Mechanism of molecular regulation of nuclear -encoded mitochondrial gene expression by electrical stimulation in the cultured neonatal rat cardiac myocytes

To answer the question whether increased energy demand resulting from myocyte hypertrophy and enhanced $\beta$-myosin heavy chain mRNA, contractile protein synthesis and assembly leads to mitochondrial proliferation and differentiation, we set up an electrical stimulation model of cultured neonatal rat cardiac myocytes. We describe, as a result of increased contractile activity, increased mitochondrial profiles, cytochrome oxidase mRNA, and activity, as well as a switch in mitochondrial carnitine palmitoyltransferase-I (CPT-I) from the liver to muscle isoform. We investigate physiological pathways that lead to accumulation of gene transcripts for nuclear encoded mitochondrial proteins in the heart. Cardiomyocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation (c-fos, c-jun, junB, nuclear respiratory factor 1 (Nrf-1)), mitochondrial proliferation (cytochrome c (Cyt c), cytochrome oxidase), and mitochondrial differentiation (carnitine palmitonyltransferase I (CPT-I) isoforms) were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25-3 hr) and followed by c-jun (0.5-3 hr), junB (0.5-6 hr), NRF-1 (1-12 hr), Cyt c (12-72 hr), cytochrome c oxidase (12-72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA. Electrical stimulation increased c-fos, $\beta$-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element (CRE), and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the Nrf-1 and CRE sites inhibited the induction by electrical stimulation or by transfection of c-jun into non-paced cardiac myocytes whereas mutation of the Sp-1 site maintained or increased the fold induction. This is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c. Overexpression of c-jun by transfection also activates the Nrf-1 and Cyt c mRNA sequentially. Electrical stimulation of cardiac myocytes activates the c-Jun-N-terminal kinase so that the fold-activation of the cyt c promoter is increased by pacing when either c-jun or c-fos/c-jun are cotransfected. We have identified physical association of Nrf-1 protein with the Nrf-1 enhancer element and of c-Jun with the CRE binding sites on the Cyt c promoter. This is the first demonstration that induction of Nrf-1 and c-Jun by pacing of cardiac myocytes directly mediates Cyt c gene expression and mitochondrial proliferation in response to hypertrophic stimuli in the heart.^ Subsequent to gene activation pathways that lead to mitochondrial proliferation, we observed an isoform switch in CPT-I from the liver to muscle mRNA. We have found that the half-life for the muscle CPT-I is not affected by electrical stimulation, but electrical decrease the T1/2 in the liver CPT-I by greater than 50%. This suggests that the liver CPT-I switch to muscle isoform is due to (1) a decrease in T1/2 of liver CPT-I and (2) activation of muscle CPT-Itranscripts by electrical stimulation. (Abstract shortened by UMI.) ^

Regulation of Spec genes in aboral ectoderm cells of sea urchin {\it Strongylocentrotus purpuratus}

The Spec genes of the sea urchin Stronylocentrotus purpuratus serves as an excellent model for studying cell type-specific gene expression during early embryogenesis. The Spec1/Spec2 genes encode cytosolic calcium-binding proteins related to the calmodulin/troponin C/myosin light chain superfamily. Members of the Spec gene family are activated shortly after the sixth cleavage as the lineage-specific founder cells giving rise to aboral ectoderm are established, and the accumulation of the Spec mRNAs is limited exclusively to aboral ectoderm cell lineages. In this dissertation, the transcriptional regulation of the Spec genes was studied. Sequence comparisons of the Spec gene 5$\sp\prime$ flanking regions showed that a DNA block of approximately 800 bp from the 3$\sp\prime$ end of the first exon to the 5$\sp\prime$ end of a repetitive DNA element, termed RSR, was highly conserved. In Spec2a, the conserved region was a continuous stretch of DNA, but in Spec1 and Spec2c, DNA insertions interrupt the conserved sequence block and alter the relative placement of the RSR element and other 5$\sp\prime$ flanking DNA. Thus, drastic rearrangements have occurred within the putative control regions of the Spec genes. In vivo expression experiments using the sea urchin embryo gene-transfer system showed that while the 5$\sp\prime$ flanking regions of all three Spec genes conferred proper temporal activation to the reporter CAT gene, only the Spec2a 5$\sp\prime$ flanking region could restrict lacZ gene expression to aboral ectoderm cells. However, the Spec2a conserved region alone was not sufficient to confer proper spatial expression, suggesting that negative spatial elements are also associated with the proper activation of Spec2a. A major positive regulatory region, defined as the RSR enhancer, was identified between base pairs $-$631 and $-$443 on Spec2a. The RSR enhancer was essential for maximal activity and conferred preferential aboral ectoderm expression to a lacZ reporter gene. DNaseI footprinting and band-shift analysis of the RSR enhancer revealed multiple DNA-elements. One of the elements, an A/T-rich sequence called the A/T palindrome was studied in detail. This element binds a single 45-kDa nuclear protein, the A/T palindrome binding protein (A/TBP), whose DNA-binding specificity suggests a possible relationship with the bicoid-class homeodomain proteins. Mutated A/T palindromes are incapable of binding the 45-kDa protein and lower promoter activity by 8-fold. DNA-binding activity for A/TBP is low in unfertilized eggs, increases by the 16-cell stage and continues rising in blastulae. These data suggest that A/TBP plays a major role in the activation of the Spec2a gene in aboral ectoderm cells. ^

Regulation of {\it myogenin\/} transcription during mouse embryogenesis

The formation of skeletal muscle during vertebrate development involves the induction of mesoderm and subsequent generation of myoblasts that ultimately differentiate into mature muscles. The recent identification of a group of myogenic regulators that can convert fibroblasts to myoblasts has contributed to our understanding of the molecular events that underlie the establishment of the skeletal muscle phenotype. Members of this group of myogenic regulators share a helix-loop-helix (HLH) motif that mediates DNA binding. The myogenic HLH proteins bind to the consensus sequence CANNTG, referred to as an E-box, and activate muscle-specific transcription. In addition to E-boxes, other motifs, such as the MEF-2 binding site, have been shown to mediate muscle-specific transcription. The myogenic HLH proteins are expressed in the myogenic precursors in somites and limb buds, and in differentiated muscle fibers during embryogenesis, consistent with their roles as regulators for muscle development. The myogenic HLH proteins appear to auto-activate their own and cross-activate one another's expression in cultured cells. Myogenin is one of the myogenic HLH proteins and likely the regulator for terminal muscle differentiation. Myogenin is a common target of diverse regulatory pathways. To search for upstream regulators of myogenin, we studied regulation of myogenin transcription during mouse embryogenesis. We showed that the myogenin promoter contains a binding site for MEF-2, which can mediate indirectly the autoregulation of myogenin transcription. We found that a transgene under the control of a 1.5 kb 5$\sp\prime$ flanking sequence can recapitulate the temporal and spatial expression pattern of the endogenous myogenin gene during mouse embryogenesis. By tracing embryonic cells that activate myogenin-lacZ during embryogenesis, we found no evidence that lacZ was expressed in myogenic precursors migrating from somites to limb buds, suggesting the existence of regulators other than myogenic HLH proteins that can maintain cells in the myogenic lineage. Mutations of an E-box and a MEF-2 site in the myogenin promoter suppressed transcription in subsets of myogenic precursors in mouse embryos. These results suggest that myogenic HLH proteins and MEF-2 participate in separable regulatory pathways controlling myogenin transcription and provide evidence for positional regulation of myogenic regulators in the embryo. ^

REGULATION OF THE EXPRESSION OF NAR OPERON ENCODING NITRATE REDUCTASE IN ESCHERICHIA COLI

The nar operon, which encodes the nitrate reductase in Escherichia coli, can be induced under anaerobic conditions without nitrate to a low level and with nitrate to a maximum level. The anaerobic formation of nitrate reductase is dependent upon the fnr gene product while the narL gene product is required for further induction by nitrate. The sequence was determined across the entire promoter and regulatory region of the nar operon. The translational start site of the first structural gene of the nar operon, narG gene, was established by identifying the nucleotide sequence for the first 20 N-terminal amino acid residues of the alpha subunit of nitrate reductase. The transcriptional start site and the level of the transcript was determined by S1 mapping procedure. One major transcript was identified which was initiated 50 base pair (bp) upstream from the translational start site of the first structural gene. The synthesis of the transcript was repressed aerobically, fully induced by nitrate anaerobically, and greatly reduced in a ${\rm Fnr\sp-}$ mutant. Deletions were created in the 5$\sp\prime$ nar regulatory sequence with either an intact nar operon or a nar::lacZ fusion. The expression of the plasmids with deletions were determined in a strain with wild type fnr and narL loci, a Fnr- mutant strain and a NarL- mutant strain. These experiments demonstrated that the $5\sp\prime$ limit of the nar operon lies at about $-210$ bp from the transcription start site. The region required for anaerobic induction by the fnr gene product is located around $-60$ bp. Two putative narL recognition sites were identified, one of which is around $-200$ and another immediately adjacent to the fnr recognition region. The deletion of the sequences around $-200$ rendered the remaining narL complex repressive and thus decreased the expression of nar operon, suggesting that the two potential narL sites interact with each other over a significant length of DNA. ^

Cloning, characterization and regulation of the soluble guanylyl cyclase alpha1 and beta1 genes

Cell signaling by nitric oxide (NO) through soluble guanylyl cyclase (sGC) and cGMP production regulates physiological responses such as smooth muscle relaxation, neurotransmission, and cell growth and differentiation. Although the NO receptor, sGC, has been studied extensively at the protein level, information on regulation of the sGC genes remains elusive. In order to understand the molecular mechanisms involved at the level of gene expression, cDNA and genomic fragments of the murine sGCα1 subunit gene were obtained through library screenings. Using the acquired clones, the sGCα 1 gene structure was determined following primer extension, 3 ′RACE and intron/exon boundary analyses. The basal activity of several 5′-flanking regions (putative promoter regions) for both the α1 and β1 sGC subunits were determined following their transfection into mouse N1E-115 neuroblastoma and rat RENE1Δ14 uterine epithelial cells using a luciferase reporter plasmid. Using the sGC sequences, real-time RT-PCR assays were designed to measure mRNA levels of the sGC α1 and β1 genes in rat, mouse and human. Subsequent studies found that uterine sGC mRNA and protein levels decreased rapidly in response to 17β-estradiol (estrogen) in an in vivo rat model. As early as 1 hour following treatment, mRNA levels of both sGC mRNAs decreased, and reached their lowest level of expression after 3 hours. This in vivo response was completely blocked by the pure estrogen receptor antagonist, ICI 182,780, was not seen in several other tissues examined, did not occur in response to other steroid hormones, and was due to a post-transcriptional mechanism. Additional studies ex vivo and in various cell culture models suggested that the estrogen-mediated decreased sGC mRNA expression did not require signals from other tissues, but may require cell communication or paracrine factors between different cell types within the uterus. Using chemical inhibitors and molecular targeting in other related studies, it was revealed that c-Jun-N-terminal kinase (JNK) signaling was responsible for decreased sGC mRNA expression in rat PC12 and RFL-6 cells, two models previously determined to exhibit rapid decreased sGC mRNA expression in response to different stimuli. To further investigate the post-transcriptional gene regulation, the full length sGCα1 3′-untranslated region (3′UTR) was cloned from rat uterine tissue and ligated downstream of the rabbit β-globin gene and expressed as a chimeric mRNA in the rat PC12 and RFL-6 cell models. Expression studies with the chimeric mRNA showed that the sGCα 1 3′UTR was not sufficient to mediate the post-transcriptional regulation of its mRNA by JNK or cAMP signaling in PC12 and RFL-6 cells. This study has provided numerous valuable tools for future studies involving the molecular regulation of the sGC genes. Importantly, the present results identified a novel paradigm and a previously unknown signaling pathway for sGC mRNA regulation that could potentially be exploited to treat diseases such as uterine cancers, neuronal disorders, hypertension or various inflammatory conditions. ^

Regulation of Alix by an autoinhibitory intramolecular interaction

In eukaryotic cells, the ESCRTs (endosomal sorting complexes required for transport) machinery is required for cellular processes such as endosomal sorting, retroviral budding and cytokinesis. The ALG-2 interacting protein Alix is a modular adaptor protein that is critically involved in these ESCRTs-associated cellular processes and consists of an N-terminal Bro1 domain, a middle V domain and C-terminal Pro-rich domain (PRD). In these cellular processes, Alix interacts with the ESCRT-III component CHMP4 at the Bro1 domain, with HIV-1 p6 Gag or EIAV p9Gag at the V domain, and with the ESCRT-I component TSG101 at the Pro-rich domain. Here we demonstrate that the N-terminal Bro1 domain forms an intramolecular interaction with C-terminal PRD within Alix. This Bro1-PRD intramolecular interaction forms a closed conformation of Alix that autoinhibits Alix interaction with all of these partner proteins. Moreover, the binding of Ca2+-activated ALG-2 to the PRD of Alix relieves the autoinhibitory intramolecular interaction, resulting in an open conformation of Alix which is able to interact with all of these partner proteins. The partner proteins bound to Alix in turn maintain Alix in the open conformation after ALG-2 dissociation with Alix. Consistent with the effect of Ca2+-activated ALG-2 on opening/activating Alix in these ESCRTs-associated functions, ALG-2 overexpression accelerates EGF-induced degradation of EGFR in an Alix-dependent manner. These findings discover an intrinsic autoinhibitory mechanism of Alix and a two-step process to activate/open Alix and then keep Alix active/open. This study has solved long-standing issues on the regulations of Alix in ESCRTs-associated functions and the role of ALG-2-Alix interaction, and may serve as the structural basis for further studies about Alix regulations. ^

Regulation of chromatin structure, Smad binding and AFP expression by the Forkhead box transcription factor: Foxa1

Transcription factors must be able to access their DNA binding sites to either activate or repress transcription. However, DNA wrapping and compaction into chromatin occludes most binding sites from ready access by proteins. Pioneer transcription factors are capable of binding their DNA elements within a condensed chromatin context and then reducing the level of nucleosome occupancy so that the chromatin structure is more accessible. This altered accessibility increases the probability of other transcription factors binding to their own DNA binding elements. My hypothesis is that Foxa1, a ‘pioneer’ transcription factor, activates alpha-fetoprotein (AFP) expression by binding DNA in a chromatinized environment, reducing the nucleosome occupancy and facilitating binding of additional transcription factors.^ Using retinoic-acid differentiated mouse embryonic stem cells, we illustrate a mechanism for activation of the tumor marker AFP by the pioneer transcription factor Foxa1 and TGF-β downstream effector transcription factors Smad2 and Smad4. In differentiating embryonic stem cells, binding of the Foxa1 forkhead box transcription factor to chromatin reduces nucleosome occupancy and levels of linker histone H1 at the AFP distal promoter. The more accessible DNA is subsequently bound by the Smad2 and Smad4 transcription factors, concurrent with activation of transcription. Chromatin immunoprecipitation analyses combined with siRNA-mediated knockdown indicate that Smad protein binding and the reduction of nucleosome occupancy at the AFP distal promoter is dependent on Foxa1. In addition to facilitating transcription factor binding, Foxa1 is also associated with histone modifications related to active gene expression. Acetylation of lysine 9 on histone H3, a mark that is associated active transcription, is dependent on Foxa1, while methylation of H3K4, also associated with active transcription, is independent of Foxa1. I propose that Foxa1 potentiates a region of chromatin to respond to Smad proteins, leading to active expression of AFP.^ These studies demonstrate one mechanism whereby a transcription factor can alter the accessibility of additional transcription factors to chromatin, by altering nucleosome positions. Specifically, Foxa1 exposes DNA so that Smad4 can bind to its regulatory element and activate transcription of the tumor-marker gene AFP.^

Regulation of mRNA splicing in Moloney murine sarcoma virus mutants

Cells infected with the conditionally defective MuSVts110 mutant of Moloney murine sarcoma virus are transformed at 33$\sp\circ$C but appear morphologically normal at 39$\sp\circ$C. The molecular basis for this phenotype is as follows: MuSVts110 contains a 1487 nucleotide central deletion that has truncated the 3$\sp\prime$ end to the gag gene and the 5$\sp\prime$ end of the mos gene. The resulting gag-mos junction is out-of-frame and the v-mos protein is not expressed. At 33$\sp\circ$C or lower, a splicing event is activated such that a 431 base intron is removed to realign the gag and mos gene in-frame, allowing the expression of a transforming protein P85$\sp{gag-mos}$. Temperature-dependent splicing appeared to be an intrinsic property of MuSVts110 transcripts and not a general feature of pre-mRNA splicing in 6m2 cells since splicing activity of a heterologous transcript in the same cells did not vary with temperature. The possibility that the splice event was not temperature-sensitive, but that the accumulation of spliced transcript at the lower growth temperatures was due to its selective thermolability was ruled out as stability studies revealed that the relative turnover rates of the unspliced and spliced MuSVts110 transcripts were not affected by temperature.^ The consensus sequences containing the splice sites activated in the MuSVts110 mutant (5$\sp\prime$ gag and 3$\sp\prime$ mos) are present, but not utilized, in wild-type MuSV-124. To test the hypothesis that it was the reduction of the 1919 base intervening sequence in MuSV-124 to 431 bases in MuSVts110 which activated splicing, the identical 1487 base deletion was introduced into cloned wild-type MuSV-124 DNA to create the MuSVts110 equivalent, ts32.^ To examine conditions permissive for splicing, we assayed splice site activation in a series of MuSV-124 "intron-modification" mutants. Data suggest that splicing in wild-type MuSV-124 may be blocked due to the lack of a proximal branchpoint sequence, but can be activated by those intron mutations which reposition a branch site closer to the 3$\sp\prime$ splice site. (Abstract shortened with permission of author.) ^

Regulation of myogenesis by growth signals -growth factors, oncogenes and protein kinases

Establishment of a myogenic phenotype involves antagonism between cell proliferation and differentiation. The recent identification of the MyoD family of muscle-specific transcription factors provides opportunities to dissect at the molecular level the mechanisms through which defined cell type-specific transcription factors respond to environmental cues and regulate differentiation programs. This project is aimed at elucidation of the molecular mechanism whereby growth factors repress myogenesis. Initial studies demonstrated that nuclear oncogenes such as c-fos, junB and c-jun are immediate early genes that respond to serum and TGF-$\beta$. Using the muscle creatine kinase (MCK) enhancer linked to the reporter gene CAT as a marker for differentiation, we showed that transcriptional function of myogenin can be disrupted in the presence of c-Fos, JunB and cjun. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, failed to show the inhibition. The repression by Fos and Jun is targeted at KE-2 motif, the same sequence that mediates myogenin-dependent activation and muscle-specific transactivation. Deletion analysis indicated that the transactivation domain of c-Jun at the N-terminus is responsible for the repression. Considering that myogenin is a phosphoprotein and cAMP and TPA are able to regulate myogenesis, we examined whether constitutively active protein kinase C (PKC) and protein kinase A (PKA) could substitute for exogenous growth factors and prevent transcription activation by myogenin. Indeed, the basic region of myogenin is phosphorylated by PKC at a threonine that is conserved in all members of the MyoD family. Phosphorylation at this site attenuates DNA binding activity of myogenin. Protein kinase A can also phosphorylate myogenin in a region adjacent to the DNA binding domain. However, phosphorylation at this site is insufficient to abrogate myogenin's DNA binding capacity, suggesting that PKA and PKC may affect myogenin transcriptional activity through different mechanisms. These findings provide insight into the mechanisms through which growth factor signals negatively regulate the muscle differentiation program and contribute to an understanding of signal transducing pathways between the cell membrane and nucleus. ^

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. ^

MOLECULAR MECHANISMS UNDERLYING THE TRANSCRIPTIONAL REGULATION OF T HELPER 17 AND REGULATORY T CELLS

CD4+ T helper (Th) lymphocytes are vital for integrating immune responses by orchestrating the function of other immune cell types. Naïve Th cells can differentiate into different effector subsets that are characterized by their cytokine profile and immune regulatory functions. These subsets include Th1, Th2, Th17, natural and inducible regulatory T cells (nTreg and iTreg respectively), among others. We focused our investigation on two Th lineages, Th17 and regulatory T cells, with opposing functions in the immune system. These subsets have been suggested to be reciprocally regulated since they both require TGF-b for their development. We investigated the role of the Treg-associated master transcription factor Foxp3, and found that Foxp3 inhibits Th17 cell generation by preventing the transcriptional activity of the two main Th17-specific transcription factors, nuclear orphan receptors RORa and RORgt. At the molecular level, we identified two different functional domains in Foxp3 required for such inhibition: the LQALL sequence in exon 2 and the TIP60/HDAC7 binding domain. These domains could be crucial to either prevent the association of the nuclear receptors to coactivators or to recruit histone deacetylases to RORa- or RORgt-target genes. Since TGF-b is a common cytokine required for the commitment towards both Th lineages, we determined the role of the TGF-b-dependent signaling pathway in the generation of each subset. By using mice with deficiencies in signaling molecules downstream of TGF-b, we found that while Smad2, Smad3 and Smad4 are required for the generation of iTreg cells, only Smad2 is indispensable for the induction of IL-17-producing cells, suggesting that TGF-b induces these T helper lineages through differential signaling pathways. Thus, our findings describe novel transcriptional regulatory mechanisms that control the generation of two T helper lineages with opposing functions. These findings could provide novel therapeutic targets to treat diseases where the balance of these T cells is dysregulated, such as in autoimmunity, chronic infectious diseases and cancer.

The molecular mechanism of retinoic acid action: Regulation of tissue transglutaminase gene expression

Retinoic acid is a small lipophilic molecule that exerts profound effects on the growth and differentiation of both normal and transformed cells. It is also a natural morphogen that is critical in the development of embryonic structures. The molecular effects of retinoic acid involve alterations in the expression of several proteins and these changes are presumably mediated in part by alterations in gene expression. For instance, retinoic acid causes a rapid induction of tissue transglutaminase, an enzyme involved in protein cross-linking. The molecular mechanisms responsible for the effects of retinoic acid on gene expression have not been characterized. To approach this question, I have isolated and characterized tissue transglutaminase of cDNA clones. The deduced amino acid sequences of tissue transglutaminase and of factor XIIIa showed a relatively high degree of homology in their putative calcium binding domains.^ To explore the mechanism of induction of this enzyme, both primary (macrophages) and cultured cells (Swiss 3T3-C2 and CHO fibroblasts) were used. I found that retinoic acid is a general inducer of tissue transglutaminase mRNA in these cells. In murine peritoneal macrophages retinoic acid causes a rapid accumulation of this mRNA and this effect is independent of concurrent protein synthesis. The retinoic acid effect is not mediated by a post-transcriptional increase in the stability of the tissue transglutaminase mRNA, but appears to involve an increase in the transcription rate of the tissue transglutaminase gene. This provides the first example of regulation by retinoic acid of a specific gene, supporting the hypothesis that these molecules act by directly regulating the transcriptional activity of specific genes. A molecular model for the effects of retinoic acid on the expression of genes linked to cellular proliferation and differentiation is proposed. ^

Molecular mechanisms by which p53/LSD1 and ERalpha/Trim24 complexes mediate gene regulation

In this dissertation, I identify two molecular mechanisms by which transcription factors cooperate with their co-regulators to mediate gene regulation. In the first part, I demonstrate that p53 directly recruits LSD1, a histone demethylase, to AFP chromatin to demethylate methylated H3K4 and actively mediate transcription repression. Loss of p53 and LSD1 interaction at chromatin leads to derepression of AFP in hepatic cells. In the second part, I reveal that Trim24 functions as an important co-activator in ERα-mediated gene activation in response to estrogen stimulation. Trim24 is recruited by ligand-bound ERα to chromatin and stabilizes ERα-chromatin interactions by binding to histone H3 via its PHD finger, which preferentially recognizes unmethylated H3K4. ^

Molecular mechanisms involved in the regulation of cell polarity and genome stability by the p21 -activated kinase, Shk1, in the fission yeast, Schizosaccharomyces pombe

The essential p21-activated kinase (PAK), Shk1, is a critical component of a Ras/Cdc42/PAK complex required for cell viability, normal cell polarity, proper regulation of cytoskeletal dynamics, and sexual differentiation in the fission yeast, Schizosaccharomyces pombe. While cellular functions of PAKs have been described in eukaryotes from yeasts to mammals, the molecular mechanisms of PAK regulation and function are poorly understood. This study has characterized a novel Shk1 inhibitor, Skb15, and, in addition, identified the cell polarity regulator, Tea1, as a potential biological substrate of Shk1 in S. pombe. Skb15 is a highly conserved WD repeat protein that was discovered from a two-hybrid screen for proteins that interact with the catalytic domain of Shk1. Molecular data indicate that Skb15 negatively regulates Shk1 kinase activity in S. pombe cells. A null mutation in the skb15 gene is lethal and results in deregulation of actin polymerization and localization, microtubule biogenesis, and the cytokinetic machinery, as well as a substantial uncoupling of these processes from the cell cycle. Loss of Skb15 function is suppressed by partial loss of Shk1, demonstrating that negative regulation of Shk1 by Skb15 is required for proper execution of cytoskeletal remodeling and cytokinetic functions. A mouse homolog of Skb15 can substitute for its counterpart in fission yeast, demonstrating that Skb15 protein function has been substantially conserved through evolution. ^ Our laboratory has recently demonstrated that Shk1, in addition to regulating actin cytoskeletal organization, is required for proper regulation of microtubule dynamics in S. pombe cells. The Shk1 protein localizes to interphase and mitotic microtubules, the septum-forming region, and cell ends. This pattern of localization overlaps with that of the cell polarity regulator, Tea1, in S. pombe cells. The tea1 gene was identified by Paul Nurse's laboratory from a screen for genes involved in the control of cell morphogenesis in S. pombe. In contrast to wild type S. pombe cells, which are rod shaped, tea1 null cells are often bent and/or branched in shape. The Tea1 protein localizes to the cell ends, like Shk1, and the growing tips of interphase microtubules. Thus, experiments were performed to investigate whether Tea1 interacts with Shk1. The tea1 null mutation strongly suppresses the loss of function of Skb15, an essential inhibitor of Shk1 function. All defects associated with the skb15 mutation, including defects in F-actin organization, septation, spindle elongation, and chromosome segregation, are suppressed by tea1Δ, suggesting that Tea1 may function in these diverse processes. Consistent with a role for Tea1 in cytokinesis, tea1Δ cells have a modest cell separation defect that is greatly exacerbated by a shk1 mutation and, like Shk1, Tea1 localizes to the septation site. Molecular analyses showed that Tea1 phosphorylation is significantly dependent on Shk1 function in vivo and that bacterially expressed Tea1 protein is directly phosphorylated by recombinant Shk1 kinase in vitro. Taken together, these results identify Tea1 as a potential biological substrate of Shk1 in S. pombe. ^ In summary, this study provides new insights into a conserved regulatory mechanism for PAKs, and also begins to uncover the molecular mechanisms by which the Ras/Cdc42/PAK complex regulates the microtubule and actin cytoskeletons and cell growth polarization in fission yeast. ^

Regulation of survivin gene expression in the human endometrium and endometrial cancer

In the United States, endometrial cancer is the leading cancer of the female reproductive tract. There are 40,100 new cases and 7,470 deaths from endometrial cancer estimated for 2008 (47). The average five year survival rate for endometrial cancer is 84% however, this figure is substantially lower in patients diagnosed with late stage, advanced disease and much higher for patients diagnosed in early stage disease (47). Endometrial cancer (EC) has been associated with several risk factors including obesity, diabetes, hypertension, previously documented occurrence of hereditary non-polyposis colorectal cancer (HNPCC), and heightened exposure to estrogen (25). As of yet, there has not been a dependable molecular predictor of endometrial cancer occurrence in women with these predisposing factors. The goal of our lab is to identify genes that are aberrantly expressed in EC and may serve as molecular biomarkers of EC progression. One candidate protein that we are exploring as a biomarker of EC progression is the cell survival protein survivin.