Proximal E-box Research Articles

CREB activates the MafA promoter through proximal E-boxes and a CCAAT motif in pancreatic β-cells.

MafA is a key transcriptional regulator of pancreatic islet β-cell function. Its target genes include those encoding preproinsulin and the glucose transporter Glut2 (Slc2a2); thus, MafA function is essential for glucose-stimulated insulin secretion. Expression levels of MafA are reduced in β-cells of diabetic mouse models and human subjects, suggesting that β-cell dysfunction associated with type 2 diabetes is attributable to the loss of MafA. On the other hand, MafA is transcriptionally upregulated by incretin hormones through activation of CREB and its co-activator CRTC2. β-cell-specific expression of MafA relies on a distal enhancer element. However, the precise mechanism by which CREB-CRTC2 regulates the enhancer and proximal promoter regions of MafA remains unclear. In this report, we analyzed previously published ChIP-seq data and found that CREB and NeuroD1, a β-cell-enriched transactivator, bound to both the promoter and enhancer regions of human MAFA. A series of reporter assays revealed that CREB activated the enhancer through a conserved cAMP-responsive element (CRE) but stimulated MAFA promoter activity even when the putative CRE was deleted. Two E-box elements and a CCAAT motif, which bind NeuroD1 and ubiquitous NF-Y transcription factors, respectively, were necessary for transcriptional activation of the MAFA promoter by CREB. Genome-wide analysis of CREB-bound loci in β-cells revealed that they were enriched with CCAAT motifs. Furthermore, promoter analysis of the Isl1 gene encoding a β-cell-enriched transcription factor revealed that a CRE-like element and two CCAAT motifs, but not the E-box, were necessary for activation by CREB. These results provide clues to elucidate the detailed mechanism by which CREB regulates MafA as well as β-cell-specific genes.

ABCA1 Expression Is Upregulated in an EMT in Breast Cancer Cell Lines via MYC-Mediated De-Repression of Its Proximal Ebox Element.

The ATP-Binding Cassette transporter A1 (ABCA1) reverse cholesterol transport channel has been associated with a number of phenotypes in breast cancer, including reduced proliferation and increased metastatic capacity. It is induced in an epithelial–mesenchymal transition (EMT), but little is known about how this occurs, and whether it is sufficient to promote metastatic phenotypes. To address these questions, we have deciphered the transcriptional regulation of ABCA1 across EMT states and found that it is repressed by MYC via an E-box element in its P1 alternative promoter. De-repression of the promoter by MYC knockdown leads to induction of ABCA1 expression. This indicates that ABCA1 expression is regulated in an EMT, revealing another link between ABCA1 and malignant phenotypes.

TMPRSS4 promotes cancer stem\u2013like properties in prostate cancer cells through upregulation of SOX2 by SLUG and TWIST1

BackgroundTransmembrane serine protease 4 (TMPRSS4) is a cell surface–anchored serine protease. Elevated expression of TMPRSS4 correlates with poor prognosis in colorectal cancer, gastric cancer, prostate cancer, non–small cell lung cancer, and other cancers. Previously, we demonstrated that TMPRSS4 promotes invasion and proliferation of prostate cancer cells. Here, we investigated whether TMPRSS4 confers cancer stem–like properties to prostate cancer cells and characterized the underlying mechanisms.MethodsAcquisition of cancer stem–like properties by TMPRSS4 was examined by monitoring anchorage-independent growth, tumorsphere formation, aldehyde dehydrogenase (ALDH) activation, and resistance to anoikis and drugs in vitro and in an early metastasis model in vivo. The underlying molecular mechanisms were evaluated, focusing on stemness-related factors regulated by epithelial–mesenchymal transition (EMT)-inducing transcription factors. Clinical expression and significance of TMPRSS4 and stemness-associated factors were explored by analyzing datasets from The Cancer Genome Atlas (TCGA).ResultsTMPRSS4 promoted anchorage-independent growth, ALDH activation, tumorsphere formation, and therapeutic resistance of prostate cancer cells. In addition, TMPRSS4 promoted resistance to anoikis, thereby increasing survival of circulating tumor cells and promoting early metastasis. These features were accompanied by upregulation of stemness-related factors such as SOX2, BMI1, and CD133. SLUG and TWIST1, master EMT-inducing transcription factors, made essential contributions to TMPRSS4-mediated cancer stem cell (CSC) features through upregulation of SOX2. SLUG stabilized SOX2 via preventing proteasomal degradation through its interaction with SOX2, while TWIST1 upregulated transcription of SOX2 by interacting with the proximal E-box element in the SOX2 promoter. Clinical data showed that TMPRSS4 expression correlated with the levels of SOX2, PROM1, SNAI2, and TWIST1. Expression of SOX2 was positively correlated with that of TWIST1, but not with other EMT-inducing transcription factors, in various cancer cell lines.ConclusionsTogether, these findings suggest that TMPRSS4 promotes CSC features in prostate cancer through upregulation of the SLUG- and TWIST1-induced stem cell factor SOX2 beyond EMT. Thus, TMPRSS4/SLUG–TWIST1/SOX2 axis may represent a novel mechanism involved in the control of tumor progression.

Hypoxia Inhibits Myogenic Differentiation through p53 Protein-dependent Induction of Bhlhe40 Protein

Satellite cells are muscle-resident stem cells capable of self-renewal and differentiation to repair injured muscles. However, muscle injury often leads to an ischemic hypoxia environment that impedes satellite cell differentiation and reduces the efficiency of muscle regeneration. Here we performed microarray analyses and identified the basic helix-loop-helix family transcription factor Bhlhe40 as a candidate mediator of the myogenic inhibitory effect of hypoxia. Bhlhe40 is strongly induced by hypoxia in satellite cell-derived primary myoblasts. Overexpression of Bhlhe40 inhibits Myog expression and mimics the effect of hypoxia on myogenesis. Inhibition of Bhlhe40, conversely, up-regulates Myog expression and promotes myogenic differentiation. Importantly, Bhlhe40 knockdown rescues myogenic differentiation under hypoxia. Mechanistically, Bhlhe40 binds to the proximal E-boxes of the Myog promoter and reduces the binding affinity and transcriptional activity of MyoD on Myog. Interestingly, hypoxia induces Bhlhe40 expression independent of HIF1α but through a novel p53-dependent signaling pathway. Our study establishes a crucial role of Bhlhe40 in mediating the repressive effect of hypoxia on myogenic differentiation and suggests that inhibition of Bhlhe40 or p53 may facilitate muscle regeneration after ischemic injuries.

Achaete scute-like 2 suppresses CDX2 expression and inhibits intestinal neoplastic epithelial cell differentiation.

The role of Achaete scute-like 2 (Ascl2) in colorectal cancer (CRC) cell differentiation is unknown. LS174T, HT-29 and Caco-2 cells have high Ascl2 expression, while Lovo and SW480 cells have low Ascl2 expression. LS174T and HT-29 cells with Ascl2 knockdown were transfected with caudal type homeobox 2 (CDX2) promoter constructs and used for luciferase assays and chromatin immunoprecipitation (ChIP) assays. Ascl2 knockdown promoted differentiation of CRC cells into a goblet cell phenotype, as determined by increased expression of MUC2, TFF3, and CDX2. Ascl2 knockdown activated CDX2 expression through a transcriptional mechanism via direct binding of Ascl2 to the proximal E-box of the CDX2 promoter. Ascl2 over-expression in Lovo and SW480 cells inhibited a goblet cell phenotype, as determined by reduced CDX2 and MUC2 expression. Inverse correlations between Ascl2 and CDX2, and Ascl2 and MUC2 mRNA levels, as well as Ascl2 and CDX2 protein levels were observed in CRC cancerous samples. This study demonstrates CDX2 repression by Ascl2 and highlights a role for Ascl2 in CRC cell differentiation. These findings suggest that the Ascl2/CDX2 axis may serve as a potential therapeutic target in colorectal cancer.

Method for trapping affinity chromatography of transcription factors using aldehyde–hydrazide coupling to agarose

The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP–C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP–C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3′ end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide–agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA–protein complexes form. The complex is then coupled to hydrazide–agarose for trapping the DNA–protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach.

Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility

Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22bp apart and within 70bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells.

Expression of the Myosin Heavy Chain IIB Gene in Porcine Skeletal Muscle: The Role of the CArG-Box Promoter Response Element

Due to its similarity to humans, the pig is increasingly being considered as a good animal model for studying a range of human diseases. Despite their physiological similarities, differential expression of the myosin heavy chain (MyHC) IIB gene (MYH4) exists in the skeletal muscles of these species, which is associated with a different muscle phenotype. The expression of different MyHC isoforms is a critical determinant of the contractile and metabolic characteristics of the muscle fibre. We aimed to elucidate whether a genomic mechanism was responsible for the drastically different expression of MYH4 between pigs and humans, thus improving our understanding of the pig as a model for human skeletal muscle research. We utilized approximately 1 kb of the MYH4 promoter from a domestic pig and a human (which do and do not express MYH4, respectively) to elucidate the role of the promoter sequence in regulating the high expression of MYH4 in porcine skeletal muscle. We identified a 3 bp genomic difference within the proximal CArG and E-box region of the MYH4 promoter of pigs and humans that dictates the differential activity of these promoters during myogenesis. Subtle species-specific genomic differences within the CArG-box region caused differential protein-DNA interactions at this site and is likely accountable for the differential MYH4 promoter activity between pigs and humans. We propose that the genomic differences identified herein explain the differential activity of the MYH4 promoter of pigs and humans, which may contribute to the differential expression patterns displayed in these otherwise physiologically similar mammals. Further, we report that both the pig and human MYH4 promoters can be induced by MyoD over-expression, but the capacity to activate the MYH4 promoter is largely influenced by the 3 bp difference located within the CArG-box region of the proximal MYH4 promoter.

MicroRNA-200 (miR-200) Cluster Regulation by Achaete Scute-like 2 (Ascl2): IMPACT ON THE EPITHELIAL-MESENCHYMAL TRANSITION IN COLON CANCER CELLS

Ascl2, a basic helix-loop-helix transcription factor, is a downstream target of WNT signaling that controls the fate of intestinal cryptic stem cells and colon cancer progenitor cells. However, its involvement in colon cancer and downstream molecular events is largely undefined; in particular, the mechanism by which Ascl2 regulates the plasticity of epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) programs in colon cancer cells remains unknown. In this study, we systematically demonstrate that Ascl2 loss of function in colon cancer cells promotes MET by derepressing the expression of microRNA (miR)-200s (i.e. miR-200b, miR-200a, miR-429, miR-200c, and miR-141) and further activating their expression through a transcriptional mechanism that involves direct binding to the most proximal E-box (E-box2) in the miR-200b-a-429 promoter. Activation of miR-200s due to Ascl2 deficiency led to the inhibition of ZEB1/2 expression and the alteration of epithelial and mesenchymal features. Transfection of miR-200b, miR-200a, and miR-429 inhibitors into Ascl2-deficient colon cancer cells promoted the epithelial-mesenchymal transition in a reversible manner. Transfection of miR-200a or miR-429 inhibitors into Ascl2-deficient colon cancer cells increased cellular proliferation and migration. Ascl2 mRNA levels and the miR-200a, miR-200b, miR-200c, miR-141, or miR-429 levels in the colon cancerous samples were inversely correlated. These results provide the first evidence of a link between Ascl2 and miR-200s in the regulation of EMT-MET plasticity in colon cancer.

Abstract 2260: c-Myc induced senescence is dependent on transactivation of actin depolymerizing factor (ADF)/cofilin to mediate potent bystander effect

Abstract Oncogene-induced senescence may prevent unlimited proliferation and lead to cell death. This phenomenon is likely to be associated with up-regulation of p53 and p16INK4 tumor suppressor. Morphological transformation is also an important feature of senescence that exhibits enlarged cell volume and flat morphology. In this study, we found that c-Myc was co-upregulated with cofilin-1, an actin-binding protein essential for actin dynamics and morphological maintenance in replicative senescence. Additionally, oxidative stress induced senescent phenotypes also accompanied by up-regulation of both c-myc and cofilin-1. Knockdown of cofilin-1 was sufficient to minimize the cell shapes and senescence associated β-galactosidase marker that was induced by over-expressed c-myc or oxidative stress. Over-expression of cofilin-1 was also sufficient to induce senescence in the absence of p53 and p16INK4, but was unable to influence c-Myc level. On the other hand, over-expression of c-Myc could induce cofilin-1 expression. We next found that c-myc was able to induce the transactivation of cofilin-1 gene according to results of quantitative polymerase chain reaction (qPCR) and luciferase reporter gene assay. Furthermore, the results of ChIP assay provided evidence that c-myc can bind to two proximal E-box elements nearby the transcriptional initiation on cofilin-1 gene promoter. Although c-myc promoted cellular senescence in vitro, the conditional medium from c-myc over-expressing A549 cells accelerated migration and proliferation in separate cultured cells without c-myc over-expression. Knockdown of cofilin-1 also alleviated this bystander effect caused by over-expression of c-myc. Therefore, c-myc oncogene may require cofilin-1 for cell senescence that influences the growth of other tumor cells. Taken together, current data provide evidence that c-myc/cofilin-1 signaling pathway is a novel mechanism on promoting oncogene-induced cellular senescence and tumor progression. Citation Format: Shih-Ting Lin, Cheng-Han Tsai, Yi-Jang Lee, Chia-Chien Lo, Yen-Ting Chou. c-Myc induced senescence is dependent on transactivation of actin depolymerizing factor (ADF)/cofilin to mediate potent bystander effect. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2260. doi:10.1158/1538-7445.AM2014-2260

Dual roles of c-Myc in the regulation of hTERT gene.

Human telomerase gene hTERT is important for cancer and aging. hTERT promoter is regulated by multiple transcription factors (TFs) and its activity is dependent on the chromatin environment. However, it remains unsolved how the interplay between TFs and chromatin environment controls hTERT transcription. In this study, we employed the recombinase-mediated BAC targeting and BAC recombineering techniques to dissect the functions of two proximal E-box sites at −165 and +44 nt in regulating the hTERT promoter in the native genomic contexts. Our data showed that mutations of these sites abolished promoter binding by c-Myc/Max, USF1 and USF2, decreased hTERT promoter activity, and prevented its activation by overexpressed c-Myc. Upon inhibition of histone deacetylases, mutant and wildtype promoters were induced to the same level, indicating that the E-boxes functioned to de-repress the hTERT promoter and allowed its transcription in a repressive chromatin environment. Unexpectedly, knockdown of endogenous c-Myc/Max proteins activated hTERT promoter. This activation did not require the proximal E-boxes but was accompanied by increased promoter accessibility, as indicated by augmented active histone marks and binding of multiple TFs at the promoter. Our studies demonstrated that c-Myc/Max functioned in maintaining chromatin-dependent repression of the hTERT gene in addition to activating its promoter.

CD10 expression is enhanced by Twist1 and associated with poor prognosis in esophageal squamous cell carcinoma with facilitating tumorigenicity in vitro and in vivo.

CD10 expression was identified as a contributor to cancer progression in several cancers; however, the exact biological significance and mechanism of CD10 expression remains unclear. In addition, CD10 expression in esophageal squamous cell carcinoma (ESCC) has not been studied. We investigated the relationship between CD10 and Twist1. Furthermore, we examined the effect of CD10 on tumorigenicity using in vivo and in vitro systems as well as establishing the clinical significance of CD10 expression in ESCC using large clinical samples. CD10 expression was upregulated by Twist1 and there was a strong correlation between mRNA and protein expression. Twist1 can specifically upregulate CD10 at the transcriptional level via an interaction with the promoter region of CD10 and the proximal E-box CAGGTG in the CD10 promoter was identified as a binding site for Twist1. CD10 is frequently expressed in ESCC cell lines and silencing CD10 suppresses migration/invasion and anchorage-independent tumor growth of ESCC cells. Knockdown of CD10 inhibits the growth of ESCC xenograft in nude mice, suggesting that CD10 plays a role in enhancing the tumorigenesis of ESCC. From among 153 ESCC samples, 46 (30.0%) showed varying degrees of CD10 expression in cancer cells. In addition, stromal fibroblasts also showed varying amounts of CD10 expression in 92 (60.9%) tumor samples. CD10 overexpression in cancer cells as well as in stromal fibroblasts was an independent poor prognostic factor in both overall survival and disease-free survival. CD10 could be a promising target for the treatment of ESCC.

Regulation of major vault protein expression by upstream stimulating factor 1 in SW620 human colon cancer cells

Major vault protein (MVP) is the main constituent of the vault ribonucleoprotein particle and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several types of normal tissues, little is known about its physiological role. In the present study, we identified the crucial MVP promoter elements that regulate MVP expression. An examination of tissue expression profiles revealed that MVP was expressed in the heart, placenta, lung, liver, kidney and pancreas. Elements of the MVP promoter contain binding sites for transcription factors, STAT, p53, Sp1, E-box, GATA, MyoD and Y-box. By deletion analysis, a conserved proximal E-box binding site was demonstrated to be important for human MVP promoter transactivation. Introduction of siRNA against upstream stimulating factor (USF) 1, which is known to bind the E-box binding site, decreased the expression of MVP in SW620 and ACHN cells. Using a chromatin immunoprecipitation (ChIP) assay, USF1 bound the MVP promoter in SW620 cells. These findings suggest that USF1 binding to an E-box element may be critical for basal MVP promoter activation. The results of the present study are useful in understanding the molecular mechanisms regulating MVP gene expression, and may aid in elucidating the physiological functions of MVP.

The transcription factor Snail enhanced the degradation of E-cadherin and desmoglein 2 in oral squamous cell carcinoma cells

Epithelial–mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell–cell junctions and cell polarity as well as the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail is a regulator of EMT that represses E-cadherin transcription through its interaction with proximal E-boxes in the promoter region of target genes. To investigate the role of Snail in EMT, we generated stable Snail transfectants using the oral squamous cell carcinoma cell line HSC-4 (Snail/HSC-4). Snail/HSC-4 cells had a spindle-shaped mesenchymal morphology, and enhanced migration and invasiveness relative to control cells. Consistent with these EMT changes, the downregulation of epithelial marker proteins, E-cadherin and desmoglein 2, and the upregulation of mesenchymal marker proteins, vimentin and N-cadherin were detected. Despite these observations, the mRNA levels of E-cadherin and desmoglein 2 did not decrease significantly. Although E-cadherin and desmoglein 2 proteins were stable in parental HSC-4 cells, these proteins were rapidly degraded in Snail/HSC-4 cells. The degradation of E-cadherin, but not desmoglein 2, was inhibited by dynasore, an inhibitor of dynamin-dependent endocytosis. Therefore, in HSC-4 cells Snail regulates levels of these proteins both transcriptionally and post-translationally.

The transcription factor Snail expressed in cutaneous squamous cell carcinoma induces epithelial–mesenchymal transition and down-regulates COX-2

Cutaneous spindle cell squamous cell carcinoma (SCC) is a rare, but highly malignant variant of SCC. The presence of spindle-shaped cells with a sarcomatous appearance, which are derived from squamous cells, suggests that these cells are produced as a result of epithelial–mesenchymal transition (EMT). EMT is a complex process in which epithelial cells lose their polarity and cell–cell contacts, while also acquiring increased motility and invasiveness. Snail regulates EMT by binding to proximal E-boxes in the promoter region of E-cadherin and repressing its transcription. When examining the expression of EMT markers and Snail in spindle cell SCCs, we found that cyclooxygenase-2 (COX-2) expression was down-regulated. Since it has been shown that COX-2 is constitutively overexpressed in a variety of malignancies, including colon, gastric, and lung carcinomas, the down-regulation of COX-2 expression was unexpected. The presence of E-box-like sequences in the promoter region of COX-2 prompted us to perform a more detailed analysis. We introduced a Snail expression vector into keratinocyte-derived cell lines (HaKaT, HSC5, and A431 cells), and isolated stable transfectants. We determined that COX-2 expression was down-regulated in cells expressing Snail. Consistent with these observations, reporter assays revealed that COX-2 promoter activity was repressed upon Snail overexpression. Thus Snail down-regulates COX-2 in these cells.

Polycomb Protein EZH2 Regulates Tumor Invasion via the Transcriptional Repression of the Metastasis Suppressor RKIP in Breast and Prostate Cancer

Epigenetic modifications such as histone methylation play an important role in human cancer metastasis. Enhancer of zeste homolog 2 (EZH2), which encodes the histone methyltransferase component of the polycomb repressive complex 2 (PRC2), is overexpressed widely in breast and prostate cancers and epigenetically silences tumor suppressor genes. Expression levels of the novel tumor and metastasis suppressor Raf-1 kinase inhibitor protein (RKIP) have been shown to correlate negatively with those of EZH2 in breast and prostate cell lines as well as in clinical cancer tissues. Here, we show that the RKIP/EZH2 ratio significantly decreases with the severity of disease and is negatively associated with relapse-free survival in breast cancer. Using a combination of loss- and gain-of-function approaches, we found that EZH2 negatively regulated RKIP transcription through repression-associated histone modifications. Direct recruitment of EZH2 and suppressor of zeste 12 (Suz12) to the proximal E-boxes of the RKIP promoter was accompanied by H3-K27-me3 and H3-K9-me3 modifications. The repressing activity of EZH2 on RKIP expression was dependent on histone deacetylase promoter recruitment and was negatively regulated upstream by miR-101. Together, our findings indicate that EZH2 accelerates cancer cell invasion, in part, via RKIP inhibition. These data also implicate EZH2 in the regulation of RKIP transcription, suggesting a potential mechanism by which EZH2 promotes tumor progression and metastasis.

Interplay between two myogenesis-related proteins: TBP-interacting protein 120B and MyoD

Gene expression in myogenesis is governed by multiple myogenic factors including MyoD. Previously, we demonstrated that TBP-interacting protein 120B (TIP120B) promotes in vitro myogenesis through its anti-ubiquitination ability. In this study, we investigated interplay between MyoD and TIP120B. Mouse C2C12 cells subjected to myotube differentiation contained increased amounts of TIP120B and MyoD. Dexamethasone, which inhibits myogenic signaling, decreased the amounts of those proteins. Mouse and human TIP120B promoters, which carry multiple E-box motifs, were potentiated by MyoD. In the human TIP120B, a proximal E-box binds to MyoD in vitro and exhibits MyoD-dependent transcription activation function. Expression of the endogenous TIP120B gene was correlated with the level of MyoD in different types of muscle-related cells. Furthermore, MyoD binds specifically to a proximal E-box-carrying promoter region in chromatin. Proteasome-sensitive MyoD was increased and decreased by overexpression and knockdown of TIP120B, respectively. Moreover, stability of MyoD was increased by TIP120B. The results suggest that MyoD and TIP120B potentiate each other at gene expression and post-translation levels, respectively, which may promote myogenesis cooperatively.

An Ebox Element in the Proximal Gata4 Promoter Is Required for Gata4 Expression In Vivo

GATA4 is an essential transcription factor required for the development and function of multiple tissues, including a major role in gonadogenesis. Despite its crucial role, the molecular mechanisms that regulate Gata4 expression in vivo remain poorly understood. We recently found that the Gata4 gene is expressed as multiple transcripts with distinct 5′ origins. These co-expressed alternative transcripts are generated by different non-coding first exons with transcripts E1a and E1b being the most prominent. Moreover, we previously showed that an Ebox element, located in Gata4 5′ flanking sequences upstream of exon 1a, is important for the promoter activity of these sequences in cell lines. To confirm the importance of this element in vivo, we generated and characterized Gata4 Ebox knockout mice. Quantitative PCR analyses realized on gonads, heart and liver at three developmental stages (embryonic, pre-pubertal and adult) revealed that the Ebox mutation leads to a robust and specific decrease (up to 89%) of Gata4 E1a transcript expression in all tissues and stages examined. However, a detailed characterization of the gonads revealed normal morphology and GATA4 protein levels in these mutants. Our qPCR data further indicate that this outcome is most likely due to the presence of Gata4 E1b mRNA, whose expression levels were not decreased by the Ebox mutation. In conclusion, our work clearly confirms the importance of the proximal Ebox element and suggests that adequate GATA4 protein expression is likely protected by a compensation mechanism between Gata4 E1a and E1b transcripts operating at the translational level.

Abstract 2162: Inhibition of the metastasis suppressor RKIP by EZH2 and YY1

Abstract The Raf kinase inhibitory protein (RKIP) acts as an activator of apoptosis and inhibitor of metastasis in prostate cancer. Diminished expression of RKIP has been reported in several cancers including prostate carcinomas; however, the underlying mechanism of RKIP regulation remains largely unknown. RKIP transcription is inhibited by the EMT inducer Snail which also represses the expression of the metastasis suppressor E-cadherin. E-cadherin and RKIP are co-expressed in many tumors. E-cadherin transcription is also suppressed by the polycomp repressive complex (PRP) transcription repressor, enhancer of zeste homolog 2 (EZH2), which mediates an H3 lysine 27 trimethylation in target promoters. EZH2 is highly expressed in several metastatic tumors including prostate cancer and its levels are inversely correlated with E-cadherin and RKIP expressions. The zinc-finger transcription factor Yin-Yang 1 (YY1) is also overexpressed in prostate tumors and has been associated with tumor progression. Silencing of YY1 by siRNA reverses the EMT phenotype of metastatic prostate cancer cell lines by induction of E-cadherin and RKIP. Here, we explored the direct roles of YY1 and EZH2 in the transcriptional regulation of RKIP in prostate cancer. We used as experimental models the metastatic and non-metastatic prostate cancer cell lines DU145 and LNCaP, respectively, which differ in the expression levels of EZH2, YY1 and RKIP. DU145 cells express low RKIP and high EZH2 and YY1 levels, whereas LNCaP cells express the opposite. Putative binding sites were identified for both YY1 and EZH2 (proximal E-boxes) within the RKIP promoter. CHiP analyses showed that both factors can physically associate with the RKIP promoter. Loss and gain of function reporter approaches showed that ectopic expression of EZH2 in LNCaP cells inhibits the wild type, but not the E-box mutant RKIP basal promoter activity. Similarly, a mutated EZH2 vector that lacks efficient binding to the target promoter couldn't inhibit the RKIP promoter activity. EZH2-mediated inhibition of RKIP promoter activity could also be inhibited by HDAC inhibitors, suggesting that HDAC recruitment and deacetylation of histone H3 is perquisite for EZH2 suppressive activity. Silencing of EZH2 or YY1 in DU145 also resulted in induction of RKIP promoter activity. The negative effect of YY1 on RKIP promoter activity was corroborated by inhibition of RKIP mRNA and protein levels. The above observations were further validated in prostate cancer tissue microarrays whereby YY1 and RKIP protein expressions were found to be inversely correlated. The present findings support the roles of EZH2 and YY1 as transcription repressors of RKIP in prostate cancer and suggest that their overexpression may be associated, in part, with the high cell metastatic potential through downregulation of RKIP. We propose that EZH2 and YY1 might be potential prognostic markers of prostate cancer progression and targets for therapeutic intervention. 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 2162. doi:10.1158/1538-7445.AM2011-2162

Expression of Insulinoma-Associated 2 (INSM2) in Pancreatic Islet Cells Is Regulated by the Transcription Factors Ngn3 and NeuroD1

The insulinoma-associated 2 (Insm2) gene is a member of the Snail/Gfi1/Insm1 transcriptional repressor superfamily. However, little is known about how the expression of human INSM2 or mouse Insm2 in neuroendocrine tissues is regulated. Here we report the expression of INSM2/Insm2 in human fetal pancreas and mouse embryos, as well as adult pancreatic islets, and its regulation by two major islet transcription factors. Mutagenesis and chromatin immunoprecipitation analysis demonstrated that the proximal E-boxes of the mouse Insm2 promoter are direct targets of neurogenin 3 and neurogenic differentiation 1 (NeuroD1). Furthermore, we found that endogenous Insm2 expression was activated in Ngn3/NeuroD1-transduced pancreatic epithelial duct cells. Our results suggest that Insm2 plays an important role in the differentiation cascade of Ngn3/NeuroD1 signaling in pancreatic islets.