Elk-1 Binding Site Research Articles

Abstract 2923: New insights into interactions of the androgen receptor with ELK1 and the platform antagonist KCI807

Abstract Prostate cancer (PC) is generally dependent on the androgen signaling axis for growth. Advanced PC is managed by androgen deprivation therapy (ADT). However, the tumors frequently progress by restoring AR signaling that is either androgen-independent or hypersensitive to androgen, leading to castration resistant prostate cancer (CRPC). CRPC is also supported by hormone-independent actions of AR splice variants which lack the ligand binding domain. Additionally, ADT has many undesirable side effects on a variety of normal tissues that depend on androgen for non-growth related functions, including effects on the cardiovascular system, central nervous system (cognitive effects), bone and muscle. Therefore, a more strategic therapy approach would be to disrupt a functional arm of AR-signaling that is critical for PC/CRPC growth but not for the essential physiological roles of AR in normal tissues. Previous studies in this laboratory have identified ELK1 as an AR tethering protein essential for activation of a critical set of androgen/AR growth genes in various PC model cell line models, both in vitro and in vivo, including those resistant to castration and enzalutamide. It was also established that the N-terminal A/B domain of AR binds to ELK1 by co-opting the two ERK docking sites on ELK1 to constitutively activate genes enriched for cell growth functions in PC cells. A platform antagonist (KCI807) was discovered that bound to AR (Kd = 7x10-8 M), blocking its association with ELK1 and inhibiting PC/CRPC tumor growth. KCI807 has a limited target gene set (a subset of AR target genes) that is primarily and highly enriched for functions in cell cycle progression and mitosis. To further drug development, we must identify the ELK1 docking site recognition sites in AR and the KCI807 binding site. Using a variety of complimentary approaches, we have now mapped and validated the ELK1 docking site recognition sites in the AR A/B domain and also the binding site of KCI807 in AR. The two docking site recognition sites in the AR A/B domain for the two docking sites in ELK1 span amino acids 358-456 and amino acids 514-557. We have also developed a working model in which KCI807 binds in a cleft adjacent to the downstream ELK1-binding site, within the DBD, displacing an alpha helix which in turn disrupts ELK1 binding. Further structural and functional studies based on this knowledge will directly aid in design of next-generation antagonists to selectively target ELK1-dependent growth signaling by AR in PC/CRPC tumors. Citation Format: Claire L. Soave, Charles Ducker, Nathan Nicely, Yanfang Huang, Luke Pardy, Peter Shaw, Manohar Ratnam. New insights into interactions of the androgen receptor with ELK1 and the platform antagonist KCI807 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2923.

ETS1, ELK1, and ETV4 Transcription Factors Regulate Angiopoietin-1 Signaling and the Angiogenic Response in Endothelial Cells.

BackgroundAngiopoietin-1 (Ang-1) is the main ligand of Tie-2 receptors. It promotes endothelial cell (EC) survival, migration, and differentiation. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors.ObjectiveThe main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response.MethodsIn silico enrichment analyses that were designed to predict TF binding sites of the promotors of eighty-six Ang-1-upregulated genes showed significant enrichment of ETS1, ELK1, and ETV4 binding sites in ECs. Human umbilical vein endothelial cells (HUVECs) were exposed for different time periods to recombinant Ang-1 protein and mRNA levels of ETS1, ELK1, and ETV4 were measured with qPCR and intracellular localization of these transcription factors was assessed with immunofluorescence. Electrophoretic mobility shift assays and reporter assays were used to assess activation of ETS1, ELK1, and ETV4 in response to Ang-1 exposure. The functional roles of these TFs in Ang-1-induced endothelial cell survival, migration, differentiation, and gene regulation were evaluated by using a loss-of-function approach (transfection with siRNA oligos).ResultsAng-1 exposure increased ETS1 mRNA levels but had no effect on ELK1 or ETV4 levels. Immunostaining revealed that in control ECs, ETS1 has nuclear localization whereas ELK1 and ETV4 are localized to the nucleus and the cytosol. Ang-1 exposure increased nuclear intensity of ETS1 protein and enhanced nuclear mobilization of ELK1 and ETV4. Selective siRNA knockdown of ETS1, ELK1, and ETV4 showed that these TFs are required for Ang-1-induced EC survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration. Moreover, ETS1, ELK1, and ETV4 knockdown inhibited Ang-1-induced upregulation of thirteen, eight, and nine pro-angiogenesis genes, respectively.ConclusionWe conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.

Elk1 and AP-1 sites in the TBP promoter mediate alcohol-induced deregulation of Pol III-dependent genes

The major risk factors for hepatocellular carcinoma (HCC) are chronic liver diseases that include hepatitis B, hepatitis C, alcoholic liver disease and non-alcoholic steatohepatitis. However, the mechanisms of alcohol-associated HCC remain to be elucidated. The products of RNA Pol III (RNA polymerase III) dependent genes are elevated in both transformation cells and tumor cells. TBP (TATA-box binding protein) is a central transcription factor, which regulates Pol I, Pol II and Pol III gene activity. Our studies have demonstrated that alcohol increases TBP expression and Pol III gene transcription to promote liver tumor formation. We continue to investigate how ethanol mediates TBP expression. Here, we report that ethanol induces TBP promoter activity and the induction is ethanol dose dependent. Blocking the JNK1 pathway by a chemical inhibitor and siRNA reduces this ethanol-induced activity. Furthermore, mutating G>A at a −46bp Elk1 binding site of the TBP promoter or mutating AP-1 binding site at −37bp (A>G) and −38bp (C>T) reduces the TBP promoter activity. Mutation of both Elk1 and AP-1 binding sites dramatically represses this induction. Together, these studies demonstrate that, for the first time, alcohol increases Pol III gene transcription through a response element, which is composed of the overlapping Elk1 and AP-1 binding sites of the TBP promoter and affected by alcohol. It suggests that these binding sites may play a critical role in alcohol-induced deregulation of Pol III genes in liver tumor development.

Transcriptional Regulation ofFucosyltransferase 1Gene Expression in Colon Cancer Cells

The α1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of H antigens, Lewis B, and Lewis Y. In this study, we clarified the transcriptional regulation of FUT1 in the DLD-1 colon cancer cell line, which has high expression of Lewis B and Lewis Y antigens, expresses the FUT1 gene, and shows α1,2-fucosyltransferase (FUT) activity. 5′-rapid amplification of cDNA ends revealed a FUT1 transcriptional start site −10 nucleotides upstream of the site registered at NM_000148 in the DataBase of Human Transcription Start Sites (DBTSS). Using the dual luciferase assay, FUT1 gene expression was shown to be regulated at the region −91 to −81 nt to the transcriptional start site, which contains the Elk-1 binding site. Site-directed mutagenesis of this region revealed the Elk-1 binding site to be essential for FUT1 transcription. Furthermore, transfection of the dominant negative Elk-1 gene, and the chromatin immunoprecipitation (CHIp) assay, supported Elk-1-dependent transcriptional regulation of FUT1 gene expression in DLD-1 cells. These results suggest that a defined region in the 5′-flanking region of FUT1 is critical for FUT1 transcription and that constitutive gene expression of FUT1 is regulated by Elk-1 in DLD-1 cells.

FGF19 Signaling Cascade Suppresses APOA Gene Expression

Lipoprotein(a) is a highly atherogenic lipoprotein, whose metabolism is poorly understood. Currently no safe drugs exists that lower elevated plasma lipoprotein(a) concentrations. We therefore focused on molecular mechanisms that influence apolipoprotein(a) (APOA) biosynthesis. Transgenic human APOA mice (tg-APO mice) were injected with 1 mg/kg of recombinant human fibroblast growth factor 19 (FGF19). This led to a significant reduction of plasma APOA and hepatic expression of APOA. Incubation of primary hepatocytes of tg-APOA mice with FGF19 induced ERK1/2 phosphorylation and, in turn, downregulated APOA expression. Repression of APOA by FGF19 was abrogated by specific ERK1/2 phosphorylation inhibitors. The FGF19 effect on APOA was attenuated by transfection of primary hepatocytes with siRNA against the FGF19 receptor 4 (FGFR4). Using promoter reporter assays, mutation analysis, gel shift, and chromatin immune-precipitation assays, an Ets-1 binding element was identified at -1630/-1615bp region in the human APOA promoter. This element functions as an Elk-1 binding site that mediates repression of APOA transcription by FGF19. These findings provide mechanistic insights into the transcriptional regulation of human APOA by FGF19. Further studies in the human system are required to substantiate our findings and to design therapeutics for hyper lipoprotein(a).

Resection of Non–Small Cell Lung Cancers Reverses Tumor-Induced Gene Expression Changes in the Peripheral Immune System

To characterize the interactions of non-small cell lung cancer (NSCLC) tumors with the immune system at the level of mRNA and microRNA (miRNA) expression and to define expression signatures that characterize the presence of a malignant tumor versus a nonmalignant nodule. We have examined the changes of both mRNA and miRNA expression levels in peripheral blood mononuclear cells (PBMC) between paired samples collected from NSCLC patients before and after tumor removal using Illumina gene expression arrays. We found that malignant tumor removal significantly changes expression of more than 3,000 protein-coding genes, especially genes in pathways associated with suppression of the innate immune response, including natural killer cell signaling and apoptosis-associated ceramide signaling. Binding sites for the ETS domain transcription factors ELK1, ELK4, and SPI1 were enriched in promoter regions of genes upregulated in the presence of a tumor. Additional important regulators included five miRNAs expressed at significantly higher levels before tumor removal. Repressed protein-coding targets of those miRNAs included many transcription factors, several involved in immunologically important pathways. Although there was a significant overlap in the effects of malignant tumors and benign lung nodules on PBMC gene expression, we identified one gene panel which indicates a tumor or nodule presence and a second panel that can distinguish malignant from nonmalignant nodules. A tumor presence in the lung influences mRNA and miRNA expression in PBMC and this influence is reversed by tumor removal. These results suggest that PBMC gene expression signatures could be used for lung cancer diagnosis.

Identification of functional CCAAT/enhancer-binding protein and Ets protein binding sites in the human chorionic somatomammotropin enhancer sequences

The human chorionic somatomammotropin (CS) A and B genes (listed as CSH1 and CSH2 in the HUGO database) are highly expressed in placenta. A 241 bp potent enhancer, nucleotides (nts) 1-241, located at the 3' end of the CS-B gene (CS-Benh) stimulates promoter activity specifically in placental trophoblast cells in vitro. Strong activity is exerted by a 23 bp element within the CS-Benh (nts 117-139), shown to interact with transcription enhancer factor (TEF) members of the transcription enhancer activator (TEA) DNA-binding domain-containing family. An identical TEF element is present in the homologous (97.5%) CS-Aenh; however, a few nucleotide differences suppress its activity. Previously, we identified regulatory sequences distinct from the TEF element within an 80 bp modulatory domain (nts 1-80) in the CS-Benh. Using structural and functional assays we now show that CCAAT/enhancer-binding protein (C/EBP) binding sites exist in the 80 bp modulatory domains of both enhancers, and an Elk-1 binding site exists in the modulatory domain of the CS-Aenh. C/EBPα or C/EBPβ strongly repressed CSp.CAT activity but stimulated CSp.CAT.CS-Benh activity. In contrast, the equivalent CS-A enhancer sequences were unable to relieve promoter repression. Elk-1 overexpression also resulted in differential effects on the CS-Aenh versus CS-Benh. Finally, we provide evidence for the association of C/EBPβ with the CS-A and CS-B genes in human placental chromatin, including differential involvement of C/EBPβ with the CS-Aenh versus the CS-Benh, and therefore consistent with the notion that these are regions of regulatory significance in vivo. We conclude that members of the C/EBP and Ets families can differentially modulate CS-Benh and CS-Aenh activity.

Genome-Wide Analysis Reveals PADI4 Cooperates with Elk-1 to Activate c-Fos Expression in Breast Cancer Cells

Peptidylarginine deiminase IV (PADI4) catalyzes the conversion of positively charged arginine and methylarginine residues to neutrally charged citrulline, and this activity has been linked to the repression of a limited number of target genes. To broaden our knowledge of the regulatory potential of PADI4, we utilized chromatin immunoprecipitation coupled with promoter tiling array (ChIP-chip) analysis to more comprehensively investigate the range of PADI4 target genes across the genome in MCF-7 breast cancer cells. Results showed that PADI4 is enriched in gene promoter regions near transcription start sites (TSSs); and, surprisingly, this pattern of binding is primarily associated with actively transcribed genes. Computational analysis found potential binding sites for Elk-1, a member of the ETS oncogene family, to be highly enriched around PADI4 binding sites; and coimmunoprecipitation analysis then confirmed that Elk-1 physically associates with PADI4. To better understand how PADI4 may facilitate gene transactivation, we then show that PADI4 interacts with Elk-1 at the c-Fos promoter and that, following Epidermal Growth Factor (EGF) stimulation, PADI4 catalytic activity facilitates Elk-1 phosphorylation, histone H4 acetylation, and c-Fos transcriptional activation. These results define a novel role for PADI4 as a transcription factor co-activator.

Abstract 3685: C-Jun modulates alcohol-induced RNA Pol III-dependent transcription through Brf1 and TBP pathway

Abstract Cancer cells have a consistent cytological feature of nucleolar hypertrophy, enlarged nucleoli, where RNA polymerase III genes (Pol III genes) are transcripted. This implies that transformation in situ is closely linked to the deregulation of Pol III gene transcription, because the size of the nucleolus reflects the levels of rRNA synthesis. Deregulation of Pol III genes is tightly associated cell transformation and tumorgenesis. However, very little is known about whether alcohol consumption affects Pol III gene transcription. By using cell culture model and animal model, we have found, for the first time that alcohol induces Pol III gene transcription in JNK1 and ADH dependent manner. Alcohol increases c-Jun expression in HepG2-ADH cells. To determine whether the alcohol-mediated increases c-jun required to stimulate TBP and Brf1 expression are due to enhance occupancy of c-jun at their promoters, chromatin immunoprecipitation (ChIP) assays were performed. Ethanol induces a marked increase in the recruitment of c-jun to the TBP promoter, while a modest increase in the occupancy of both c-fos and Elk-1 was observed. These results support the idea that alcohol mediates an increase in TBP expression primarily through enhanced expression of c-jun and its recruitment to the TBP promoter. Sequence analysis reveals both putative AP-1 and Elk-1 binding sites in regions 5’ of the transcription start site of the Brf1 gene. ChIP analysis reveals that ethanol induces an increase in the occupancy of c-jun to the region containing an AP-1 binding site, while no significant increase in binding of Elk-1 to the Elk-1 binding site is observed. Interestingly, c-jun, c-fos and Elk-1 are also found to occupy the tRNALeu gene. Upon ethanol treatment, an increase in TBP recruitment to the tRNALeu gene is observed, as well as a marked increase in the occupancy of c-jun. However, alcohol does not induce any apparent change in recruitment of either c-fos or Elk-1 to the tRNA gene. These results suggest that c-jun may function directly at sequences near tRNA genes to induce transcription. Together, these results support the idea that alcohol enhances expression of TBP, Brf1, and tRNA gene by facilitating c-jun recruitment to these promoters. *: This project was supported by NIH grant AA017288 to S.Z. **: Corresponding author: szhong@usc.edu. Tel: 626-628-7693 or 323-442-114 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3685. doi:10.1158/1538-7445.AM2011-3685

Haplotype association mapping of acute lung injury in mice implicates activin a receptor, type 1.

Because acute lung injury is a sporadic disease produced by heterogeneous precipitating factors, previous genetic analyses are mainly limited to candidate gene case-control studies. To develop a genome-wide strategy in which single nucleotide polymorphism associations are assessed for functional consequences to survival during acute lung injury in mice. To identify genes associated with acute lung injury, 40 inbred strains were exposed to acrolein and haplotype association mapping, microarray, and DNA-protein binding were assessed. The mean survival time varied among mouse strains with polar strains differing approximately 2.5-fold. Associations were identified on chromosomes 1, 2, 4, 11, and 12. Seven genes (Acvr1, Cacnb4, Ccdc148, Galnt13, Rfwd2, Rpap2, and Tgfbr3) had single nucleotide polymorphism (SNP) associations within the gene. Because SNP associations may encompass "blocks" of associated variants, functional assessment was performed in 91 genes within ± 1 Mbp of each SNP association. Using 10% or greater allelic frequency and 10% or greater phenotype explained as threshold criteria, 16 genes were assessed by microarray and reverse real-time polymerase chain reaction. Microarray revealed several enriched pathways including transforming growth factor-β signaling. Transcripts for Acvr1, Arhgap15, Cacybp, Rfwd2, and Tgfbr3 differed between the strains with exposure and contained SNPs that could eliminate putative transcriptional factor recognition sites. Ccdc148, Fancl, and Tnn had sequence differences that could produce an amino acid substitution. Mycn and Mgat4a had a promoter SNP or 3'untranslated region SNPs, respectively. Several genes were related and encoded receptors (ACVR1, TGFBR3), transcription factors (MYCN, possibly CCDC148), and ubiquitin-proteasome (RFWD2, FANCL, CACYBP) proteins that can modulate cell signaling. An Acvr1 SNP eliminated a putative ELK1 binding site and diminished DNA-protein binding. Assessment of genetic associations can be strengthened using a genetic/genomic approach. This approach identified several candidate genes, including Acvr1, associated with increased susceptibility to acute lung injury in mice.

GnRH Induces the c-Fos Gene via Phosphorylation of SRF by the Calcium/Calmodulin Kinase II Pathway

Despite extensive studies on GnRH regulation of the gonadotropin subunit genes, very little is known about mechanism of induction of intermediary immediate early genes, such as c-Fos, that are direct targets of GnRH signaling and that upon induction, activate transcription of gonadotropin genes. Although c-Fos is induced by a variety of stimuli in other cell types, in the gonadotropes, only GnRH induces c-Fos and through it FSHβ. Thus, understanding the specificity of c-Fos induction by GnRH will provide insight into GnRH regulation of FSHβ gene expression. GnRH induction of c-Fos in LβT2 cells requires the serum response factor (SRF)-binding site, but not the Ets/ELK1 site. This is in contrast to c-Fos induction by growth factors in other cells, which activate c-Fos transcription via phosphorylation of ELK1 and require the ELK1-binding site. The SRF site alone is sufficient for induction by GnRH, whereas induction by 12-tetradecanoylphorbol-13-acetate (TPA) requires both the ELK1 and SRF sites. Although ELK1 site is not required, upon GnRH stimulation, ELK1 interacts with SRF and is recruited to the SRF site. GnRH phosphorylates ELK1 through ERK1/2 and p38 MAPK, which correlates with the signaling pathways necessary for c-Fos and FSHβ induction. GnRH also causes phosphorylation of SRF through calmodulin-dependent kinase II (CamKII), which leads to increased binding to its site. CamKII activation is sufficient for phosphorylation of SRF and for induction of the c-Fos gene through the SRF site. Thus, GnRH uses a combination of growth factor signaling and the CamKII pathway to induce c-Fos to regulate FSHβ gene expression in gonadotrope cells.

Alcohol Induces RNA Polymerase III-dependent Transcription through c-Jun by Co-regulating TATA-binding Protein (TBP) and Brf1 Expression

Chronic alcohol consumption is associated with steatohepatitis and cirrhosis, enhancing the risk for hepatocellular carcinoma. RNA polymerase (pol) III transcribes a variety of small, untranslated RNAs, including tRNAs and 5S rRNAs, which determine the biosynthetic capacity of cells. Increased RNA pol III-dependent transcription, observed in transformed cells and human tumors, is required for oncogenic transformation. Given that alcohol consumption increases risk for liver cancer, we examined whether alcohol regulates this class of genes. Ethanol induces RNA pol III-dependent transcription in both HepG2 cells and primary mouse hepatocytes in a manner that requires ethanol metabolism and the activation of JNK1. This regulatory event is mediated, at least in part, through the ability of ethanol to induce expression of the TFIIIB components, Brf1, and the TATA-binding protein (TBP). Induction of TBP, Brf1, and RNA pol III-dependent gene expression is driven by enhanced c-Jun expression. Ethanol promotes a marked increase in the direct recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. Chronic alcohol administration in mice leads to enhanced expression of TBP, Brf1, tRNA, and 5S rRNA gene transcription in the liver. These alcohol-dependent increases are more pronounced in transgenic animals that express the HCV NS5A protein that display increased incidence of liver tumors. Together, these results identify a new class of genes that are regulated by alcohol through the co-regulation of TFIIIB components and define a central role for c-Jun in this process.

Integrative genomic analyses on Ikaros and its expression related to solid cancer prognosis

Ikaros is a member of the Kruppel family of zinc finger DNA-binding proteins. The Ikaros protein contains two separate regions of zinc-finger domains: 4 DNA-binding zinc fingers near the N-terminus and 2 zinc fingers for protein-protein interactions near the C-terminus. Here, we identified the Ikaros gene from 14 vertebrate genomes and found Ikaros existed in all kinds of vertebrate including fish, amphibians, birds and mammals. Moreover, except rat and Xenopus tropicalis Ikaros proteins, which lack the first C2H2-type 1 Zinc finger region, all identified Ikaros proteins contain six C2H2-type 1 Zinc finger regions. We found human Ikaros gene showed a predominant expression in the liver, lymph node, thymus, intestine, lung, mammary gland, bone marrow, brain, heart, placenta and prostate. Moreover, four available SNPs disrupted an existing exonic splicing enhancer were identified in Ikaros. Besides the reported acute lymphoblastic leukemia (ALL), the expression of Ikaros was related to the prognosis of 13 cases of cancers including blood cancers, breast, lung, ovarian and skin cancer. Moreover, the relationship between the expression of Ikaros and prognosis varied in different cancers, even in the same cancer from different database. Two tumor-related transcriptional factor (c-Fos and Elk-1) binding sites were identified within the 1.5-kb regions upstream of the transcriptional start site of human Ikaros, which may be involved in the effect of Ikaros in tumors.

Abstract 4864: EGF-induced RNA pol III transcription mediated by epigenetic modification of histone H3

Abstract Cancer cells have a consistent cytological feature of nucleolar hypertrophy, where RNA Pol I and Pol III genes are transcripted. This implies that transformation in situ is closely linked to the deregulation of rRNA transcription, because the size of the nucleolus reflects the levels of rRNA synthesis. RNA Pol III genes encode a variety of untranslated RNAs, including tRNAs and 5S rRNAs. Deregulation of RNA Pol III transcription enhances cellular tRNAs and 5S rRNA production, leading to an increase in translational capacity to promote cell proliferation, transformation, and tumor formation. tRNA gene promoters require the transcription factor complexes, TFIIIB (TBP, Brf1 and Bdp1) and TFIIIC in addition to RNA pol III, to specify accurate and efficient transcription. The TFIIIB subunits, Brf1 and Bdp1, are specifically used in the RNA pol III transcription. Recent study demonstrated that the induction of Brf1 caused an increase in cell proliferation and oncogenic transformation, whereas depletion of Brf1 impeded transformation. These results demonstrate that increases in RNA Pol III transcription are necessary for cellular transformation. Our previous study showed that EGF induces TBP expression through MAP kinases, mediating RNA Pol III transcription. We had identified that MAK kinases mediated phosphorylation of histone H3 at serine 10 and 28. However, it is not clear which upstream of MAP kinases involves in EGF-induced RNA Pol III transcription and whether epigenetic modifications of H3 mediate the induction genes. Our recent studies showed that EGF strongly induces phosphorylation of H3 serine 28 (H3S28). Furhermore, we found that EGFR inhibitor AG1517 reduced EGF-induced both RNA Pol III transcription and phosphorylation of H3S28, but PI3K inhibitor LY 294002 did not decrease the inductions. EGFR deficient blocked the induction of RNA Pol III genes and phosphorylation of H3S28. In contrast, dominant negative mutant PI3K, Δp85 did not repress the inductions by EGF. To further explore the mechanism underlying EGF-induced RNA Pol III transcription, we found that EGF increases TFIIIB subunit, Brf1 expression. To investigate whether the epigenetic modification phosphor-H3S28 mediates RNA Pol III transcription, we determined phosphorylated H3S28 occupancy on Brf1 promoter by using CHIP assay. The result indicates that phospho-H3S28 specifically occupant on Elk-1 binding site of mouse Brf1 promoter at −507/−346, but not at −71/+98, regulating RNA Pol III transcription. Taken together, these results indicate that EGF activates EGFR and induces phosphorylation of H3S28, which mediate Brf1 expression to upregulate RNA Pol III transcription. This project was supported by NIH grant AA017288 to S.Z. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4864.

The Elk-1 and Serum Response Factor Binding Sites in the Major Immediate-Early Promoter of Human Cytomegalovirus Are Required for Efficient Viral Replication in Quiescent Cells and Compensate for Inactivation of the NF-κB Sites in Proliferating Cells

The major immediate-early promoter (MIEP) region of human cytomegalovirus (HCMV) plays a critical role in the regulation of lytic and latent infections by integrating multiple signals supplied by the infecting virus, the type and physiological state of the host cell, and its extracellular surroundings. The interaction of cellular transcription factors with their cognate binding sites, which are present at high densities within the enhancer upstream from the MIEP core promoter, regulate the rate of IE gene transcription and thus affect the outcome of HCMV infection. We have shown previously that the NF-kappaB binding sites within the MIEP enhancer and cellular NF-kappaB activity induced by HCMV infection are required for efficient MIEP activity and viral replication in quiescent cells (P. Caposio, A. Luganini, G. Hahn, S. Landolfo, and G. Gribaudo, Cell. Microbiol. 9:2040-2054, 2007). We now show that the inactivation of either the Elk-1 or serum response factor (SRF) binding site within the enhancer also reduces MIEP activation and viral replication of recombinant HCMV viruses in quiescent fibroblasts. In these cells, we show that the expression of either Elk-1 or SRF is required for optimal IE gene expression, and that the HCMV-stimulated activation of the MEK1/2-ERK1/2 signaling axis leads to Elk-1 transcriptional competency. Furthermore, the replication kinetics of recombinant viruses in which NF-kappaB, Elk-1, and SRF binding sites all are inactivated demonstrate that the higher levels of Elk-1 and SRF binding to MIEP in proliferating cells can compensate even for a lack of HCMV-induced NF-kappaB-mediated MIEP transactivation. These observations highlight the importance of the combination of different MIEP binding sites to optimize IE gene expression in cells in different physiological states.

Regulatory Effect of Extracellular Signal-Regulated Kinases (ERK) on Type I Collagen Synthesis in Human Dermal Fibroblasts Stimulated by IL-4 and IL-13

Synthesis of collagen is up-regulated by pro-fibrogenic growth factors and cytokines such as TGF-β 1, IL-4, and IL-13 binding to their corresponding cell membrane receptors of fibroblasts. The ERK pathway is an important MAPK signaling pathway that is involved in regulating cell function. The aim of our studies was to examine effects of IL-4 and IL-13 on the ERK signaling pathway and its function in regulating type I collagen gene expression in human fibroblasts. We found that human dermal fibroblasts treated with IL-4 and IL-13 exhibited an increase in the activated ERK1/2 pathway. As well, pro-fibrogenic cytokines increased the promoter activity of type I collagen, and this activity decreased with cells that were co-transfected with dominant negative plasmids of ERK1 and 2. RT-PCR confirmed that collagen transcript levels decreased when cells were transfected with dn ERK1 and 2 and then further stimulated with IL-4 and IL-13. These results were also mirrored with collagen secretion assays. In addition, we studied the role for transcription factor Elk-1 known to be activated via the ERK pathway. Dominant negative Elk-1 showed inhibition of collagen promoter activity in fibroblasts transfected with full collagen type I promoter or two fragments which contain the Elk-1 binding site. Our results suggest that the modulation of collagen gene expression may occur via the ERK pathway and is mediated by Elk-1.

Elk1 and SRF transcription factors convey basal transcription and mediate glucose response via their binding sites in the human LXRB gene promoter

The nuclear receptors LXRα (NR1H3) and LXRβ (NR1H2) are attractive drug targets for the treatment of diabetes and cardiovascular disease due to their established role as regulators of cholesterol and lipid metabolism. A large body of literature has recently indicated their important roles in glucose metabolism and particularly LXRβ is important for proper insulin production in pancreas. In this study, we report that glucose induces transcription via the LXRB gene promoter. The transcription start site of the human LXRB gene was determined and we identified two highly conserved, and functional, ETS and Elk1 binding sites, respectively, in the LXRB gene promoter. The Elk1 binding site also bound the serum responsive factor (SRF). Mutation of these sites abolished binding. Furthermore, mutation of the binding sites or siRNA knockdown of SRF and Elk1 significantly reduced the promoter activity and impaired the glucose response. Our results indicate that the human LXRB gene is controlled by glucose, thereby providing a novel mechanism by which glucose regulates cellular functions via LXRβ.

Long‐term depression activates transcription of immediate early transcription factor genes: involvement of serum response factor/Elk‐1

Long-term depression (LTD) is one of the paradigms used in vivo or ex vivo for studying memory formation. In order to identify genes with potential relevance for memory formation we used mouse organotypic hippocampal slice cultures in which chemical LTD was induced by applications of 3,5-dihydroxyphenylglycine (DHPG). The induction of chemical LTD was robust, as monitored electrophysiologically. Gene expression analysis after chemical LTD induction was performed using cDNA microarrays containing >7,000 probes. The DHPG-induced expression of immediate early genes (c-fos, junB, egr1 and nr4a1) was subsequently verified by TaqMan polymerase chain reaction. Bioinformatic analysis suggested a common regulator element [serum response factor (SRF)/Elk-1 binding sites] within the promoter region of these genes. Indeed, here we could show a DHPG-dependent binding of SRF at the SRF response element (SRE) site within the promoter region of c-fos and junB. However, SRF binding to egr1 promoter sites was constitutive. The phosphorylation of the ternary complex factor Elk-1 and its localization in the nucleus of hippocampal neurones after DHPG treatment was shown by immunofluorescence using a phosphospecific antibody. We suggest that LTD leads to SRF/Elk-1-regulated gene expression of immediate early transcription factors, which could in turn promote a second broader wave of gene expression.

Smooth Muscle-Specific Genes Are Differentially Sensitive to Inhibition by Elk-1

Understanding the mechanism of smooth muscle cell (SMC) differentiation will provide the foundation for elucidating SMC-related diseases, such as atherosclerosis, restenosis, and asthma. In the current study, overexpression of Elk-1 in SMCs down-regulated expression of several endogenous smooth muscle-restricted proteins, including telokin, SM22alpha, and smooth muscle alpha-actin. In contrast, down-regulation of endogenous Elk-1 in smooth muscle cells increased the expression of only telokin and SM22alpha, suggesting that smooth muscle-specific promoters are differentially sensitive to the inhibitory effects of Elk-1. Consistent with this, overexpression of the DNA binding domain of Elk-1, which acts as a dominant-negative protein by displacing endogenous Elk-1, enhanced the expression of telokin and SM22alpha without affecting expression of smooth muscle alpha-actin. Elk-1 suppressed the activity of smooth muscle-restricted promoters, including the telokin promoter that does not contain a consensus Elk-1 binding site, through its ability to block myocardin-induced activation of the promoters. Gel mobility shift and chromatin immunoprecipitation assays revealed that Elk-1 binds to a nonconsensus binding site in the telokin promoter and Elk-1 binding is dependent on serum response factor (SRF) binding to a nearby CArG box. Although overexpression of the SRF-binding B-box domain of Elk-1 is sufficient to repress the myocardin activation of the telokin promoter, this repression is not as complete as that seen with an Elk-1 fragment that includes the DNA binding domain. In addition, reporter gene assays demonstrate that an intact Elk-1 binding site in the telokin promoter is required for Elk-1 to maximally inhibit promoter activity. Together, these data suggest that the differential sensitivity of smooth muscle-specific genes to inhibition by Elk-1 may play a role in the complex changes in smooth muscle-specific protein expression that are observed under pathological conditions.

Comparative genomics on Vangl1 and Vangl2 genes

WNT signals are transduced to the beta-catenin pathway or the planar cell polarity (PCP) pathway. WNT - beta-catenin pathway is implicated in carcinogenesis, while WNT-PCP pathway is implicated in cell motility and metastasis. Drosophila Van Gogh (Vang), Frizzled (Fz), Starry night (Stan), Prickle (Pk) and Diego (Dgo) are PCP signaling molecules. Vangl1 (Strabismus 2) and Vangl2 (Strabismus 1 or Ltap) are mammalian homologs of Drosophila Vang interacting with PRICKLE1, PRICKLE2, ANKRD6, DVL1, DVL2, DVL3, KAI1 and MAGI3. Here we identified and characterized rat Vangl1 and Vangl2 genes by using bioinformatics. Rat Vangl1 gene, consisting of eight exons, was located within AC098913.7 and AC108524.6 genome sequences. Rat Vangl2 gene, consisting of eight exons, was located within AC118856.3 and AC115243.5 genome sequences. Exon-intron structure of mammalian Vangl1 and Vangl2 orthologs was well conserved. E47 and double ELK1-binding sites were conserved among promoters of mammalian Vangl1 orthologs. PAX4, NFkappaB, HNF4, SOX9, RFX1, and POU2F1 (OCT1)-binding sites were conserved among promoters of mammalian Vangl2 orthologs. Rat Vangl1 (526 aa) and Vangl2 (521 aa) were four-transmembrane proteins with 71.5% total-amino-acid identity. Ser cluster motif (SxxSxxSxxSxxSxxS) in the N-terminal cytoplasmic region and PDZ-binding motif in the C-terminal cytoplasmic tail were evolutionarily conserved among vertebrate Vangl1 and Vangl2 orthologs. This is the first report on rat Vangl1 and Vangl2 genes as well as on comparative genomics for Vangl1 and Vangl2 orthologs.