{\it cis\/} -acting elements and {\it trans\/} -acting factors that control serum amyloid A gene expression during inflammation

To understand how the serum amyloid A (SAA) genes are regulated, the cis-acting elements and trans-acting factors involved in the regulation of mouse SAA3 and rat SAA1 genes expression during inflammation were analyzed.^ To identify DNA sequences involved in the liver-specific expression of the mouse SAA3 gene, the 5$\sp\prime$ flanking region of this gene was analyzed by transient transfection studies. Results suggest that C/EBP, a liver-enriched transcription factor, plays an important role for the enhanced expression of the mouse SAA3 gene in hepatocytes.^ Transfection studies of the regulation of the expression of rat SAA1 gene indicated that a 322 bp fragment ($-$304 to +18) of the gene contains sufficient information for cytokine-induced expression of the reporter gene in a liver cell-specific manner. Further functional analysis of the 5$\sp\prime$ flanking region of the rat SAA1 gene demonstrated that a 65 bp DNA fragment ($-$138/$-$73) can confer cytokine-inducibility onto a heterologous promoter both in liver and nonliver cells. DNase I footprint and gel retardation assays identified five putative cis-regulatory elements within the 5$\sp\prime$ flanking region of the gene: one inducible element, a NF$\kappa$B binding site and four constitutive elements. Two constitutive elements, footprint regions I and III, were identified as C/EBP binding sites with region III having over a 10-fold higher affinity for C/EBP binding than region I. Functional analysis of the cis-elements indicated that C/EBP(I) and C/EBP(III) confer liver cell-specific activation onto a heterologous promoter, while sequences corresponding to the NF$\kappa$B element and C/EBP(I) impart cytokine responsiveness onto the heterologous promoter. These results suggest that C/EBP(I) possesses two functions: liver-specific activation and cytokine responsiveness. The identification of two cytokine responsive elements (NF$\kappa$B and C/EBP(I)), and two liver-specific elements (C/EBP(I) and C/EBP(III)) implies that multiple cis-acting elements are involved in the regulation of the expression of the rat SAA1 gene. The tissue-specific and cytokine-induced expression of rat SAA1 gene is likely the result of the interactions of these cis-acting elements with their cognate trans-acting factors as well as the interplay between the different cis-acting elements and their binding factors. (Abstract shortened with permission of author.) ^

Cloning and molecular analysis of paraxis: A {\it trans\/}-acting factor required for somite maturation

During vertebrate embryogenesis, cells from the paraxial mesoderm coalesce in a rostral-to-caudal progression to form the somites. Subsequent compartmentalization of the somites yields the sclerotome, myotome and dermatome, which give rise to the axial skeleton, axial musculature, and dermis, respectively. Recently, we cloned a novel basic-Helix-Loop-Helix (bHLH) protein, called scleraxis, which is expressed in the sclerotome, in mesenchymal precursors of bone and cartilage, and in connective tissues. This dissertation focuses on the cloning, expression and functional analysis of a bHLH protein termed paraxis, which is nearly identical to scleraxis within the bHLH region but diverges in both its amino and carboxyl termini. During the process of mouse embryogenesis, paraxis transcripts are first detected at about day 7.5 post coitum within the primitive mesoderm lying posterior to the head and heart primordia. Subsequently, paraxis expression progresses caudally through the paraxial mesoderm, immediately preceding somite formation. Paraxis is expressed at high levels in newly formed somites before the first detectable expression of the myogenic bHLH genes, and as the somite becomes compartmentalized, paraxis becomes downregulated within the myotome.^ To determine the function of paraxis during mammalian embryogenesis, mice were generated with a null mutation in the paraxis locus. Paraxis null mice survived until birth, but exhibited severe foreshortening along the anteroposterior axis due to the absence of vertebrae caudal to the midthoracic region. The phenotype also included axial skeletal defects, particularly shortened bifurcated ribs which were detached from the vertebral column, fused vertebrae and extensive truncation and disorganization caudal to the hindlimbs. Mutant neonates also lacked normal levels of trunk muscle and exhibited defects in the dermis as well as the stratification of the epidermis. Analysis of paraxis -/- mutant embryos has revealed a failure of the somites to both properly epithelialize and compartmentalize, resulting in defects in somite-derived cell lineages. These results suggest that paraxis is an essential component of the genetic pathway regulating somitogenesis. ^

A urokinase receptor promoter element (-152/-135) bound with an AP-2-alpha-related protein and SP1 is required for the induction of gene expression by PMA and SRC

The urokinase-type plasminogen activator receptor (u-PAR) promotes extracellular matrix degradation, invasion and metastasis. A first objective of this dissertation was to identify cis-elements and trans-acting factors activating u-PAR gene expression through a previously footprinted (–148/–124) promoter region. Mobility shifting experiments on nuclear extracts of a high u-PAR-expressing colon cancer cell line (RKO) indicated Sp1, Sp3 and a factor similar to, but distinct from, AP-2α bound to an oligonucleotide spanning –152/–135. Mutations preventing the binding of the AP-2α-related factor reduced u-PAR promoter activity. In RKO, the expression of a dominant negative AP-2 (AP-2αB) diminished u-PAR promoter activity, protein and u-PAR mediated laminin degradation. Conversely, u-PAR promoter activity in low u-PAR-expressing GEO cells was increased by AP-2αA expression. PMA treatment, which induces u-PAR expression, caused an increased amount of the AP-2α-related factor-containing complex in GEO, and mutations preventing AP-2α-like and Sp1/Sp3 binding reduced the u-PAR promoter stimulation by PMA. In resected colon cancers, u-PAR protein amounts were related to the amount of the AP-2α-related factor-containing complex. In conclusion, constitutive and PMA- inducible u-PAR gene expression and -proteolysis are mediated partly through transactivation via a promoter sequence (–152/435) bound with an AP-2α-related factor and Sp1/Sp3. ^ A second interest of this dissertation was to determine if a constitutively active Src regulates the transcription of the u-PAR gene, since c-src expression increases invasion in colon cancer. Increased u-PAR protein and laminin degradation paralleling elevated Src activity was evident in SW480 colon cancer cells stably expressing a constitutively active Src (Y- c-src527F). Nuclear run-on experiments indicated that this was due largely to transcriptional activation. While transient transfection of SW480 cells with Y-c-src527F induced a u-PAR-CAT-reporter, mutations preventing Sp1-binding to promoter region –152/435 abolished this induction. Mobility shift assays revealed increased Sp1 binding to region –152/135 with nuclear extracts of Src-transfected SW480 cells. Finally, the amounts of endogenous u-PAR in resected colon cancers significantly correlated with Src-activity. These data suggest that u-PAR gene expression and proteolysis are regulated by Src, this requiring the promoter region (–152/–135) bound with Sp1, thus, demonstrating for the first time that transcription factor Sp1 is a downstream effector of Src. ^

Control of the master virulence regulatory gene atxA in Bacillus anthracis

Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule are positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In batch culture, multiple signals impact atxA transcript levels, and the timing and steady state level of atxA expression is critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to directly interact with the atxA promoter. The AbrB-binding site has been described, but additional cis-acting control sequences have not been defined. Using transcriptional lacZ fusions, electrophoretic mobility shift assays, and Western blot analysis, the cis-acting elements and trans-acting factors involved in regulation of atxA in B. anthracis strains containing either both virulence plasmids, pXO1 and pXO2, or only one plasmid, pXO1, were studied. This work demonstrates that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA, and an A+T-rich upstream element (UP-element) for RNA polymerase (RNAP). In addition, the data show that a trans-acting protein(s) other than AbrB negatively impacts atxA transcription when it binds specifically to a 9-bp palindrome within atxA promoter sequences located downstream of P1. Mutation of the palindrome prevents binding of the trans-acting protein(s) and results in a corresponding increase in AtxA and anthrax toxin production in a strain- and culture-dependent manner. The identity of the trans-acting repressor protein(s) remains elusive; however, phenotypes associated with mutation of the repressor binding site have revealed that the trans-acting repressor protein(s) indirectly controls B. anthracis development. Mutation of the repressor binding site results in misregulation and overexpression of AtxA in conditions conducive for development, leading to a marked sporulation defect that is both atxA- and pXO2-61-dependent. pXO2-61 is homologous to the sensor domain of sporulation sensor histidine kinases and is proposed to titrate an activating signal away from the sporulation phosphorelay when overexpressed by AtxA. These results indicate that AtxA is not only a master virulence regulator, but also a modulator of proper B. anthracis development. Also demonstrated in this work is the impact of the developmental regulators AbrB, Spo0A, and SigH on atxA expression and anthrax toxin production in a genetically incomplete (pXO1+, pXO2-) and genetically complete (pXO1+, pXO2+) strain background. AtxA and anthrax toxin production resulting from deletion of the developmental regulators are strain-dependent suggesting that factors on pXO2 are involved in control of atxA. The only developmental deletion mutant that resulted in a prominent and consistent strain-independent increase in AtxA protein levels was an abrB-null mutant. As a result of increased AtxA levels, there is early and increased production of anthrax toxins in an abrB-null mutant. In addition, the abrB-null mutant exhibited an increase in virulence in a murine model for anthrax. In contrast, virulence of the atxA promoter mutant was unaffected in a murine model for anthrax despite the production of 5-fold more AtxA than the abrB-null mutant. These results imply that AtxA is not the only factor impacting pathogenesis in an abrB-null mutant. Overall, this work highlights the complex regulatory network that governs expression of atxA and provides an additional role for AtxA in B. anthracis development.

Regulation of the {\it neu\/} oncogene by the GTG element binding proteins

A previous study in our lab has shown that the transforming neu oncogene ($neu\sp\*$) was able to initiate signals that lead to repression of the neu promoter activity. Further deletion mapping of the neu promoter identified that the GTG element (GGTGGGGGGG), located between $-$243 and $-$234 relative to the translation initiation codon, mediates such a repression effect. I have characterized the four major protein complexes that interact with this GTG element. In situ UV-crosslinking indicated that each complex contains proteins of different molecular weights. The slowest migrating complex (S) contain Sp1 or Sp1-related proteins, as indicated by the data that both have similar molecular weights, similar properties in two affinity chromatographies, and both are antigenically related in gel shift analysis. Methylation protection and interference experiments demonstrated these complexes bind to overlapping regions of the GTG element. Mutations within the GTG element that either abrogate or enhance complex S binding conferred on the neu promoter with lower activity, indicating that positive factors other than Sp1 family proteins also contribute to neu promoter activity. A mutated version (mutant 4) of the GTG element, which binds mainly the fastest migrating complex that contains a very small protein of 26-kDa, can repress transcription when fused to a heterologous promoter. Further deletion and mutation studies suggested that this GTG mutant and its binding protein(s) may cooperate with some DNA element within a heterologous promoter to lock the basal transcription machinery; such a repressor might also repress neu transcription by interfering with the DNA binding of other transactivators. Our results suggest that both positive and negative trans-acting factors converge their binding sites on the GTG element and confer combinatorial control on the neu gene expression. ^

Transcriptional regulation of cell lineage-specific expression of the murine type I collagen genes and of osteoblast differentiation

The aim of my project is to examine the mechanisms of cell lineage-specific transcriptional regulation of the two type I collagen genes by characterizing critical cis-acting elements and trans-acting factors. I hypothesize that the transcription factors that are involved in the cell lineage-specific expression of these genes may have a larger essential role in cell lineage commitment and differentiation. I first examined the proximal promoters of the proα1(I) and the proα2(I) collagen genes for cell type-specific DNA-protein interactions, using in vitro DNaseI and in vivo DMS footprinting. These experiments demonstrated that the cis-acting elements in these promoters are accessible to ubiquitous DNA-binding proteins in fibroblasts that express these genes, but not in other cells that do not express these genes. I speculate that in type I collagen-expressing cells, cell type-specific enhancer elements facilitate binding of ubiquitous proteins to the proximal promoters of these genes. Subsequently, examination of the upstream promoter of the proα(I) collagen gene by transgenic mice experiments delineated a 117 bp sequence (-1656 to -1540 bp) as the minimum element required for osteoblast-specific expression. This 117 bp element contained two segments that appeared to have different functions: (1) the A-segment, which was necessary to obtain osteoblast-specific expression and (2) the C-segment, which was dispensable for osteoblast-specific expression, but was necessary to obtain high-level expression. In experiments to identify trans-acting factors that bind to the 117 bp element, I have demonstrated that the cell lineage-restricted homeodomain proteins, Dlx2, Dlx5 and mHOX, bound to the A-segment and that the ubiquitous transcription factor, Sp1, bound to the C-segment of this element. These results suggested a model where the binding of cell lineage-restricted proteins to the A-segment and of ubiquitous proteins to the C-segment of the 117 bp element of the proα1 (I) collagen gene activated this gene in osteoblasts. These results, combined with additional evidence that Dlx2, Dlx5 and mHOX are probably involved in osteoblast differentiation, support my hypothesis that the transcription factors involved in osteoblast-specific expression of type I collagen genes may have essential role in osteoblast lineage commitment and differentiation. ^

Regulatory factors and control of anthrax toxin gene expression

Bacillus anthracis plasmid pXO1 carries genes for three anthrax toxin proteins, pag (protective antigen), cya (edema factor), and lef (lethal factor). Expression of the toxin genes is enhanced by two signals: CO$\sb2$/bicarbonate and temperature. The CO$\sb2$/bicarbonate effect requires the presence of pXO1. I hypothesized that pXO1 harbors a trans-acting regulatory gene(s) required for CO$\sb2$/bicarbonate-enhanced expression of the toxin genes. Characterization of such a gene(s) will lead to increased understanding of the mechanisms by which B. anthracis senses and responds to host environments.^ A regulatory gene (atxA) on pXO1 was identified. Transcription of all three toxin genes is decreased in an atxA-null mutant. There are two transcriptional start sites for pag. Transcription from the major site, P1, is enhanced in elevated CO$\sb2$. Only P1 transcripts are significantly decreased in the atxA mutant. Deletion analysis of the pag upstream region indicates that the 111-bp region upstream of the P1 site is sufficient for atxA-mediated increase of this transcript. The cya and lef genes each have one apparent transcriptional start site. The cya and lef transcripts are significantly decreased in the atxA mutant. The atxA mutant is avirulent in mice. The antibody response to all three toxin proteins is significantly decreased in atxA mutant-infected mice. These data suggest that the atxA gene product activates expression of the toxin genes and is essential for virulence.^ Since expression of the toxin genes is dependent on atxA, whether increased toxin gene expression in response to CO$\sb2$/bicarbonate and temperature is associated with increased atxA expression was investigated. I monitored steady state levels of atxA mRNA and AtxA protein in different growth conditions. The results indicate that expression of atxA is not influenced by CO$\sb2$/bicarbonate. Steady state levels of atxA mRNA and AtxA protein are higher at 37$\sp\circ$C than 28$\sp\circ$C. However, increased pag expression at high temperature can not be attributed directly to increased atxA expression.^ There is evidence that an additional factor(s) may be involved in regulation of pag. Expression of pag in strains overproducing AtxA is significantly decreased compared to the wildtype strain. A specific interaction of tagged-AtxA with the pag upstream DNA has not been demonstrated. Furthermore, four proteins in B. anthracis extract can be co-immunoprecipitated with tagged-AtxA. Amino-terminal sequence of one protein has been determined and found highly homologous to chaperonins of GroEL family. Studies are under way to determine if this GroEL-like protein interactions with AtxA and plays any role in atxA-mediated activation of toxin genes. ^

Investigation of the deregulation of interleukin-6 in two non-Hodgkin's lymphoma cell lines

Increased serum interleukin-6 (IL-6) is a poor prognostic factor for patients with lymphoma. This may be related to the fact that IL-6 has been shown to be an autocrine and paracrine growth factor for lymphoma cells. We have investigated the regulation of IL-6 in two lymphoma cell lines which produce IL-6 as an autocrine growth factor. The cell lines, LY3 and LY12, were established from two patients with non-Hodgkin's lymphoma. One patient had diffuse large cell lymphoma (LY3), whereas the other had small noncleaved cell lymphoma (LY12). There was no rearrangement or amplification of the IL-6 gene, but we detected IL-1 alpha and TNF production in addition to IL-6. We investigated the effect of inhibitors of IL-1 and TNF on IL-6 production in LY3 and LY12. Our results show that IL-6 production is mainly secondary to endogenous IL-1 production in LY3 cells, however LY12 cells produce IL-6 via a different mechanism since neither anti-IL-1 nor anti-TNF significantly inhibited IL-6 production.^ Transfection of LY12 cells with wildtype and mutant IL-6 promoter-chloramphenicol acetyl transferase constructs, showed increased activity of a trans-acting factor that binds to the NF-kB motif. Therefore, we determined whether there were abnormalities in members of the NF-kB family of transcription factors, such as p65, p50, p52/lyt-10 or rel, which bind to kB motifs. We found increased expression of the p52/lyt-10 transcription factor and activation of the NF-kB pathway in LY12. However, expression of p50, p65 and rel was not increased in LY12 cells. Future investigations could be aimed at determining the effect of inhibitors of NF-kB on IL-6 production. ^

A FUNCTIONAL VARIANT OF HLA-DR1 DELINEATED BY T-LYMPHOCYTE CLONE REACTIVITY, PROTEIN CONFORMATION, AND GENOMIC RESTRICTION SITES

This research characterized a serologically indistinguishable form of HLA-DR1 that: (1) cannot stimulate some DR1-restricted or specific T-lymphocyte clones; (2) displays an unusual electrophoretic pattern on two dimensional gels; and (3) is marked by a polymorphic restriction site of the alpha gene. Inefficient stimulation of some DR1-restricted clones was a property of DR1$\sp{+}$ cells that shared HLA-B14 on the same haplotype and/or were carriers of 21-hydroxylase (21-OH) deficiency. Nonclassical 21-OH deficiency frequently demonstrates genetic linkage with HLA-B14;DR1 haplotypes and associates with duplications of C4B and one 21-OH gene. Cells having both stimulatory (DR1$\sb{\rm n}$) and nonstimulatory (DR1$\sb{\rm x}$) parental haplotypes did not mediate proliferation of these clones. However, heterozygous DR1$\sb{\rm x}$, 2 and DR1$\sb{\rm x}$, 7 cells were efficient stimulators of DR2 and DR7 specific clones, respectively, suggesting that a trans acting factor may modify DR1 alleles or products to yield a dominant DR1$\sb{\rm x}$ phenotype. Incompetent stimulator populations did not secrete an intercellular soluble or contact dependent suppressor factor nor did they express interleukin-2 receptors competing for T-cell growth factors. Two dimensional gel analysis of anti-DR immunoprecipitates revealed, in addition to normal DR$\alpha$ and DR$\beta$ chains, a 50kD species from DR1$\sb{\rm x}$ but not from the majority of DR1$\sb{\rm n}$ or non-DR1 cells. The 50kD structure was stable under reducing conditions in SDS and urea, had antigenic homology with DR, and dissociated after boiling into 34kD and 28kD peptide chains apparently identical with DR$\alpha$ and DR$\beta$ as shown by limited digest peptide maps. N-linked glycosylation and sialation of DRgp50 appeared to be unchanged from normal DR$\alpha$ and DR$\beta$. Bg1II digestion and $DR\alpha$ probing of DR1$\sb{\rm x}$ genomic DNA revealed a 4.5kb fragment while DR1$\sb{\rm n}$ DNA yielded 3.8 and 0.76kb fragments; all restriction sites mapped to the 3$\sp\prime$ untranslated region of $DR\alpha$. Collectively, these data suggest that DRgp50 represents a novel combinatorial association between constitutive chains of DR that may interfere with or compete for normal T cell receptor recognition of DR1 as both an alloantigen and restricting element. Furthermore, extensive chromosomal abnormalities previously mapped to the class III region of B14;DR1 haplotypes may extend into the adjacent class II region with consequent intrusion on immune function. ^

Genetic mechanisms of Na+, K+-ATPase regulation in hypertension

The spontaneously hypertensive rat (SHR) is a model of essential hypertension. During the early development of hypertension, the SHR demonstrates increased proximal tubule (PT) Na+ reabsorption. I hypothesized that the increased PT Na+ reabsorption exhibited by the young SHR was due to altered sub-cellular distribution of Na+, K +-ATPase compared to the normotensive Wistar Kyoto (WKY). The hypothesis is supported, herein, by observations of greater Na+, K +-ATPase α 1 abundance in PT plasma membrane and lower abundance in late endosomes of 4wk SHR despite no difference in total PT α 1 abundance. There is a greater amount of Ser-18 unphosphorylated α 1 in the 4wk SHR PT. Total PT Na+, K+-ATPase γ abundance is greater in SHR at 4wk and 16wk but γ abundance in plasma membrane is greater only at 4wk. The phosphatase, calcineurin, was chosen for study because it is involved in the stimulation of Na+, K +-ATPase. No difference in calcineurin coding sequence, expression, or activity was observed in SHR. Gene expression arrays were next used to find candidate genes involved in the regulation of Na+, K +-ATPase. The first candidate analyzed was soluble epoxide hydrolase (sEH). The gene encoding sEH (EPHX2) showed lower expression in SHR. There was also a reduction in sEH protein abundance but there was no correlation between protein abundance and blood pressure in F2 progeny. Two EPHX2 alleles were identified, an ancestral allele and a variant allele containing four polymorphisms. sEH activity was greater in animals carrying the variant allele but the inheritance of the variant allele did not correlate with blood pressure. Gene expression arrays also led to the examination of genes involved in redox balance/Na+, K+-ATPase regulation. A pattern of lower expression of genes involved in reactive radical detoxification in SHR was discerned. Six transcription factor binding sites were identified that occurred more often in these genes. Three transcription factors that bind to the HNF1 site were expressed at lower levels in SHR. This points to the HNF1 transcriptional complex as an important trans-acting regulator of a wide range of genes involved in altered redox balance in SHR. ^

Molecular and genetic analyses of Fgf signaling during cardiac outflow tract development

Epithelial-mesenchymal tissue interactions regulate the development of derivatives of the caudal pharyngeal arches (PAs) to govern the ultimate morphogenesis of the aortic arch and outflow tract (OFT) of the heart. Disruption of these signaling pathways is thought to contribute to the pathology of a significant proportion of congenital cardiovascular defects in humans. In this study, I tested whether Fibroblast Growth Factor 15 (Fgf15), a secreted signaling molecule expressed within the PAs, is an extracellular mediator of tissue interactions during PA and OFT development. Analyses of Fgf15−/− mouse embryonic hearts revealed abnormalities primarily localized to the OFT, correlating with aberrant cardiac neural crest cell behavior. The T-box-containing transcription factor Tbx1 has been implicated in the cardiovascular defects associated with the human 22q11 Deletion Syndromes, and regulates the expression of other Fgf family members within the mouse PAs. However, expression and genetic interaction studies incorporating mice deficient for Tbx1, its upstream regulator, Sonic Hedgehog (Shh), or its putative downstream effector, Fgf8, indicated that Fgf15 functions during OFT development in a manner independent of these factors. Rather, analyses of compound mutant mice indicated that Fgf15 and Fgf9, an additional Fgf family member expressed within the PAs, genetically interact, providing insight into the factors acting in conjunction with Fgf15 during OFT development. Finally, in an effort to further characterize this Fgf15-mediated developmental pathway, promoter deletion analyses were employed to isolate a 415bp sequence 7.1Kb 5′ to the Fgf15 transcription start site both necessary and sufficient to drive reporter gene expression within the epithelium of the PAs. Sequence comparisons among multiple mammalian species facilitated the identification of evolutionarily conserved potential trans-acting factor binding sites within this fragment. Subsequent studies will investigate the molecular pathway(s) through which Fgf15 functions via identification of factors that bind to this element to govern Fgf15 gene expression. Furthermore, targeted deletion of this element will establish the developmental requirement for pharyngeal epithelium-derived Fgf15 signaling function. Taken as a whole, these data demonstrate that Fgf15 is a component of a novel, Tbx1-independent molecular pathway, functioning within the PAs in a manner cooperative with Fgf9, required for proper development of the cardiac OFT. ^

Cis-acting elements and developmental regulators govern toxin gene expression in Bacillus anthracis

Expression of the structural genes for the anthrax toxin proteins is coordinately controlled by host-related signals such as elevated CO2 , and the trans-acting positive regulator, AtxA. No specific binding of AtxA to the toxin gene promoters has been demonstrated and no sequence-based similarities are apparent in the promoter regions of toxin genes. We hypothesized that the toxin genes possess common structural features that are required for positive regulation. To test this hypothesis, I performed an extensive characterization of the toxin gene promoters. I determined the minimal sequences required for atxA-mediated toxin gene expression and compared these sequences for structural similarities. In silico modeling and in vitro experiments indicated significant curvature within these regions. Random mutagenesis revealed that point mutations associated with reduced transcriptional activity, mostly mapped to areas of high curvature. This work enabled the identification of two potential cis-acting elements implicated in AtxA-mediated regulation of the toxin genes. In addition to the growth condition requirements and AtxA, toxin gene expression is under growth phase regulation. The transition state regulator AbrB represses atxA expression to influence toxin synthesis. Here I report that toxin gene expression also requires sigH, a gene encoding the RNA polymerase sigma factor associated with development in B. subtilis. In the well-studied B. subtilis system, σH is part of a feedback control pathway that involves AbrB and the major response regulator of sporulation initiation, Spo0A. My data indicate that in B. anthracis, regulatory relationships exist between these developmental regulators and atxA . Interestingly, during growth in toxin-inducing conditions, sigH and abrB expression deviates from that described for B. subtilis, affecting expression of the atxA gene. These findings, combined with previous observations, suggest that the steady state level of atxA expression is critical for optimal toxin gene transcription. I propose a model whereby, under toxin-inducing conditions, control of toxin gene expression is fine-tuned by the independent effects of the developmental regulators on the expression of atxA . The growth condition-dependent changes in expression of these regulators may be crucial for the correct timing and uninterrupted expression of the toxin genes during infection. ^

Analysis of endogenous retinoic acid receptor expression and function in murine melanoma cells

There have been numerous reports over the past several years on the ability of vitamin A analogs (retinoids) to modulate cell proliferation, malignant transformation, morphogenesis, and differentiation in a wide variety of cell types and organisms. Two families of nuclear retinoid-inducible, trans-acting, transcription-enhancing receptors that bear strong DNA sequence homology to thyroid and steroid hormone receptors have recently been discovered. The retinoic acid receptors (RARs) and retinoid X receptors (RXRs) each have at least three types designated $\alpha,$ $\beta,$ and $\gamma,$ which are encoded by separate genes and expressed in a tissue and cell type-specific manner. We have been interested in the mechanism by which retinoids inhibit tumor cell proliferation and induce differentiation. As a model system we have employed several murine melanoma cell lines (S91-C2, K1735P, and B16-F1), which are sensitive to the growth-inhibitory and differentiation-inducing effects of RA, as well as a RA-resistant subclone of one of the cell lines (S91-C154), in order to study the role of the nuclear RARs in these effects. The initial phase of this project consisted of the characterization of the expression pattern of the three known RAR and RXR types in the murine melanoma cell lines in order to determine whether any differences exist which may elucidate a role for any of the receptors in RA-induced growth inhibition and differentiation. The novel finding was made that the RAR-$\beta$ gene is rapidly induced from undetectable levels by RA treatment at the mRNA and protein level, and that the induction of RAR-$\beta$ by other biologically active retinoids correlated with their ability to inhibit the growth of the highly RA-sensitive S91-C2 cell line. This suggests a role for RAR-$\beta$ in the growth inhibiting effect of retinoids. The second phase of this project involves the stable expression of RAR-$\beta$ in the S91-C2 cells and the RAR-$\beta$ receptor-null cell line, K1735P. These studies have indicated an inverse correlation between RAR-$\beta$ expression and proliferation rate. ^

An extracytoplasmic function sigma factor operon regulates Myxococcus xanthus developmental gene expression

Myxococcus xanthus is a Gram-negative soil bacterium that undergoes multicellular development when high-density cells are starved on a solid surface. Expression of the 4445 gene, predicted to encode a periplasmic protein, commences 1.5 h after the initiation of development and requires starvation and high density conditions. Addition of crude or boiled supernatant from starving high-density cells restored 4445 expression to starving low-density cells. Addition of L-threonine or L-isoleucine to starving low-density cells also restored 4445 expression, indicating that the high-density signaling activity present in the supernatant might be composed of extracellular amino acids or small peptides. To investigate the circuitry integrating these starvation and high-density signals, the cis- and trans-acting elements controlling 4445 expression were identified. The 4445 transcription start site was determined by primer extension analysis to be 58 by upstream of the predicted translation start site. The promoter region contained a consensus sequence characteristic of e&barbelow;xtrac&barbelow;ytoplasmic f&barbelow;unction (ECF) sigma factor-dependent promoters, suggesting that 4445 expression might be regulated by an ECF sigma factor-dependent pathway, which are known to respond to envelope stresses. The small size of the minimum regulatory region, identified by 5′-end deletion analysis as being only 66 by upstream of the transcription start site, suggests that RNA polymerase could be the sole direct regulator of 4445 expression. To identify trans-acting negative regulators of 4445 expression, a strain containing a 4445-lacZ was mutagenized using the Himar1-tet transposon. The four transposon insertions characterized mapped to an operon encoding a putative ECF sigma factor, ecfA; an anti-sigma factor, reaA; and a negative regulator, reaB. The reaA and the reaB mutants expressed 4445 during growth and development at levels almost 100-fold higher than wild type, indicating that these genes encode negative regulators. The ecfA mutant expressed 4445-lacZ at basal levels, indicating that ecfA is a positive regulator. High Mg2+ concentrations over-stimulated this ecfA pathway possibly due to the depletion of exopolysaccharides and assembled type IV pili. These data indicate that the ecfA operon encodes a new regulatory stress pathway that integrates and transduces starvation and cell density cues during early development and is also responsive to cell-surface alterations.^

The transcriptional regulation of the {\it neu\/} gene: Examples of activation, repression, and cell-specific expression

The neu oncogene encodes a growth factor receptor-like protein, p185, with an intrinsic tyrosine kinase activity. A single point mutation, an A to T transversion resulting in an amino acid substitution from valine to glutamic acid, in the transmembrane domain of the rat neu gene was found to be responsible for the transforming and tumorigenic phenotype of the cells that carry it. In contrast, the human proto-neu oncogene is frequently amplified in tumors and cell lines derived from tumors and the human neu gene overexpression/amplification in breast and ovarian cancers is known to correlate with poor patient prognosis. Examples of the human neu gene overexpression in the absence of gene amplification have been observed, which may suggest the significant role of the transcriptional and/or post-transcriptional control of the neu gene in the oncogenic process. However, little is known about the transcriptional mechanisms which regulate the neu gene expression. In this study, three examples are presented to demonstrate the positive and negative control of the neu gene expression.^ First, by using band shift assays and methylation interference analyses, I have identified a specific protein-binding sequence, AAGATAAAACC ($-$466 to $-$456), that binds a specific trans-acting factor termed RVF (for EcoRV factor on the neu promoter). The RVF-binding site is required for maximum transcriptional activity of the rat neu promoter. This same sequence is also found in the corresponding regions of both human and mouse neu promoters. Furthermore, this sequence can enhance the CAT activity driven by a minimum promoter of the thymidine kinase gene in an orientation-independent manner, and thus it behaves as an enhancer. In addition, Southwestern (DNA-protein) blot analysis using the RVF-binding site as a probe points to a 60-kDa polypeptide as a potential candidate for RVF.^ Second, it has been reported that the E3 region of adenovirus 5 induces down-regulation of epidermal growth factor (EGF) receptor through endocytosis. I found that the human neu gene product, p185, (an EGF receptor-related protein) is also down-regulated by adenovirus 5, but via a different mechanism. I demonstrate that the adenovirus E1a gene is responsible for the repression of the human neu gene at the transcriptional level.^ Third, a differential expression of the neu gene has been found in two cell model systems: between the mouse fibroblast Swiss-Webster 3T3 (SW3T3) and its variant NR-6 cells; and between the mouse liver tumor cell line, Hep1-a, and the mouse pancreas tumor cell line, 266-6. Both NR-6 and 266-6 cell lines are not able to express the neu gene product, p185. I demonstrate that, in both cases, the transcriptional repression of the neu gene may account for the lack of the p185 expression in these two cell lines. ^