III repressors by a similar quenching mechanism. Although the elements within the proximal promoter exhibit the potential for complex regulation, they are unable to confer tissue-specific promoter activity when analyzed either by in vitro transcription employing nuclear extracts from tissues that do not express the endogenous gene (i.e. brain, kidney, and spleen), or by transient transfection assays in non-CPS I-expressing cells (i.e. Rat1 and C6). However, we identified a cell-type specific enhancer that lies 10 kb upstream of the promoter. Unexpectedly, the enhancer functions as well in a gene-specific manner because enhancer-stimulated promoter activity was exclusively dependent on the GAG element within the CPS I proximal promoter. As a result, the GAG element is required to couple enhancer activity to the proximal promoter and exemplifies an efficient method whereby enhancers can be constrained to activate only their cognate promoters.

Regulation of gene expression is a fundamental biological process required to adapt the full set of hereditary information (i.e., the genome) to the varied environments that any organism encounters. Here, we elucidate two distinct forms of gene regulation – of endogenous genes by binding of transcription factors to information-containing genomic sequences and of selfish genes (“transposons”) by targeting of small RNAs to repetitive genomic sequences – using a wide array of approaches.

To study regulation by transcription factors, we used glucocorticoid receptor (GR), a hormone-activated, DNA-binding protein that controls inflammation, metabolism, stress responses and other physiological processes. In vitro, GR binds as an inverted dimer to two imperfectly palindromic “half sites” separated by a “spacer”. Moreover, GR binds different sequences with distinct conformations, as demonstrated by nuclear magnetic resonance spectroscopy (NMR) and other biophysical methods.

In vivo, GR employs different functional surfaces when regulating different genes. We investigated whether sequences bound by GR in vivo might be a composite of several motifs, each biased toward utilization of a particular pattern of functional surfaces of GR. Using microarrays and deep sequencing, we characterized gene expression and genomic occupancy by GR, with and without glucocorticoid treatment, of cells expressing GR alleles bearing differences in three known functional surfaces. We found a “sub-motif”, the GR “half site”, that relates to utilization of the dimerization interface and directs genomic binding by GR in a distinct conformation.

To study repression of tranposons, we characterized the production and function of small RNAs in the yeast Cryptococcus neoformans. We found that target transcripts are distinguished by suboptimal introns and inefficient splicing. We identified a complex, SCANR, required for synthesis of small RNAs and demonstrate that it physically associates with the spliceosome. We propose that recognition of gene products by SCANR is in kinetic competition with splicing, thereby further promoting small RNA production from target transcripts.

To achieve these results, we developed new bioinformatics tools: twobitreader, a small Python package for efficient extraction of genomic sequences; scripter, a flexible back-end for easily creating scripts and pipeline; and seriesoftubes, a pipeline built upon scripter for the analysis of deep sequencing data.

The major group of HIV-1 viruses that comprises the current global pandemic evolved, during their worldwide spread, into at least 10 distinct subtypes or clades. Subtype C predominates in sub-Saharan Africa and is responsible for the majority of the worldwide HIV-1 infections, while subtype B predominates in North America and Europe and subtype A/E is prevalent in Southeast Asia. Functional distinctions in the arrangement of NF-kappaB elements within the long terminal repeat (LTR) among HIV-1 subtypes have been identified, thus raising the possibility that transcriptional divergence amongst the subtypes of HIV-1 has occurred. In addition, significant amino acid variations have been observed amongst the clade-specific Tat proteins. In the present study, we sought to examine clade specific interactions between Tat, TAR and cellular proteins as well as to determine how these interactions may modulate the efficiency of HIV-1 gene transcription. Our results indicate that while sequence variation in the NF-kappaB region of the clade-specific LTRs plays a modest role in altering HIV-1 expression, clade-specific Tat proteins significantly modify viral gene expression.

In order to identify potential cis-acting elements responsible for the correct expression of the mosquito trypsin genes we have resorted to an evolutionary approach. This approach is based on the identification of DNA footprints that are conserved in homologous genes isolated from different species. Several trypsin clones have been isolated from Aedes species specific genomic DNA libraries, and by sequencing been shown to contain an ORF coding for the late trypsin gene. Analysis of the 5' FLR's of the species specific late trypsin genes, reveals the presence of a conserved TATA-box and transcriptional initiator. A phylogenetic footprinting analysis detected some evolutionarily conserved sequence elements in the 5' regulatory regions of the late trypsin gene. A cis-element that bears sequence similarity with the target of the transcription factor Tinman has been identified. Analysis of the trypsin coding region shows that the late trypsin from the most distant species retains approximately 83% amino acid identity with late trypsin from Ae aegypti. Furthermore, unique features of the late trypsin specificity pocket from Ae aegypti are retained in all species examined making this a unique evolutionary molecular tag for this serine protease. The dicotomy of a positive/negative charge observed in the specificity pocket of trypsin members of the chymotrypsin family of serine proteases is retained; however, the conserved aspartate is replaced by a glutamate. The position of this glutamate is displaced towards the N-terminus by a serine, which is characteristic of chymotrypsin-like enzymes from the same family. Moreover, there is an insertion of a proline after the serine amino acid at the C-terminal end of the pocket. Alignment of this trypsin to other members of the same protease family strongly suggests that it is related to a unique group of proteases called serine collagenases. The most important enzymatic characteristic of enzymes belonging to this group is there lack of substrate specificity. Outside of the typical metalloproteases, these are the only proteases known to cleave collagen. What these observations mean in terms of the evolution of enzyme specificity aid structural fold remain to be elucidated.

RAR$\beta$ has been shown to be expressed in the developing heart at the 8 somite stage. Differentiation of EC cells with DMSO produces a mixture of embryonic cardiac, skeletal, endodermal and other mesodermal derivatives. The RAR$\beta$-2 isoform is predominantly expressed in the differentiated cardiac muscle cells. RT-PCR with isoform specific primers has been used to identify RAR$\beta$-2 as the predominant isoform. These results show, that like RA-treated EC cells, expression of the RAR$\beta$-2 isoform in DMSO differentiated P19 cells predominates. Stable transformants of the RARE$\beta$-2-CAT reporter gene construct were pooled and differentiated with either RA or DMSO. The results obtained demonstrate that RA induced CAT activities increased to a maximum of 188 fold by day 3. The increase in RAR$\beta$-2 mRNA levels was not due to another enhancer element as evidenced by nuclear run-on analysis. RAR$\beta$2 mRNA levels seem to be regulated by a post-transcriptional mechanism. The stability of the DMSO induced RAR$\beta$2 message was determined using the transcriptional inhibitor actimomycin-D on day 4 and 7 of differentiation. The half life of the message was found to be approximately equal on both days. We postulate that stabilization of the message occurs at an earlier time point, and that accumulation of the message occurs up until Day 7 of differentiation. Simultaneous administration of RA and DMSO to aggregating P19 cells blocked the up-regulation of Brachyury expression in these cells. No beating cardiac muscle was formed, however neuroectoderm was found to be the predominant cell type, indicating that the DMSO differentiating effect was usurped by the presence of RA. (Abstract shortened by UMI.)

Several evidence indicate that presenilin-2 gene is rhythmically regulated in the mouse suprachiasmatic nuclei (SCN), which is the master circadian clock in mammals, as well as in the liver. We investigated the mechanism of circadian modulation behind presenilin-2 oscillation. CLOCK and BMAL1 are the two major transcription factors for circadian gene expression. We demonstrated that CLOCK and BMAL1 partially modulate presenilin-2 gene expression in vitro. In vivo, we showed that the gene does not oscillate in the muscle, spleen, thymus, heart and kidney. On the other hand, we demonstrated a circadian oscillation of presenilin-2 in the liver. We suspect another posttranscriptional mechanism to act in cooperation with the transcriptional mechanism regulated by CLOCK/BMAL1 for the circadian modulation of presenilin-2. We showed that the full-length transcript of presenilin-2 does not oscillate in the liver, but shorter transcripts, potentially issued from alternative spliced mRNA, do oscillate.

Enhancers are regulatory elements that orchestrate cell type specific gene expression patterns. They can be separated from their target genes by large distances and activate transcription by coming into physical proximity with promoters in three-dimensional nuclear space. Complex regulatory networks with multiple enhancers often cooperate to control the same target gene. Antigen receptor loci have proved to be a rich ground for understanding enhancer-mediated gene regulation. The loci undergo somatic recombination of their V, (D), J segments to create a diverse repertoire of antigen receptors that can counteract a wide range of foreign antigens. V(D)J recombination relies on the presence of proximal enhancers that activate the antigen receptor loci in a lineage and cell stage specific manner. Unexpectedly we find that both active and inactive antigen receptor loci enhancers cooperate to disseminate their effects in a localized and long-range mode. We demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each enhancer. We further establish that in T cells, the Igk enhancer MiEκ exerts a long-range effect on another antigen receptor locus Tcrb located 29MB away on chromosome 6. MiEκ deletion leads to inefficient Tcrb recombination resulting in a block in T cell development, an effect that is associated with a long-range contact and cooperation between the MiEκ and the Tcrb enhancer, Eβ. MiEκ deletion alters enrichment of the transcription factor CBFβ on Eβ in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer sharing between active and inactive elements in lineage and stage specific control. Finally, we expand our assessment of enhancer cooperation to the entire chromosome 6. We detect nearly nine hundred putative regulatory elements that are active in either DP or pre-B cells with less than 20% common to the two cell types. We also demonstrate how long-range contacts between enhancers and promoters coincide with target gene expression status, and provide a resource for identifying the regulatory elements that control T and B cell specific gene expression patterns.

Molecular Biology and Genetics
Ph.D.
Opioid receptor modulation of pro-inflammatory cytokine production is vital for host defense and the inflammatory response. Previous results have shown the mu-opioid receptor (MOR) selective agonist, DAMGO, has the capacity to increase the expression of the pro-inflammatory chemokines, CCL2/MCP-1, CCL5/RANTES and CXCL10/IP-10 in peripheral blood mononuclear cells (PBMCs). We have shown that MOR activation is able to induce the expression of TGF-β, and TGF-β appears to be required for induction of CCL5 following MOR activation. This work suggests a novel role for TGF-β in the inflammatory response. NF-κB is a transcription factor that plays a pivotal role in inflammation and the immune response. We have found that NF-kB inhibitors can prevent the MOR-induced activation of CCL2 and CCL5, and that the NF-kB subunit, p65, is phosphorylated at serine residues 311 and 536 in response to μ-opioid receptor activation. In vivo, DAMGO administration can induce binding of p. 65 to the enhancer region of the CCL2 promoter. Furthermore, we demonstrate that PKCζ is phosphorylated following DAMGO-induced MOR activation and, is essential for NF-kB activity as well as CCL2 expression and transcriptional activity. In conclusion, these data suggest a pro-inflammatory role for MOR which involves NF-κB activation and PKCζ as well as a novel role for TGF-β as a regulator of pro-inflammatory chemokines.
Temple University--Theses

The Interferon Regulatory Factors (IRFs) are a family of interferon-inducible proteins which play distinct roles in diverse processes such as pathogen response, cytokine signalling, cell growth regulation and hematopoietic development. The objective of this research was to investigate the mechanisms by which IRF-1 and IRF-2 regulate gene expression and cell growth. Structure-function analyses of the IRF-2 protein demonstrate that transcriptional repression by IRF-2 is contained within the first 125 N-terminal amino acids and correlates directly with IRF-2 DNA binding. Overexpression of functional IRF-2 deletion mutant proteins in NIH3T3 cells results in oncogenic transformation and tumorigenesis, suggesting that IRF-2 oncogenicity correlates directly with transcriptional repression. Similar structure-function analyses localize IRF-1 transactivation to the C-terminus. Like IRF-1, hybrid constructs which fuse the DNA binding domain of IRF-1 and IRF-2 to the transactivation domain of NF-kappaB RelA(p65) are transcriptional activators. Inducible expression of IRF-1 and IRF/RelA in NIH3T3 cells results in reduced cellular growth and induction of apoptosis. Furthermore, expression of the PKR, STAT1(p91), and WAF1 growth regulatory proteins are elevated following induction of IRF-1 or IRF/RelA, correlating transactivation function and tumor suppressor activity of IRF-1 or IRF/RelA. By RNA fingerprinting, the secretory leukocyte protease inhibitor (SLPI) was identified as the first gene whose expression is downregulated by IRF-1 or IRF-1/RelA. A region in the SLPI promoter was identified that bound IRF-1, suggesting a direct mechanism for IRF-1 regulation of SLPI expression.

Glucocorticoids and somatostatin both influence a broad spectrum of biological activities and their actions are cooperative in growth control, pancreatic islet function, immune suppression, and stress response, e.g. in vivo studies indicate that glucocorticoids may act through somatostatin to suppress growth, growth hormone secretion and inflammation. Recent studies have suggested that glucocorticoids influence somatostatin production but the precise nature of this effect has remained unclear. In this thesis, I characterized the actions of glucocorticoids on somatostatin gene expression and their molecular mechanisms of action in three consecutive studies. (1) I started with an investigation of the in vivo and in vitro effects of glucocorticoids and found that dexamethasone exerts significant effects on somatostatin peptide and steady state mRNA levels in normal somatostatinoma (1027B$ sb2$) cells. Glucocorticoids stimulate somatostatin production in peripheral tissues (stomach, pancreas, and jejunum) and suppress its biosynthesis in cerebral cortex and hypothalamus. Glucocorticoids induce dose-dependent biphasic effects on steady state somatostatin-mRNA levels in normal rat islet and 1027B$ sb2$ cells, characterized by stimulation at low doses (10$ sp{-10}$ M) and marked inhibition at high doses ($ geq$10$ sp{-7}$ M). This suggests a complex molecular mechanisms of glucocorticoid action on the somatostatin gene involving multi-level regulation. (2) I further discovered that glucocorticoids stimulate somatostatin gene transcription in PC12 (pheochromocytoma) cells transfected with somatostatin promoter-CAT (chloramphenicol acetyl transferase) reporter gene. Dexamethasone induces a dose-dependent 2.2 fold stimulation of somatostatin-CAT expression in PC12 cells and exerts an additive effect on cAMP-induced gene transcription. The dexamethasone effect is abolished in A126-1B2 (protein kinase A-deficient mutant PC12) cells and with CRE (cAMP response element) mutant

Binding of epidermal growth factor (EGF) to the EGF receptor stimulates the tyrosine kinase activity of the receptor and initiates a signal transduction cascade culminating in a mitogenic response. In many tumor derived cell lines which overexpress EGF receptor, exposure to EGF results in growth inhibition. The mechanism for this is unclear. This work involves analysis of growth inhibition by EGF, mechanisms of EGF receptor overexpression and regulation of the EGF receptor gene. The first two studies utilize a cell line (PC-10) that overexpresses EGF receptor without gene amplification. PC-10 cells appear to adopt a novel mechanism to overexpress EGF receptor; one that involves stabilization of the receptor message rather than the more common gene amplification. PC-10 cells were found to be killed by EGF in a cell density dependent manner. However, chronic exposure to EGF subsequently allowed proliferation under conditions which previously resulted in cell death. These "adapted" cells had similar levels of EGF receptor on the cell surface and similar EGF binding parameters. The tyrosine kinase activity of the receptor in response to EGF in the adapted cells was significantly reduced both in vitro and in vivo. Evidence was also found that the signal transduction cascade initiated by EGF was altered by adaptation. These data provide evidence for a unique mechanism for EGF receptor-overexpressing cells to survive EGF toxicity, one that involves a reduction in the tyrosine kinase activity of the EGF receptor in the absence of a decrease in the number of EGF receptor. Finally, additional studies were carried out on the response of the EGF receptor gene to activation of protein kinase C (PKC) in A549 cells. Activation of PKC resulted in increased levels of EGF receptor mRNA. No induction of a transfected reporter gene containing EGF receptor regulatory DNA could be seen. Repression of the reporter gene, however, was consistently seen. The cis-element for repression was mapped to 233 base pairs in the EGF receptor regulatory region. Additional data support the hypothesis that a previously characterized repressor protein called GCF may be responsible for this repression.

Phosphate plays a critical role in the body as a constituent of bone and tooth for body development, and as a urinary buffer for pH in body acid-base balance regulation. The phosphorus level in blood in human is between 3.0 to 4.5 mg/dl. When the blood phosphate concentration is lower than 2.5 mg/dl, the person develops hypophosphatemia. When the blood phosphate concentration is higher than 4.5 mg/dl, the person develops hyperphosphatemia. It is critically important for the body to control the phosphate level in blood and maintain the phosphate homeostasis. The kidney and the intestine are the important sites to regulate phosphate homeostasis. This dissertation research was to explore the transporter gene(s) involved in the intestinal sodium-dependent phosphate absorption in human and to investigate the role of sodium-phosphate (NaPi) transporters in phosphate homeostatic regulation. The research was performed to test the hypothesis that the sodium-dependent phosphate (NaPi) cotransporter is involved in the phosphate absorption in intestine and various physiological regulators modulate the activity of this transporter. A cDNA encoding a novel human small intestinal Na⁺-P i transporter was isolated from a human intestinal cDNA library. This cDNA encodes a 689 amino acid polypeptide which is different from the renal NaPi cotransporters. This human intestinal NaPi cotransporter gene was mapped to human chromosome 4p15.1--p15.3 by the F̲luorescence I̲n-S̲itu H̲y̲bridization (FISH) method. The human intestinal NaPi cotransporter gene structure was studied by screening a human genomic DNA library. This gene contains 12 exons and 11 introns. There were two transcription initiation sites identified by primer extension. In vivo and in vitro studies showed that the intestinal NaPi cotransporter gene expression is regulated by EGF and vitamin D3. EGF inhibits NaPi-cotransporter gene expression, while vitamin D3 stimulates NaPi-cotransporter gene expression. From these studies, I concluded that the intestinal sodium-dependent phosphate absorption is mainly mediated by intestinal NaPi cotransporters (NaPi-IIb), and this transporter is modulated by various physiological regulators in order to maintain the phosphate homeostasis.

TAp63, a member of the p53 family, has been shown to regulate energy metabolism. Here, we report coiled coil domain-containing 3 (CCDC3) as a new TAp63 target. TAp63, but not ΔNp63, p53 or p73, induces the expression of CCDC3 mRNA level by directly binding to the p63 consensus DNA binding sequence within the CCDC3 enhancer region. The CCDC3 expression is markedly reduced in TAp63-null mouse embryonic fibroblasts and brown adipose tissues and by tumor necrosis factor alpha that reduces p63 transcriptional activity but induced by metformin, an anti-diabetic drug that activates p63. Also, the expression of CCDC3 is positively correlated with TAp63 levels, but inversely with ΔNp63 levels, during adipocyte differentiation. Interestingly, CCDC3, as a secreted protein, targets liver cancer cells and increases long chain polyunsaturated fatty acids, but decreases ceramide in the cells. CCDC3 alleviates glucose intolerance, insulin resistance, and fatty liver (steatosis) formation in transgenic CCDC3 mice on the high-fat diet by markedly reducing hepatic PPARγ expression and consequently leading to a drastic decrease of the PPARγ target gene, CIDEA, and other genes involved in de novo lipogenesis and of lipid droplets formation in their livers. Similar results are reproduced by hepatic expression of ectopic CCDC3 in mice on high-fat diet. Altogether, these results demonstrate that CCDC3 modulates liver lipid metabolism by inhibiting liver de novo lipogenesis as a downstream player of the p63 network.

Nuclear factor-erythroid 2 (NF-E2) is a basic leucine zipper (bZIP) transcription factor that recognizes the DNA binding site [(T/C)GCTGA(G/C)TCA (T/C)] that contains a central AP-1 motif (underlined), but is distinct from AP-1 factors (Jun, Fos) due to the requirement of residues outside of the AP-1 core. NF-E2 is a heterodimer comprised of a cap'n'collar factor (p45) and a small Maf protein (MafF, G or K). It has been shown that NF-E2 promotes the opening of chromatin throughout the beta-globin loci, promoting gene expression and erythroid differentiation. Also, the p45 subunit is restricted to hematopoietic progenitor, erythroid, megakaryocytic and mast cells while the small Mafs are ubiquitously expressed.
I have used the erythroleukemia cell lines, MEL and CB3, as model systems to study the role of NF-E2. MEL cells can be induced to differentiate, while CB3 cells, which do not express p45 due to a Friend leukemia virus insertion in one allele and the loss of the other, express only minimal levels of alpha- and beta-globin mRNA upon induction.

Caspases play an important role in shaping the developing organism. They are required to eliminate unwanted or damaged cells and therefore are able to prevent disease. Several reports have shown increased caspase-3 expression in different cell types undergoing apoptosis. We undertook to examine the role of T cell activation through the T cell receptor (TCR) in regulating caspase-3 gene expression. The KOX-14 murine T cell hybridoma was initially used as a model for activation-induced cell death. Caspase-3 mRNA levels increased by 3-fold following T cell activation and was independent of STAT1 activation and therefore of IFN-gamma signaling in KOX-14 cells suggesting that the increase occurs early during T cell activation. Naive T cells were then isolated from the lymph nodes of mice to determine the extent of the increase in caspase-3 mRNA levels in cells undergoing proliferation rather than apoptosis. Caspase-3 mRNA levels were selectively induced (13-fold) following TCR triggering. Furthermore, caspase-3 mRNA levels were the highest in effector T cells which are destined to undergo AICD, when compared to long-lived memory T cells. Interestingly, the levels of procaspase-3 were also induced (6-fold) in activated peripheral T cells. In addition, T cells deficient for caspase-3 were more resistant to different apoptosis inducing molecules when compared to T cells containing normal levels of caspase-3. Altogether, these results demonstrate that the levels of caspase-3 must be maintained in a cell to ensure efficient apoptosis. The caspase-3 promoter region was subsequently cloned to identify transcription factors responsible for the observed increased in caspase-3 mRNA levels during T cell activation. Several regions within the promoter had either positive or negative regulatory effects on reporter activity when deleted. TCR stimulation of KOX-14 cells containing the different caspase-3 promoter constructs did not show changes in reporter ac

Copper (Cu) deficiency was induced in rats and Hep G2 cells by the use of a Cu-deficient diet and a cupruretic chelator, respectively. In the rat liver, Cu-deficiency did not alter the apo A-I mRNA abundance, but shifted significant amounts of mRNA to translationally more active fractions. These findings indicated that an increase in translational efficiency may contribute to the increase in hepatic apo A-I synthesis observed in Cu-deficient rats. In Hep G2 cells, Cu-depletion elevated the cytoplasmic apo A-I mRNA abundance by 1.5-fold. A 2.5-fold increased transcription rate and a 2-fold accelerated mRNA decay were also established in Cu-depleted cells. These changes appeared to be specific to Cu depletion, because they were reversed by Cu repletion. Moreover, the cytoplasmic mRNA abundance of HNF-4, a major transcription activator of apo A-I gene, was elevated by 1.6-fold in Cu-depleted cells. Thus the elevated cellular apo A-I mRNA level may have resulted from an accelerated mRNA turnover, and subsequently contributed to the enhanced apo A-I synthesis and secretion observed in Cu-depleted cells. Zinc (Zn) deficiency was induced in animals and Hep G2 cells by the use of Zn-deficient diet and medium, respectively. Plasma HDL apo A-I levels was reduced 18% in hamsters and 13% in rats. Whereas Zn repletion normalized plasma apo A-I to the control level in hamsters and increased it by 34% in rats. No treatment difference was detected in the intestinal apo A-I mRNA abundance in both species, although the hepatic abundance was reduced by 18% and 55% in Zn-deficient hamsters and rats, respectively. Subsequent Zn-repletion normalized the mRNA abundance to the control level in hamsters and elevated it by 41% in rats. As compared to control Hep G2 cells, the cellular Zn content and apo A-I mRNA abundance were reduced by 55% and 20% in Zn-depleted cells, but increased by 64% and 11% in Zn-supplemented cells, respectively. Furthermore, Zn-repletion completely normalized the effects of Zn-depletion. Thus the depletion of hepatic Zn content may cause the reductions in hepatic apo A-I mRNA abundance and plasma apo A-I pool observed in Zn deficiency.

The epigenetic mechanisms that connect hormone signaling to chromatin remain largely unknown. Here we show that LSD1/KDM1A is a critical glucocorticoid receptor (GR) coactivator and report a previously unexplored mechanism where LSD1 activates gene transcription through H3K4me2 demethylation. We demonstrate that direct interaction of GR with LSD1 primarily inhibit its activity against H3K4me1 in vitro. While this interaction enables GR to recruit LSD1 in vivo and allows loss of H3K4me2, it impedes further demethylation. Thus resulting in conversion of H3K4me2 to H3K4me1 at enhancers and promotes H3K27 acetylation and gene activation. We also find that H3K4me2 is an early enhancer mark predicting GR and LSD1 recruitment. These findings differ from the reported mechanism for ER and AR-mediated gene activation, providing a novel mechanism for LSD1 coactivator function as well as shed light on the role of H3K4me2 and enhancers in hormone-mediated gene regulation. In addition we present evidence supporting never before characterized H3K79me3 demethylase activity by members of the JMJD2 family of proteins.

The studies included in this thesis were aimed at identifying the mechanisms that lead to aberrant expression of the PTHRP gene in cancer.
We have used the hepatocyte growth factor receptor oncogene, Tpr-Met, as a model and examined the effect of this oncogene on PTHRP expression. When transfected into Fisher rat 313 (Fr3T3) fibroblasts, Tpr-Met increased the transcription of PTHRP mRNA and secretion of the protein. To identify the signaling pathways involved we analyzed a mutant of Tpr-Met, Tyr489, that was impaired in activating a number of downstream effectors, including Phosphatidylinositol-3 kinase, Grb2 and Shc. The ability of Tpr-Met/Tyr 489 mutant to induce PTHRP expression was significantly reduced. Furthermore, inhibiting Ras using lovastatin, in wild-type Tpr-Met transfected cells, Completely Suppressed PTHRP levels, suggesting that the mechanism was Ras-dependent.
We next directly investigated the effect of Ras on PTHRP expression in vitro, and on hypercalcemia of malignancy in vivo. When transfected PTHRP cells the activated mutant of Ras (RasV12) potently increased PTHRP mRNA and protein levels. When RasV12 expressing cells were subcutaneously injected into BALB/c/nu/ nu mice, the tumors developed rapidly, and signs of hypercalcemia were detected within 2 weeks. Inhibiting Ras using a specific inhibitor, B-1096, completely blocked expression of PTHRP, in vitro, and suppressed the sips of hypercalcemia in vivo. These results show that inhibiting Ras was sufficient to block tumor expression of PTHRP and development of hypercalcemia.
Using rat Leydig tumor H-500 cells, we next investigated effector pathways downstream of Ras that mediate serum stimulated PTHRP expression. PTHRP mRNA was decreased by a dominant negative mutant of Raf (Raf C4B) and by a MEK inhibitor (PD 098059), implicating the involvement of Ras-Raf-MEK pathway in the serum response. In addition, stimulation with UV light or expression of an activated form of Rac (Rac V12) was sufficient to increase PTHRP mRNA. Furthermore, a dominant negative mutant Of Rac (Rac N17) also blocked serum induced expression of mRNA. This suggests that the stress-activated pathways may provide alternative mechanisms that can regulate the PTHRP gene. These pathways also appear to be important in the serum induced response. Collectively, the results from these studies contribute to our limited knowledge of the mechanisms governing PTHRP expression in cancer. The findings also provide novel targets to explore for improved therapy of hypercalcemia.

Small non-coding RNAs (sRNAs) are being increasingly recognized as critical regulators of a wide variety of processes in bacteria. To investigate the contribution of unknown sRNAs to virulence gene regulation in Vibrio cholerae, we undertook a screen to identify previously uncharacterized sRNAs under the control of the major virulence gene activator in V. cholerae, ToxT. Using a combination of direct sRNA cloning and sequencing together with a genome-wide ToxT in vitro binding assay, we identified 18 putative ToxT-regulated sRNAs. Two of these ToxT regulated sRNAs were located within the Vibrio Pathogenicity Island-1 (VPI-1), the genetic element that encodes ToxT and the Toxin Co-regulated Pilus (TCP). We verified regulation of these sRNAs by ToxT and showed that deletion of one of them, now designated tarB, caused a variable colonization phenotype when competed against the parental strain in an infant mouse model of V. cholerae infection. Infections progressing for 18 hours or less showed the ΔtarB strain was out-competed by the wild type strain, while those carried out longer, showed Δ tarB out-competing the wild type. Additionally, if inoculated from a resource poor environment the ΔtarB strain also showed decreased colonization relative to wild type. Using a bioinformatic approach, we identified that tarB-mediated regulation of the gene tcpF was primarily responsible for the tarB mutant's in vivo colonization phenotype. Further investigation of genes regulated by tarB using genome-wide transcriptional profiling of a tarB over-expressing strain revealed that tarB also directly regulates genes involved in iron and amino acid uptake. We determined that tarB has a repressive effect on many genes within the VPI-1, but has an activating effect on tcpP/tcpH, encoding regulators upstream of ToxT. Taken together, the data suggest that tarB plays an important role in regulating virulence and metabolic genes early after V. cholerae infection, but that this repressive effect on virulence genes later in infection may lead to reduced replication in vivo.

The secosteroid hormone 1, 25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) exerts its effects through a receptor protein which is a member of a superfamily of steroid receptor proteins. The 1,25(OH)₂D₃ receptor protein (VDR) binds both hormone and DNA with high affinity , thereby transcriptionally regulating the expression of a number of genes. A fragment of the cDNA to the human VDR containing essentially the entire open reading frame was transcribed and translated in vitro. demonstrated to exhibit The resulting protein was then the physical and functional features, i.e. molecular weight, immunoreactivity, 1,25(OH)₂D₃ binding, and DNA-cellulose binding, of the native human receptor from the T47D cell line. This validates the authenticity of the cDNA in a cell free system and provides a biochemical means of generating this rare and labile macromolecule to use in heretofore difficult structure/function studies. The gene for rat bone gla protein (BGP) was isolated and 1250 bp including 1100 bp of 5' flanking DNA were placed upstream of the human growth hormone reporter gene. Following transient transfection into the osteoblast-like rat osteosarcoma cell line, ROS 17/2.8, the BGP promoter demonstrated a low level of basal activity that was increased approximately ten-fold by addition of 10⁻⁸ M 1,25(OH)2D3. A single 249 bp fragment (-523 to -274) was sufficient to confer hormone inducibility upon both heterologous and homologous promoters. Deletion studies, complemented by evaluation with synthetic oligomers, enabled localization of the 1,25(OH)₂D₃ response element (VDRE) to within 19 bp (-456 to -438), containing an element with an imperfect direct repeat (GGTGA(N₄)GGACA) and homology to other steroid responsive elements. This enhancer element was capable of strong synergism when present in multiple copies. Gel retardation assays demonstrated that partially purified VDR from chicken or rat bound specifically and with high affinity to a DNA fragment containing the putative VDRE and this binding was perturbed by monoclonal antibodies to the VDR. surprisingly, the 249 bp fragment, when linked in an antisense orientation with respect to the BGP promoter, blocked the basal and hormone dependent gene expression. However, a 245 bp fragment 5' to the 249 bp element (-1100 to -855) restored 20-fold inducibility when linked to the first fragment in the same orientation. The presence or absence of this distal element also modulated the 1,25(OH)₂D₃ response within the milieu of the native gene. Thus, the VDRE apparently cooperates with other elements to achieve the hormonal response observed in this gene.

One-carbon (C₁) metabolism. C₁ metabolism is central to all organisms, because C₁ units have essential roles in biosyntheses of basic materials for living cells, such as protein, nucleic acids, choline and its derivatives. One unique feature of plant C₁ metabolism is that it channels significant C₁ flux from primary carbon metabolism to methylated metabolites. Part I of this dissertation presents functional analysis of plant methyane-THF reductases (MTHFRs) in Arabidopsis and maize, and regulation of the plant C₁ metabolic pathway and glycine betaine (GlyBet) biosynthesis in maize GlyBet near isogenic lines (NILs). Plant MTHFRs were isolated from Arabidopsis and maize and functionally characterized in yeast. Unlike mammalian MTHFRs, the plant enzymes strongly prefer NADH to NADPH and are not inhibited by S-adenosyl-methionine. An NADH dependent MTHFR reaction could be reversible in the cytosol, supported by radiotracer labeling data using [methyl-¹⁴C]methyltetrahydrofolate. Systematic analyses of profiling transcript abundance of plant C₁ genes using microarrays indicated that the regulation of the plant C₁ metabolic pathway may be "fine-tuned", or at a downstream "outlet" point in maize. Comparison of GlyBet NILs through analyses of metabolites, [¹⁴C]formaldehyde labeling and northern blotting of phosphoethanolamine methyltransferase (P-EAMT) indicated that the regulation glycine betaine biosynthesis is at P-EAMT, whose transcript is probably repressed by its product, phospho-choline. Global gene responses to salt stress. To understand how genes respond to salt stress is very important for the genetic improvement of salt tolerance in plants. Part II of this dissertation presents a systematic analysis of global gene responses to salt stress in maize roots, using microarrays, that contained ∼8,000 ESTs. Microarray expression analysis revealed that 916 different ESTs were up- or down-regulated under salt stress (611 up-regulated, 305 down-regulated), representing 11% of ∼8,000 ESTs printed on slides. These up- or down-regulated ESTs were clustered into various subgroups based on repression or induction of the timing, amplitude and duration of their transcripts. Contig analysis assigned 916 up- or down-regulated ESTs into 472 tentative unique genes (51.6%). These responsive genes are involved in a broad range of cellular processes, biochemical pathways and signal transduction cascades etc. The functions of some regulated genes are discussed.

Wilms' tumour, a pediatric kidney cancer that affects 1 in 10 000 children, is an excellent paradigm for studying the relationship between cancer and development. The Wilms' tumour suppressor 1 ( WT1) gene was identified through the study of hereditary cases of Wilms' tumour showing cytogenetic deletions at chromosome position 11p13. The WT1 gene encodes a zinc finger transcription factor necessary for the development of the genitourinary system. WT1 functions as an activator or a repressor, interacts with a number of different protein partners and regulates the expression of several genes important for cellular growth and differentiation. WT1 mRNA is present in tissues of mesodermal origin that undergo a mesenchymal to epithelial transition. Expression of WT1 is tightly regulated both temporally and spatially during development of the urogenital system.
We have identified a novel trans-acting factor, named complex D, which shows sequence specific binding to the WT1 promoter. By electrophoretic mobility shift assays (EMSA), we demonstrate that the transcription factor Sp1 binds the WT1 promoter at a site overlapping the complex D binding site. Molecular mass determination experiments and in situ UV crosslinking indicate that complex D is approximately 130 kDa and consists of at least two proteins. Transient transfection assays show that the integrity of the complex D binding site is necessary for maximal activation of a reporter gene, suggesting that complex D may function as an activator.
Similar to WT1, the ETS-domain transcription factor Pea3 is expressed in tissues where mesenchymal-epithelial interactions occur and both gene products are implicated in regulating the expression of genes necessary for the epithelialization of common organs. Transient transfection assays using WT1 promoter-reporter gene constructs identified a Pea3 responsive element in the WT1 promoter. Overexpression of Pea3 transactivates the WT1 promoter and the presence of the intact Pea3 responsive element is necessary for the transactivation. We demonstrate, by EMSA, the sequence specific binding of Pea3 to the responsive element.
WT1 and the paired box domain transcription factor Paired box 2 (Pax2) are expressed at the initial stages of metanephric kidney development and are critical for the initiation of nephrogenesis. We generated WT1/Pax2 compound heterozygous mutant mice to provide an in vivo model for studying the interplay between WT1 and Pax2 during nephrogenesis. WT1+/-/Pax2 1Neu/+ kidneys were 50% smaller that wild type kidneys and displayed a more severe underdevelopment of the medulla, renal calyces and renal pelvis compared to Pax21Neu/+ kidneys. We demonstrate that WT1 and Pax2 proteins physically interact in vitro and in vivo. Our data suggest that WT1 is a modifier of the Pax2 mutant phenotype and that both proteins may be implicated in a common pathway in the transcriptional network governing metanephric development.

Myeloid cells are important reservoirs of HIV-1 infection. In response to pathogenic agents, macrophages secrete inflammatory cytokines which can stimulate viral replication and contribute to AIDS pathogenesis. Two promonocytic cell models were used to investigate the relationship between NF-$ kappa$B dependent cytokine gene expression, differentiation, and HIV infection: the U937 and PLB-985 cell lines and their chronically infected counterparts U9-IIIB and PLB-IIIB. Chronic HIV-1 infection of U9-IIIB cells did not generally lead to constitutive transcription of cytokine genes. However, when co-infected with Sendai virus or stimulated with lipopolysaccharide, the induction of IFN$ beta$, IL-1$ beta$, IL-6 and TNF$ alpha$ mRNA occurred more rapidly and was increased to levels 10 to 20-fold higher in U9-IIIB cells compared to similarly induced U937 cells.
Chronic infection of PLB-985 cells led to irreversible differentiation along the monocytic pathway. In PLB-985 cells, TNF$ alpha$ or PMA treatment led to induction of NF-$ kappa$B p50 subunit binding, whereas chronic HIV or acute Sendai virus infection led to a distinct NF-$ kappa$B-like binding activity composed of 70, 90 and 100 kDa proteins with high specificity for binding the PRDII domain of IFN$ beta$. UV cross-linking, shifted-shift and immunoprecipitation analyses indicated that p50 and the strong transcriptional activator p65 represented the major constituents of this NF-$ kappa$B DNA-binding activity. (Abstract shortened by UMI.)

The c-myc gene was previously shown to be regulated by a conditional block to transcription elongation and sequences from its promoter were implicated in this regulation. The objective of this project was to define the promoter elements involved in the control of transcription elongation. Using heterologous promoter constructs, the ME1a1 protein binding site located in the c-myc promoter was shown to be required for the block to transcription elongation. From mutation analysis, a correlation was established between the binding of nuclear factors to ME1a1 sites in vitro and the ability of these sites to confer block to transcription elongation in vivo, strongly implicating trans-acting factors in this process. Fractionation studies demonstrated that three nuclear factors interact with the ME1a1 site, thus generating three protein-DNA complexes termed "a", "b", and "c". These factors were characterized and a cDNA encoding the protein responsible for complex "c" was isolated. This protein was shown to represent the human homologue of the Drosophila Cut homeodomain protein (hu-Cut) and to repress expression from the c-myc promoter in transient transfection assays.