07-107 Sigma-AldrichAnti-Sp3 Antibody

The epithelial Na+ channel (ENaC) in the distal nephron constitutes the rate-limiting step for renal sodium reabsorption. Aldosterone increases tubular sodium absorption in large part by increasing αENaC transcription in collecting duct principal cells. We previously reported that Af9 binds to +78/+92 of αENaC and recruits Dot1a to repress basal and aldosterone-sensitive αENaC transcription in mouse inner medullary collecting duct (mIMCD)3 cells. Despite this epigenetic repression, basal αENaC transcription is still evident and physiologically necessary, indicating basal operation of positive regulators. In the present study, we identified Sp1 as one such regulator. Gel shift and antibody competition assays using a +208/+240 probe revealed DNA-Sp1-containing complexes in mIMCD3 cells. Mutation of the +222/+229 element abrogated Sp1 binding in vitro and in promoter-reporter constructs stably expressed in mIMCD3 cells. Compared with the wild-type promoter, an αENaC promoter-luciferase construct with +222/+229 mutations exhibited much lower activity and impaired trans-activation in Sp1 overexpression experiments. Conversely, Sp1 knockdown inhibited endogenous αENaC mRNA and the activity of the wild-type αENaC promoter but not the mutated construct. Aldosterone triggered Sp1 recruitment to the αENaC promoter, which was required for maximal induction of αENaC promoter activity and was blocked by spironolactone. Sequential chromatin immunoprecipitation assays and functional tests of +78/+92 and +222/+229 αENaC promoter mutants indicated that while Sp1, Dot1a, and Af9 co-occupy the αENaC promoter, the Sp1 effects are functionally independent from Dot1a and Af9. In summary, Sp1 binding to a cis-element at +222/+229 represents the first identified constitutive driver of αENaC transcription, and it contributes to maximal aldosterone trans-activation of αENaC.

The integrin αvβ8 is a cell surface receptor for the latent domain (LAP) of the multifunctional cytokine TGF-β. Through its association with LAP, TGF-β is maintained in a latent form that must be activated to function. Binding to the integrin αvβ8 with subsequent metalloproteolytic cleavage of LAP represents a major mechanism of TGF-β activation in vivo. Altered expression of the integrin β8 subunit (ITGB8) is found in human chronic obstructive pulmonary disease, cancers, and brain vascular malformations. We have previously shown that the proinflammatory cytokine interleukin-1β (IL-1β) increases ITGB8 expression on lung fibroblasts, which increases αvβ8-mediated TGF-β activation in fibrosis and pathologic inflammation. Here we report the mechanism of increased ITGB8 expression by IL-1β. Our data support a model where the chromatin architecture of the ITGB8 core promoter is altered by nucleosomal repositioning that enhances the interaction of an AP1 complex (containing c-Jun and ATF2). This repositioning is caused by the dissociation of HDAC2 with the ITGB8 core promoter, leading to increased histone H4 acetylation and a loosening of nucleosomal-DNA interactions allowing "opening" of the chromatin structure and increased association of c-Jun and ATF-2. These changes are mediated through NFκB- and p38-dependent pathways. Ultimately, these events culminate in increasing ITGB8 transcription, αvβ8 surface expression, and αvβ8-mediated TGFβ activation.

We previously reported that BRG1, an ATPase subunit of SWI/SNF chromatin remodelling complexes, is constitutively expressed and that the alternative ATPase subunit (BRM) is inducibly expressed through differentiation in mammalian cells. In the present study, the regulatory elements that confer constitutive expression on brg1 were explored. First, we analysed the promoter proximal region surrounding its transcriptional start site. Using computer-aided analysis, a TATA-less, GC-rich promoter containing four putative binding sites for Sp1/3 was predicted. One of the putative Sp1/3-binding sites (from -21 to -15 bp) overlapped with a putative YY1-binding site. A gel-shift assay showed that YY1 but not Sp1/3 bound to this sequence and that Sp3 but not Sp1 bound to the other three predicted binding sites. Furthermore, chromatin immunoprecipitation analysis showed that Sp3 and YY1 bound to the promoter region together with TATA-binding protein in vivo. In vivo and in vitro binding assays showed that Sp3 and YY1 interacted with each other. Together, these results suggest that Sp3 and YY1 recruit general transcription factors and facilitate the assembly of a preinitiation complex.

Integrin alphavbeta8 is a critical regulator of transforming growth factor beta activation in vasculogenesis during development, immune regulation, and endothelial/epithelial-mesenchymal homeostasis. Recent studies have suggested roles for integrin beta8 in the pathogenesis of chronic obstructive pulmonary disease, brain arteriovenous malformations, and select cancers (Araya, J., Cambier, S., Markovics, J. A., Wolters, P., Jablons, D., Hill, A., Finkbeiner, W., Jones, K., Broaddus, V. C., Sheppard, D., Barzcak, A., Xiao, Y., Erle, D. J., and Nishimura, S. L. (2007) J. Clin. Invest. 117, 3551-3562; Su, H., Kim, H., Pawlikowska, L., Kitamura, H., Shen, F., Cambier, S., Markovics, J., Lawton, M. T., Sidney, S., Bollen, A. W., Kwok, P. Y., Reichardt, L., Young, W. L., Yang, G. Y., and Nishimura, S. L. (2010) Am. J. Pathol. 176, 1018-1027; Culhane, A. C., and Quackenbush, J. (2009) Cancer Res. 69, 7480-7485; Cambier, S., Mu, D. Z., O'Connell, D., Boylen, K., Travis, W., Liu, W. H., Broaddus, V. C., and Nishimura, S. L. (2000) Cancer Res. 60, 7084-7093). Here we report the first identification and characterization of the promoter for ITGB8. We show that a SP binding site and a cyclic AMP response element (CRE) in the ITGB8 core promoter are required for its expression and that Sp1, Sp3, and several AP-1 transcription factors form a complex that binds to these sites in a p38-dependent manner. Furthermore, we demonstrate the requirement for Sp3, ATF-2, and p38 for the transcription and protein expression of integrin beta8. Additionally, reduction of SP3 or inhibition of p38 blocks alphavbeta8-mediated transforming growth factor beta activation. These results place integrin beta8 expression and activity under the control of ubiquitous transcription factors in a stress-activated and pro-inflammatory pathway.

Cyclooxygenase (COX) encodes a rate-limiting enzyme in the biosynthesis of prostanoids. Although COX-1 is constitutively expressed in many tissues, we found that glucocorticoids cause elevated expression of COX-1 gene in cardiomyocytes. Corticosterone (CT) at physiologically relevant doses (0.05-1 microm) induces transcriptional activation of COX-1 gene as shown by nuclear run-on and promoter reporter assays. An antagonist of glucocorticoid receptor (GR), mifepristone, prevented CT from inducing COX-1. COX-1 gene promoter deletion and mutation studies indicate a role of Sp transcription factors in CT-induced COX-1 gene. EMSAs or chromatin immunoprecipitation assays suggest that GR and Sp3 transcription factor bind to the promoter of COX-1 gene. Coimmunoprecipitation assays found an association of GR with Sp3. Silencing Sp3 protein with small interfering RNA suppressed CT-induced COX-1 promoter activation. Our data suggest that activated GR interacts with Sp3 transcription factor in binding to COX-1 promoter to enhance COX-1 gene expression in cardiomyocytes.

The presence of tumor-specific mutations in the cancer genome represents a potential opportunity for pharmacologic intervention to therapeutic benefit. Unfortunately, many classes of oncoproteins (e.g., transcription factors) are not amenable to conventional small-molecule screening. Despite the identification of tumor-specific somatic mutations, most cancer therapy still utilizes nonspecific, cytotoxic drugs. One illustrative example is the treatment of Ewing sarcoma. Although the EWS/FLI oncoprotein, present in the vast majority of Ewing tumors, was characterized over ten years ago, it has never been exploited as a target of therapy. Previously, this target has been intractable to modulation with traditional small-molecule library screening approaches. Here we describe a gene expression-based approach to identify compounds that induce a signature of EWS/FLI attenuation. We hypothesize that screening small-molecule libraries highly enriched for FDA-approved drugs will provide a more rapid path to clinical application.A gene expression signature for the EWS/FLI off state was determined with microarray expression profiling of Ewing sarcoma cell lines with EWS/FLI-directed RNA interference. A small-molecule library enriched for FDA-approved drugs was screened with a high-throughput, ligation-mediated amplification assay with a fluorescent, bead-based detection. Screening identified cytosine arabinoside (ARA-C) as a modulator of EWS/FLI. ARA-C reduced EWS/FLI protein abundance and accordingly diminished cell viability and transformation and abrogated tumor growth in a xenograft model. Given the poor outcomes of many patients with Ewing sarcoma and the well-established ARA-C safety profile, clinical trials testing ARA-C are warranted.We demonstrate that a gene expression-based approach to small-molecule library screening can identify, for rapid clinical testing, candidate drugs that modulate previously intractable targets. Furthermore, this is a generic approach that can, in principle, be applied to the identification of modulators of any tumor-associated oncoprotein in the rare pediatric malignancies, but also in the more common adult cancers.

The protein kinase regulated by RNA (PKR) is interferon (IFN)-inducible and plays important roles in many cellular processes, including virus multiplication, cell growth, and apoptosis. The TATA-less PKR promoter possesses a novel 15-bp DNA element (kinase conserved sequence (KCS)) unique to the human and mouse PKR genes that is conserved in sequence and position. We found that Sp1 and Sp3 of the Sp family of transcription factors bind at the KCS element. Their involvement was analyzed in the activation of basal and IFN-inducible PKR promoter activity. Both the small and large isoforms of Sp3 co-purified with KCS protein binding activity (KBP) by using nuclear extracts from HeLa cells not treated with IFN. Two forms of the KCS-binding protein complex were demonstrated by electrophoretic mobility shift assay analysis; one contained Sp1 and the other Sp3. In mouse cells null for all Sp3 isoforms, PKR expression was reduced to approximately 50% that of wild-type cells in the absence of IFN. The IFN-inducible expression of PKR, however, was Sp3-independent but STAT1- and JAK1-dependent. Overexpression of Sp1 in human U cells resulted in increased PKR promoter activity. In Drosophila SL2 cells lacking Sp proteins, both Sp1 and Sp3 large but not small isoforms activated PKR promoter expression, with the Sp1-mediated activation dominant. Mutational analysis of the PKR promoter region indicated a cooperative interaction between two different Sp sites, one of which is within the KCS element. These results establish that, in the absence of IFN treatment, activation of PKR basal expression is mediated by Sp1 and Sp3 proteins in a cooperative manner.

The mouse kappa opioid receptor (KOR) gene is constitutively expressed in P19 embryonic stem cells but is first suppressed and reactivated during retinoic acid (RA)-induced neuronal differentiation. However, no RA response element (RARE) can be found in this gene regulatory region. The suppression and reactivation of the KOR gene in this neuronal differentiation model suggested chromatin remodeling occurred on this gene promoter triggered by RA induction. This study asks whether RA induces alteration in the nucleosomal structure of this gene promoter that has no apparent RARE and, if so, how RA remodels chromatin of this promoter. The results revealed two loose nucleosomes, N1 at -44 (3' boundary) from the transcription initiation site and N2 spanning the transcription initiation site, that are relevant to active transcription. RA formed a repressive chromatin configuration of this promoter by compacting nucleosome N1, followed by nucleosome N2 condensation. Chromatin immunoprecipitation assay demonstrated RA induced replacement of the c-Myc/Max complex with the Max/Mad1 complex on the E box located within nucleosome N1, coinciding with reduced Sp1 binding to GC boxes located within nucleosome N2 and recruitment of chromatin remodeling factor Brahma-related gene 1 (BRG-1) to this promoter. Consistently, histone deacetylation, Lys9 methylation, and hypophosphorylation of RNA polymerase II C-terminal domain were detected on this promoter after RA treatment. It is concluded that RA induces KOR gene suppression, as early neuronal differentiation marker, by inducing substitution of c-Myc/Max with Max/Mad on the E box and by BRG-1 involved nucleosome recruitment and chromatin condensation, thereby abolishing Sp1 binding.

To investigate the function of transcription factor Sp1 in beta-like globin gene activation, we analyzed the recruitment of Sp1, fetal Krüppel-like factor 2 (FKLF2), and related factors at the human beta-globin locus in a human fetal liver and mouse erythroleukemia hybrid cell (A181gamma cell) that contains a single copy of human chromosome 11. Sp1 binds at the GT boxes of the cis-elements throughout the beta-locus, but it is phosphorylated and lost over DNase I hypersensitive site (HS)2, HS3, HS4, and the human beta-globin gene promoter after A181gamma cell differentiation. The binding of FKLF2 at HS2 and HS3 was unchanged. Histone deacetylase 1, which could be recruited by Sp1, is also lost over HS2 and HS3 after differentiation, resulting in the acetylation of histones 3 and 4 across the human beta-globin locus. We previously detected in vivo GT footprints over the beta-globin locus after A181gamma differentiation. Here, we report that after differentiation, the p300/CREB-binding protein-associated factor is recruited by FKLF2 to the locus control region to acetylate histones 3 and 4 at the human beta-globin gene locus. Our results suggest that Sp1 is an inhibitor of beta-like globin gene transcription during erythroid terminal differentiation. Its phosphorylation and release allow the erythroid-specific FKLF2 or erythroid Krüppel-like factor to interact with other erythroid-specific transcription factors to initiate the transcription of beta-like globin genes.

Increased expression of vascular endothelial growth factor (VEGF) contributes to the growth of many tumors by increasing angiogenesis. Although hypoxia is a potent inducer of VEGF, we previously showed that epidermal growth factor receptor amplification and loss of PTEN, both of which can increase phosphatidylinositol-3-kinase (PI3K) activity, increase VEGF expression. Using both adenoviral vectors and a cell line permanently expressing constitutively active myristoylated Akt (myrAkt), we show that activation of Akt, which is downstream of PI3K, increases VEGF expression in vitro and increases angiogenesis in a Matrigel plug assay. Transient transfection experiments using reporter constructs containing the VEGF promoter showed that up-regulation of VEGF by Akt is mediated through Sp1 binding sites located in the proximal promoter. Small interfering RNA directed against Sp1 prevented the induction of VEGF mRNA in response to myrAkt but not to hypoxia. Expression of myrAkt is associated with increased phosphorylation of Sp1 and its increased binding to a probe corresponding to the -88/-66 promoter region. In conclusion, our results indicate that Sp1 is required for transactivation of the VEGF by Akt. Others have proposed that the PI3K/Akt pathway can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1); however, our results suggest an alternative mechanism can also operate.