A new fork for clinical application: Targeting forkhead transcription factors in cancer therapy

RAS-ERK-MAPK (Mitogen-activated protein kinase) pathway plays an essential role in proliferation, differentiation, and tumor progression. In this study, we showed that ERK downregulated FOXO3a through directly interacting with and phosphorylating FOXO3a at Serine 294, Serine 344, and Serine 425. ERK-phosphorylated FOXO3a was degraded by MDM2-mediated ubiquitin-proteosome pathway. FOXO3a phosphorylation and degradation consequently promoted cell proliferation and tumorigenesis. However, the non-phosphorylated FOXO3a mutant, which was resistant to the interaction and degradation by MDM2, resulted in inhibition of tumor formation. Forkhead O transcription factors (FOXOs) are important in the regulation of cellular functions including cell cycle arrest and cell death. Perturbation of FOXOs function leads to deregulated cell proliferation and cancer. Inactivation of FOXO proteins by activation of cell survival pathways, such as PI3K/AKT/IKK, is associated with tumorigenesis. Our study will further highlight FOXOs as new therapeutic targets in a broad spectrum of cancers. ^ Chemotherapeutic drug resistance is the most concerned problem in cancer therapy as resistance ultimately leads to treatment failure of cancer patients. In another study, we showed that blocking ERK activity with AZD6244, an established MEK1/2 inhibitor currently under human cancer clinical trials, enhances FOXO3a expression in various human cancer cell lines in vitro, and also in human colon cancer cell xenografts in vivo. Knocking down FOXO3a and its downstream gene Bim impaired AZD6244-induced growth suppression, whereas restoring activation of FOXO3a sensitized human cancer cell to AZD6244-induced growth arrest and apoptosis. More importantly, AZD6244-resistant cancer cells showed impaired endogenous FOXO3a nuclear translocation, reduced FOXO3a-Bim promoter association and significantly decreased Bim expression in response to AZD6244. AZD6244-resistant cancer cells can be sensitized to API-2 (an AKT inhibitor) and LY294002 (a PI3K inhibitor) in suppressing cell growth and colony formation, these inhibitors were known to enhance FOXO3a activity/nuclear translocation through inhibiting PI3K-AKT pathway. This study reveals novel molecular mechanism contributing to AZD6244-resistance through regulation of FOXO3a activity, further provides significant clinical implication of combining AZD6244 with PI3K/AKT inhibitors for sensitizing AZD6244-resistant cancer cells by activating FOXO3a. FOXO3a activation can be an essential pharmacological target and indicator to mediate and predict AZD6244 efficacy in clinical use. ^

L -Sox5 and Sox6: Architectural transcription factors in chondrogenesis

Transcription factors often determine cell fate and tissue development. Chondrogenesis is the developmental process by which cartilages form. Recently, gene targeting studies have shown that two transcription factors, L-Sox5 and Sox6, play essential and redundant roles in chondrogenesis in vivo by converting precartilaginous cell condensations into cartilages. Both are highly similar High-Mobility-Group (HMG)-domain proteins that bind and subsequently bend DNA containing the 7bp HMG site (A/T)(A/T)CAA(A/T)G. They have no transactivation domain, but homo- and hetero-dimerize and preferentially bind DNA containing two HMG sites. They are thought to play an architectural role in transactivation by facilitating long-range DNA and protein interactions. To understand their molecular mechanism of action, we investigated how phasing, orientation, and spacing between HMG sites affect L-Sox5 and Sox6 DNA-binding. We determined that L-Sox5 and Sox6 dimers bind with high affinity to paired HMG sites in DNA rather than a single HMG site. Binding of paired sites is independent of DNA helical phasing, orientation of paired HMG sites and independent of distance up to 255 base pairs between sites. Mutational analysis demonstrated that binding of L-Sox5 and Sox6, independent of orientation of the sites, is critically dependent on the presence of paired HMG sites rather than one HMG site alone. Our data support a unique and novel model whereby L-Sox5 and Sox6 dimerize and bind DNA with pronounced spatial flexibility, possibly by a flexible hinge, and act as architectural transcription factors that bring distant DNA sites and proteins together to form higher order transcriptional complexes that are essential for the activation of their target genes in chondrogenesis. ^

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure.

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

Insights into transcription enhancer factor 1 (TEF-1) activity from the solution structure of the TEA domain.

Transcription enhancer factor 1 is essential for cardiac, skeletal, and smooth muscle development and uses its N-terminal TEA domain (TEAD) to bind M-CAT elements. Here, we present the first structure of TEAD and show that it is a three-helix bundle with a homeodomain fold. Structural data reveal how TEAD binds DNA. Using structure-function correlations, we find that the L1 loop is essential for cooperative loading of TEAD molecules on to tandemly duplicated M-CAT sites. Furthermore, using a microarray chip-based assay, we establish that known binding sites of the full-length protein are only a subset of DNA elements recognized by TEAD. Our results provide a model for understanding the regulation of genome-wide gene expression during development by TEA/ATTS family of transcription factors.

Human TOB, an antiproliferative transcription factor, is a poly(A)-binding protein-dependent positive regulator of cytoplasmic mRNA deadenylation.

In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3' poly(A) tail-PABP complex.

The molecular and genetic characterization of the MADS box transcription factor {\it Drosophila\/} MEF2

The myocyte enhancer factor (MEF)-2 family of transcription factors has been implicated in the regulation of muscle transcription in vertebrates, but the precise position of these regulators within the genetic hierarchy leading to myogenesis is unclear. The MEF2 proteins bind to a conserved A/T-rich DNA sequence present in numerous muscle-specific genes, and they are expressed in the cells of the developing somites and in the embryonic heart at the onset of muscle formation in mammals. The MEF2 genes belong to the MADS box family of transcription factors, which control specific programs of gene expression in species ranging from yeast to humans. Each MEF2 family member contains two highly conserved protein motifs, the MADS domain and the MEF2-specific domain, which together provide the MEF2 factors with their unique DNA binding and dimerization properties. In an effort to further define the function of the MEF2 proteins, and to evaluate the degree of conservation shared among these factors and the phylogenetic pathways that they regulate, we sought to identify MEF2 family members in other species. In Drosophila, a homolog of the vertebrate MEF2 genes was identified and termed D-mef2. The D-MEF2 protein binds to the consensus MEF2 element and can activate transcription through tandem copies of that site. During Drosophila embryogenesis, D-MEF2 is specific to the mesoderm germ layer of the developing embryo and becomes expressed in all muscle cell types within the embryo. The role of D-mef2 in Drosophila embryogenesis was examined by generating a loss-of-function mutation in the D-mef2 gene. In embryos homozygous for this mutant allele, somatic, cardiac, and visceral muscles fail to differentiate, but precursors of these myogenic lineages are normally specified and positioned. These results demonstrate that different muscle cell types share a common myogenic differentiation program controlled by MEF2 and suggest that this program has been conserved from Drosophila to mammals. ^

Functional characterization of transcription factor USF

USF, Upstream Stimulatory Factor, is a family of ubiquitous transcription factors that contain highly conserved basic helix-loop-helix leucine zipper DNA binding domains and recognize the core DNA sequence CACGTG. In human and mouse, two members of the USF family, USF1 and USF2, encoded by two different genes, contribute to the USF activity. In order to gain insights into the mechanisms by which USFs function as transcriptional activators, different approaches were used to map the domains of USF2 responsible for nuclear localization and transcriptional activation. Two stretches of amino acids, one in the basic region of the DNA binding domain, the other in a highly conserved N-terminal region, were found to direct nuclear localization independently of one another. Two distinct activation domains were also identified. The first one, located in the conserved N-terminal region that overlaps the C-terminal nuclear localization signal, functioned only in the presence of an initiator element in the promoter of the reporter. The second, in a nonconserved region, activated transcription in the absence of an initiator element or when fused to a heterologous DNA binding domain. These results suggest that USF2 functions in different promoter contexts by selectively utilizing different activation domains.^ The deletion analysis of USF2 also identified two dominant negative mutants of USF, one lacking the activation domain, the other lacking the basic domain. The latter proved useful for testing the direct involvement of USFs in the transcriptional activation mediated by the viral protein IE62.^ To investigate the biological function of USFs, foci and colony formation assays were used to study the growth regulation by USFs. It was found that USFs had a strong antagonistic effect on cellular transformation mediated by the bHLH/LZ protein Myc. This effect required the DNA binding activity of either USF 1 or USF2. Moreover, USF2, but not USF1 or other mutants of USFs, was also found to have strong inhibitory effect on the cellular transformation by E1a and on the growth of HeLa cells. These results demonstrate that USFs could potentially regulate growth through two mechanisms, one by antagonizing the function of Myc in cellular transformation, the other by mediating a more general growth inhibitory effect. ^

Transcription factor SEF: Purification and functional characterization

Cytokine-induced transcription of the serum amyloid A3 (SAA3) gene promoter requires a transcriptional enhancer that contains three functional elements: two C/EBP-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa cell nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA-affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing, oligonucleotide competition and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression plasmid with SAA3-luciferase reporter resulted in 3- to 5-fold activation of SAA3 promoter. Interestingly, when SEF-transfected cells were treated with either conditioned medium (CM) or interleukin (IL) 1, the SAA3 promoter was synergistically activated in a dose-dependent manner. Furthermore, when SEF-binding site was mutated, the response of SAA3 promoter to IL-1 or CM stimulation was abolished or drastically decreased, suggesting that SEF may functionally cooperate with an IL-1-inducible transcription factor. Indeed, our functional studies showed that NFκB is a key transcription factor that mediates the IL-1-induced expression of SAA3 gene, and that SEF can synergize with NFκBp65 to activate SAA3 promoter. By coimmunoprecipitation experiments, we found that SEF could specifically interact with NFκBp65, and that the association of these two factors was enhanced upon IL-1 and CM stimulation. This suggests that the molecular basis for the functional synergy between SEF and NFκB may be due to the ability of SEF to physically interact with NPκB. In addition to its interaction with SEF, NFκB-dependent activation also requires the weak κB site in the C element and its interaction with C/EBP. Besides its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of α2-macroglobulin, Aα fibrinogen, and 6–16 genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes. By coimmunoprecipitation experiments, we determined that SEF could specifically associate with both Stat3 and Stat2 upon cytokine stimulation. To examine the functional roles of such interactions, we evaluated the effects of SEF on the transcriptional regulation of two reporter genes: Aα fibrinogen and 6–16, which are IL-6- and interferon-α-responsive, respectively. Our results showed that cotransfection of SEF expression plasmid can activate the expression of Aα fibrinogen gene and 6–16 gene. Moreover, SEF can dramatically enhance the interferon-α-induced expression of 6–16 gene and IL-6-induced expression of Aα fibrinogen gene, suggesting that SEF may functionally cooperate with ISGF3 and Stat3 to mediate interferon-α and IL-6 signaling. ^ Our findings that SEF can interact with multiple cytokine-inducible transcription factors to mediate the expression of target genes open a new avenue of investigation of cooperative transcriptional regulation of gene expression, and should further our understanding of differential gene expression in response to a specific stimulus. In summary, our data provide evidence that SEF can mediate the signaling of different cytokines by interacting with various cytokine-inducible transcription factors. ^

Alteration of CEACAM1 gene transcription in prostate cancer cells: The role of AR, Sp2, and HDAC

Cell to cell adhesion molecule (CEACAM1), a type II tumor suppressor, has been found to be down-regulated in prostate cancer cells. The mechanism that causes CEACAM1's down-regulation in tumorigenesis is unknown. Here we show that the transcriptional activity of CEACAM1 is decreased in prostate cancer cells. This decrease is not due to methylation of the CEACAM1's promoter, but rather to the alteration of transcription factors regulating CEACAM1 expression. ^ Since androgen/androgen receptors (AR) are potent regulators of prostate growth and differentiation, their role on CEACAM1 gene transcription was examined. The androgen receptor could directly increase CEACAM1 transcriptional activity in a ligand dependent manner by interacting with an AR consensus element that resides in the CEACAM1 promoter. However, AR binding to the CEACAM1 promoter is not related to the loss of CEACAM1 during prostate cancer progression. ^ Further analysis enabled us to determine the particular region in the CEACAM1 promoter that mediates a decrease in CEACAM1 transcriptional activity in prostate cancer cells. Upon further examination, we found that this CEACAM1 promoter region interacts with the Sp1, Sp2, and Sp3 transcription factors. However, only Sp2 expression was found to increase in prostate cancer cells. Inhibiting Sp2 from binding to the CEACAM1 promoter caused an increase in CEACAM1 transcriptional activity in prostate cancer cells. In addition, over-expressing Sp2 in normal prostate cells resulted in a decrease in CEACAM1 transcriptional activity and endogenous protein expression. These observations suggest that Sp2 is a transcription repressor of CEACAM1. Furthermore, prostate cancer cells treated with trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor, activated CEACAM1 transcriptional activity. This implies that HDACs are involved in CEACAM1 transcriptional activity. Mutation of the Sp2 DNA binding region on the CEACAM1 promoter inhibited TSA activation of CEACAM1 transcriptional activity. This indicates that HDACs inhibit CEACAM1 transcriptional activity through Sp2. Base on these results, we propose that Sp2 is critical for down-regulating CEACAM1 expression, and one mechanism by which Sp2 represses CEACAM1 expression is by recruiting HDAC to the CEACAM1 promoter in prostate cancer cells. Collectively, these findings provide novel insights into mechanisms that cause the down-regulation of CEACAM1 expression in prostate cancer cells. ^

Downstream targets of the Rhox5 homeobox gene

The X-linked mouse Rhox gene cluster contains over 30 homeobox genes that are candidates to regulate multiple steps in male and female gametogenesis. The founding member of the Rhox gene cluster, Rhox5, is an androgen-dependent gene expressed in Sertoli cells that promotes the survival and differentiation of the adjacent male germ cells. To decipher downstream signaling pathways of Rhox5, I used in vivo and in vitro microarray profiling to identify and characterize downstream targets of Rhox5 in the testis. This led to the identification of many Rhox5 -regulated genes, two of which I focused on in more detail. One of them, Unc5c, encodes a pro-apoptotic receptor with tumor suppressor activity that I found is negatively regulated by Rhox5 through a Rhox5-response element in the Unc5c 5' untranslated region (5' UTR). Examination of other mouse Rhox family members revealed that Rhox2 and Rhox3 also have the ability to downregulate Unc5c expression. The human RHOX protein RHOXF2 also had this ability, indicating that Unc5c repression is a conserved Rhox-dependent response. The repression of Unc5c expression by Rhox5 may, in part, mediate Rhox5's pro-survival function in the testis, as I found that Unc5c mutant mice have decreased germ cell apoptosis in the testis. This along with my other data leads me to propose a model in which Rhox5 is a negative regulator upstream of Unc5c in a Sertoli-cell pathway that promotes germ-cell survival. The other Rhox5-regulated gene that I studied in detail is insulin II (Ins2). Several lines of evidence, including electrophoretic mobility shift anaylsis, promoter mutagenesis, and chromatin immuoprecipitation analysis indicated that Ins2 is a direct target of Rhox5. Structure-function analysis identified homeodomain residues and the RHOX5 amino-terminal domain crucial for conferring Ins2 inducibility. Rhox5 regulates not only the Ins2 gene but also genes encoding other secreted proteins regulating metabolism (adiponectin and resistin), the rate-liming enzyme for monosaturated fatty acid biosynthesis (SCD-1), and transcription factors crucial for regulating metabolism (the nuclear hormone receptor PPARγ). I propose that the regulation of some or all of these molecules in Sertoli cells is responsible for the Rhox5-dependent survival of the adjacent germ cells. ^

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.^

NOVEL INDUCERS OF GLIOTOXIN PRODUCTION IN ASPERGILLUS FUMIGATUS

Secondary metabolites are produced by numerous organisms and can either be benign to humans or harmful. Genes involved in the synthesis and transport of these secondary metabolites are frequently found in gene clusters, which are often located in subtelomeric regions of the chromosome. These clusters are often coordinately regulated, being almost exclusively dependent on transcription factors that are located within the clusters themselves. Secondary metabolites are also regulated by a variety of factors, including nutritional factors, environmental factors and developmental processes. Gliotoxin, which is produced by a variety of Aspergillus species, Trichoderma species, and Penicillium species, exhibits immunosuppressive properties and has therefore been the subject of research for many laboratories. There have been a few proteins shown to regulate the gliotoxin cluster, most notably GliZ, a Zn2Cys6 binuclear finger transcription factor that lies within the cluster, and LaeA, a putative methyltransferase that globally regulates secondary metabolism clusters within numerous fungal organisms, although no study has demonstrated the direct binding of any protein to a promoter region in the gliotoxin cluster. I report here two novel proteins, GipA, a C2H2 transcription factor and GipB, a hybrid sensor kinase, which are involved in regulating the gliotoxin biosynthetic cluster. GipA plays an important role in gliotoxin production, as high-copy expression of gipA induces gliotoxin biosynthesis and loss of gipA reduces gliotoxin biosynthesis by 50%. GipB is also involved in regulating gliotoxin production, as high-copy expression of gipB induces gliotoxin biosynthesis, but only during certain stages of asexual development. Furthermore, loss of gipB reduces gliotoxin biosynthesis by 10%. Based on data obtained from this project, I propose a model for the regulation of gliA, the efflux pump of the gliotoxin cluster, which involves GipB signaling through both GliZ and GipA. I propose that GliZ and GipA are interdependent, as mutation of the GipA DNA binding site in the gliA promoter negatively affects both GliZ-mediated and GipA-mediated induction of gliA. This is further supported by the fact that GliZ cannot fully induce gliA in the absence of GipA and vice versa. This is the first time that anyone has shown evidence of a protein directly binding to the gliotoxin cluster. Even though biosynthetic clusters are often coordinately regulated, my model raises the possibility that gliA is independently regulated, as the layout of the binding site in the gliA promoter is not present upstream of any other genes in the gliotoxin cluster, except for gliZ.

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. ^

Analysis of p53 function and regulation

p53 is a tumor suppressor gene that is the most frequent target inactivated in cancers. Overexpression of wild-type p53 in rat embryo fibroblasts suppresses foci formation by other cooperating oncogenes. Introduction of wild-type p53 into cells that lack p53 arrests them at the G1/S boundary and reverses the transformed phenotype of some cells. The function of p53 in normal cells is illustrated by the ability of p53 to arrest cells at G1 phase of the cell cycle upon exposure to DNA-damaging agents including UV-irradiation and biosynthesis inhibitors.^ Since the amino acid sequence of p53 suggested that it may function as a transcription factor, we used GAL4 fusion assays to test that possibility. We found that wild-type p53 could specifically activate transcription when anchored by the GAL4 DNA binding domain. Mutant p53s, which have lost the ability to suppress foci formation by other oncogenes, were not able to activate transcription in this assay. Thus, we established a direct correlation between the tumor suppression and transactivation functions of p53.^ Having learned that p53 was a transcriptional activator, we next sought targets of p53 activation. Because many transcription factors regulate their own expression, we tested whether p53 had this autoregulatory property. Transient expression of wild-type p53 in cells increased the levels of endogenous p53 mRNA. Cotransfection of p53 together with a reporter bearing the p53 promoter confirmed that wild-type p53 specifically activates its own promoter. Deletion analysis from both the 5$\sp\prime$ and 3$\sp\prime$ ends of the promoter minimized the region responsible for p53 autoregulation to 45 bp. Methylation interference identified nucleotides involved in protein-DNA interaction. Mutations within this protected site specifically eliminated the response of the promoter to p53. In addition, multiple copies of this element confer responsiveness to wild-type p53 expression. Thus, we identified a F53 responsive element within the p53 promoter.^ The presence of a consensus NF-$\kappa$B site in the p53 promoter suggested that NF-KB may regulate p53 expression. Gel-shift experiments showed that both the p50 homodimer and the p50/p65 heterodimer bind to the p53 promoter. In addition, the p65 subunit of NF-$\kappa$B activates the p53 promoter in transient transfection experiments. TNF $\alpha$, a natural NF-$\kappa$B inducer, also activates the p53 promoter. Both p65 activation and TNF $\alpha$ induction require an intact NF-$\kappa$B site in the p53 promoter. Since NF-$\kappa$B activation occurs as a response to stress and p53 arrests cells in G1/S, where DNA repair occurs, activation of p53 by NF-$\kappa$B could be a mechanism by which cells recover from stress.^ In conclusion, we provided the first data that wild-type p53 functions as a transcriptional activator, whereas mutant p53 cannot. The correlation between growth suppression and transcriptional activation by p53 implies a pathway of tumor suppression. We have analyzed upstream components of the pathway by the identification of both p53 and NF-$\kappa$B as regulators of the p53 promoter. ^

Functional studies of a LIM -homeodomain transcription factor lmx1b in murine mid -hindbrain and limb development

This thesis is centered on applying molecular genetics to study pattern formation during animal development. More specifically, this thesis describes the functional studies of a LIM-homeodomain gene called lmx1b during murine embryogenesis. Lmx1b expression is restricted to the mid-hindbrain junction as well as to the dorsal mesenchyme of the limb, suggesting important functions during mid-hindbrain and limb development. To test these possibilities, lmx1b homozygous mutant mice were generated and their limb and CNS phenotypes examined. Lmx1b homozygous mutant mice exhibit a large reduction of mid-hindbrain structures, and that their limbs are symmetrical along the dorsal-ventral axis as the result of a dorsal to ventral transformation. Taken together, these studies define essential functions for lmx1b in mid-hindbrain patteming and in dorsal limb cell fate determination. However, the molecular mechanisms which accounts for these phenotypes are unknown, and whether lmx1b has same or distinctive functions during the mid-hindbrain and limb development is also unclear. ^ Recently, insight into molecular mechanisms of mid-hindbrain patterning and limb development has resulted from the identification of several factors with restricted expression patterns within these regions. These include the secreted factors wnt-1, fgf-8, wnt-7a and the transcription factors pax-2, and en-1. Targeted disruption of any of these genes in mice suggests that these genes might be involved in similar regulatory pathways. Analysis of the expression of these genes in lmx1b mutants demonstrates that lmxlb is not required for the initiation, but is required to maintain their expression at the mid-hindbrain junction. Thus, lmxlb is not required for specifying mid-hindbrain cell fates, rather, it functions to ensure the establishment or maintenance of a proper organizing center at the mid-hindbrain junction. Interestingly, lmxlb functions cell non-autonomously in chimera analysis, which indicates that lmx1b might regulate the expression of secreted factors such as wnt-1 and/or fgf-8 in the organizing center. In contrast, lmx1b functions cell autonomously in the dorsal limb to govern dorsal ventral limb development and its expression is regulated by with wnt-7a and en-1. However, single and double mutant analysis suggest that all three genes have partially overlapping functions as well as independent functions. The results point toward a complicated network of cross-talks among all three limb axes. ^