17-601 Sigma-AldrichChIPAb+ Sp1 - ChIP Validated Antibody and Primer Set

Microsomal epoxide hydrolase (mEH, EPHX1) is a critical biotransformation enzyme, catalyzing the metabolism of many xenobiotics. Human mEH is transcribed using alternative promoters. The upstream E1 promoter is active in liver while the far upstream E1b promoter drives the expression of mEH in all tissues, including liver. Although several liver-specific transcription factors have been identified in the regulation of E1 transcription, little is known regarding the mechanisms of E1b transcriptional regulation. Genome-wide mapping of DNase I hypersensitive sites revealed an open chromatin region between nucleotide -300 upstream and +400 downstream of E1b. This area coincides with a previously described promoter region responsible for maintaining high basal promoter activity. In silico analysis of this location revealed several Sp1/Sp3 binding sites. Site-directed mutagenesis of these motifs suppressed the transactivation activity of the E1b proximal promoter, indicating their importance as contributors to E1b promoter regulation. Further, E1b promoter activities were increased significantly following Sp1 and Sp3 overexpression, while Mithramycin A, a selective Sp1 inhibitor, reduced the promoter activities. EMSA studies demonstrated that Sp1 bound to two putative Sp1/Sp3 binding sites. ChIP analysis confirmed that both endogenous Sp1 and Sp3 were bound to the proximal promoter region of E1b. Knockdown of Sp1 expression using siRNA did not alter the endogenous E1b transcriptional level, while knockdown of Sp3 greatly decreased E1b expression in different human cell lines. Taken together, these results support the concept that Sp1 and Sp3 are functionally involved as transcriptional integrators regulating the basal expression of the derived mEH E1b variant transcript.

Tissue-specific basic helix-loop-helix (bHLH) transcription factor proteins often play essential roles in cellular differentiation. The bHLH proteins SOHLH2 and SOHLH1 are expressed specifically in spermatogonia and oocytes and are required for early spermatogonial and oocyte differentiation. We previously reported that knocking out Sohlh2 causes defects in spermatogenesis and oogenesis similar to those in Sohlh1-null mice, and that Sohlh1 is downregulated in the gonads of Sohlh2-null mice. We also demonstrated that SOHLH2 and SOHLH1 can form a heterodimer. These observations led us to hypothesize that the SOHLH2/SOHLH1 heterodimer regulates the Sohlh1 promoter. Here, we show that SOHLH2 and SOHLH1 synergistically upregulate the Sohlh1 gene through E-boxes upstream of the Sohlh1 promoter. Interestingly, we identified an SP1-binding sequence, called a GC-box, adjacent to these E-boxes, and found that SOHLH1 could bind to SP1. Furthermore, chromatin-immunoprecipitation analysis using testes from mice on postnatal day 8 showed that SOHLH1 and SP1 bind to the Sohlh1 promoter region in vivo. Our findings suggest that an SOHLH2/SOHLH1/SP1 ternary complex autonomously and cooperatively regulates Sohlh1 gene transcription through juxtaposed E- and GC-boxes during early spermatogenesis and oogenesis.

Platelet-derived growth factor alpha (PDGFA) is frequently upregulated in various cancers and thought to function as a key player in the development and progression of tumor growth by regulating aspects of cell proliferation, angiogenesis and metastasis. However, the mechanism by which it is upregulated is not fully understood. Previously, we demonstrated that conditional deletion of two transcription factors that signal for the bone morphogenetic proteins (Smad1 and Smad5) in ovarian granulosa cells causes metastatic granulosa cell tumors (GCTs) in female mice and phenocopies human juvenile GCTs (JGCTs). Smad1/5 double conditional knockout tumors, as well as human JGCTs, are highly vascularized, hemorrhagic and mitotically active. Expression analysis of these tumors and their metastases revealed a significant upregulation of key proliferation and pro-angiogenic factors such as Pdgfa, Pdgfb and Vegf. We examined whether these genes were direct targets of SMAD1 and SMAD5. Knockdown of SMAD1 and SMAD5 in mouse primary granulosa cells and a human GCT-derived cell line (COV434) resulted in upregulation of PDGFA, but not PDGFB nor VEGF. We identified several putative SMAD1/5-binding sites in the PDGFA promoter, and chromatin immunoprecipitation and reporter assays demonstrated that SMAD1/5 interact with the PDGFA promoter to regulate its activity. Further, SMAD1/5 antagonize the activity of the transcription factor Sp1, a well-known positive regulator of PDGFA, by inhibiting its occupancy at a key regulatory site on the proximal PDGFA promoter. Collectively, our studies establish that loss of SMAD1/5 leads to upregulation of PDGFA in ovarian granulosa cells, and that a novel regulatory interaction exists between the BR-SMADs and Sp1 in controlling PDGFA expression during granulosa cell tumorigenesis.

Hepatitis C virus (HCV) infection is the major cause of hepatocellular carcinoma (HCC) in Japan. We previously identified the association of SNP rs2596542 in the 5' flanking region of the MHC class I polypeptide-related sequence A (MICA) gene with the risk of HCV-induced HCC. In the current study, we performed detailed functional analysis of 12 candidate SNPs in the promoter region and found that a SNP rs2596538 located at 2.8 kb upstream of the MICA gene affected the binding of a nuclear protein(s) to the genomic segment including this SNP. By electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay, we identified that transcription factor Specificity Protein 1 (SP1) can bind to the protective G allele, but not to the risk A allele. In addition, reporter construct containing the G allele was found to exhibit higher transcriptional activity than that containing the A allele. Moreover, SNP rs2596538 showed stronger association with HCV-induced HCC (P = 1.82 × 10(-5) and OR = 1.34) than the previously identified SNP rs2596542. We also found significantly higher serum level of soluble MICA (sMICA) in HCV-induced HCC patients carrying the G allele than those carrying the A allele (P = 0.00616). In summary, we have identified a functional SNP that is associated with the expression of MICA and the risk for HCV-induced HCC.

The developmental stage-specific expression of the human β-like globin genes has been studied for decades, and many transcriptional factors as well as other important cis elements have been identified. However, little is known about the microRNAs that potentially regulate β-like globin gene expression directly or indirectly during erythropoiesis. In this study, we show that microRNA 23a (miR-23a) and miR-27a promote β-like globin gene expression in K562 cells and primary erythroid cells through targeting of the transcription factors KLF3 and SP1. Intriguingly, miR-23a and miR-27a further enhance the transcription of β-like globin genes through repression of KLF3 and SP1 binding to the β-like globin gene locus during erythroid differentiation. Moreover, KLF3 can bind to the promoter of the miR-23a∼27a∼24-2 cluster and suppress this microRNA cluster expression. Hence, a positive feedback loop comprised of KLF3 and miR-23a promotes the expression of β-like globin genes and the miR-23a∼27a∼24-2 cluster during erythropoiesis.

Histone deacetylase (HDAC) inhibitors have been used to promote neuronal survival and ameliorate neurological dysfunction in a host of neurodegenerative disease models. The precise molecular mechanisms whereby HDAC inhibitors prevent neuronal death are currently the focus of intensive research. Here we demonstrate that HDAC inhibition prevents DNA damage-induced neurodegeneration by modifying the acetylation pattern of the tumor suppressor p53, which decreases its DNA-binding and transcriptional activation of target genes. Specifically, we identify that acetylation at K382 and K381 prevents p53 from associating with the pro-apoptotic PUMA gene promoter, activating transcription, and inducing apoptosis in mouse primary cortical neurons. Paradoxically, acetylation of p53 at the same lysines in various cancer cell lines leads to the induction of PUMA expression and death. Together, our data provide a molecular understanding of the specific outcomes of HDAC inhibition and suggest that strategies aimed at enhancing p53 acetylation at K381 and K382 might be therapeutically viable for capturing the beneficial effects in the CNS, without compromising tumor suppression.

Aberrant epigenetic patterns are central in the pathogenesis of haematopoietic diseases such as myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). Vorinostat is a HDACi which has produced responses in these disorders. The purpose of this study was to address the functional effects of vorinostat in leukemic cell lines and primary AML and MDS myeloid cells and to dissect the genetic and molecular mechanisms by which it exerts its action.Functional assays showed vorinostat promoted cell cycle arrest, inhibited growth, and induced apoptosis and differentiation of K562, HL60 and THP-1 and of CD33(+) cells from AML and MDS patients. To explore the genetic mechanism for these effects, we quantified gene expression modulation by vorinostat in these cells. Vorinostat increased expression of genes down-regulated in MDS and/or AML (cFOS, COX2, IER3, p15, RAI3) and suppressed expression of genes over-expressed in these malignancies (AXL, c-MYC, Cyclin D1) and modulated cell cycle and apoptosis genes in a manner which would favor cell cycle arrest, differentiation, and apoptosis of neoplastic cells, consistent with the functional assays. Reporter assays showed transcriptional effect of vorinostat on some of these genes was mediated by proximal promoter elements in GC-rich regions. Vorinostat-modulated expression of some genes was potentiated by mithramycin A, a compound that interferes with SP1 binding to GC-rich DNA sequences, and siRNA-mediated SP1 reduction. ChIP assays revealed vorinostat inhibited DNA binding of SP1 to the proximal promoter regions of these genes. These results suggest vorinostat transcriptional action in some genes is regulated by proximal promoter GC-rich DNA sequences and by SP1.This study sheds light on the effects of vorinostat in AML and MDS and supports the implementation of clinical trials to explore the use of vorinostat in the treatment of these diseases.

Macrophages play a critical role in innate immunity, and the expression of early response genes orchestrate much of the initial response of the immune system. Macrophages undergo extensive transcriptional reprogramming in response to inflammatory stimuli such as Lipopolysaccharide (LPS).To identify gene transcription regulation patterns involved in early innate immune responses, we used two genome-wide approaches--gene expression profiling and chromatin immunoprecipitation-sequencing (ChIP-seq) analysis. We examined the effect of 2 hrs LPS stimulation on early gene expression and its relation to chromatin remodeling (H3 acetylation; H3Ac) and promoter binding of Sp1 and RNA polymerase II phosphorylated at serine 5 (S5P RNAPII), which is a marker for transcriptional initiation. Our results indicate novel and alternative gene regulatory mechanisms for certain proinflammatory genes. We identified two groups of up-regulated inflammatory genes with respect to chromatin modification and promoter features. One group, including highly up-regulated genes such as tumor necrosis factor (TNF), was characterized by H3Ac, high CpG content and lack of TATA boxes. The second group, containing inflammatory mediators (interleukins and CCL chemokines), was up-regulated upon LPS stimulation despite lacking H3Ac in their annotated promoters, which were low in CpG content but did contain TATA boxes. Genome-wide analysis showed that few H3Ac peaks were unique to either +/-LPS condition. However, within these, an unpacking/expansion of already existing H3Ac peaks was observed upon LPS stimulation. In contrast, a significant proportion of S5P RNAPII peaks (approx 40%) was unique to either condition. Furthermore, data indicated a large portion of previously unannotated TSSs, particularly in LPS-stimulated macrophages, where only 28% of unique S5P RNAPII peaks overlap annotated promoters. The regulation of the inflammatory response appears to occur in a very specific manner at the chromatin level for specific genes and this study highlights the level of fine-tuning that occurs in the immune response.

The AML1-ETO fusion protein, which is present in 10-15% of cases of acute myeloid leukemia, is known to repress myeloid differentiation genes through DNA binding and recruitment of chromatin-modifying proteins and transcription factors in target genes. ChIP-chip analysis of human hematopoietic stem/progenitor cells transduced with the AML1-ETO fusion gene enabled us to identify 1168 AML1-ETO target genes, 103 of which were co-occupied by histone deacetylase 1 (HDAC1) and had lost the hyperacetylation mark at histone H4, and 264 showed a K9 trimethylation at histone H3. Enrichment of genes involved in hematopoietic differentiation and in specific signaling pathways was observed in the presence of these epigenetic modifications associated with an 'inactive' chromatin status. Furthermore, AML1-ETO target genes had a significant correlation between the chromatin marks studied and transcriptional silencing. Interestingly, AML1 binding sites were absent on a large number of selected AML1-ETO promoters and an Sp1 binding site was found in over 50% of them. Reversible silencing induced by the fusion protein in the presence of AML1 and/or Sp1 transcription factor binding site was confirmed. Therefore, this study provides a global analysis of AML1-ETO functional chromatin modifications and identifies the important role of Sp1 in the DNA binding pattern of AML1-ETO, suggesting a role for Sp1-targeted therapy in this leukemia subtype.

VEGF plays a central role in angiogenesis in cancer. Non-small cell lung cancer (NSCLC) tumors have increased microvascular density, localized hypoxia, and high VEGF expression levels; however, there is a lack of understanding of how oncogenic and tumor microenvironment changes such as hypoxia lead to greater VEGF expression in lung and other cancers. We show that NSCLC cells secreted higher levels of VEGF than normal airway epithelial cells. Actinomycin D inhibited all NSCLC VEGF secretion, and VEGF minimal promoter-luciferase reporter constructs were constitutively active until the last 85 base pairs before the transcription start site containing three SP-1 transcription factor-binding sites; mutation of these VEGF promoter SP-1-binding sites eliminated VEGF promoter activity. Furthermore, dominant negative SP-1, mithramycin A, and SP-1 shRNA decreased VEGF promoter activity, whereas overexpression of SP-1 increased VEGF promoter activity. Chromatin immunoprecipitation assays demonstrated SP-1, p300, and PCA/F histone acetyltransferase binding and histone H4 hyperacetylation at the VEGF promoter in NSCLC cells. Cultured NSCLC cells expressed higher levels of SP-1 protein than normal airway epithelial cells, and double-fluorescence immunohistochemistry showed a strong correlation between SP-1 and VEGF in human NSCLC tumors. In addition, hypoxia-driven VEGF expression in NSCLC cells was SP-1-dependent, with hypoxia increasing SP-1 activity and binding to the VEGF promoter. These studies are the first to demonstrate that overexpression of SP-1 plays a central role in hypoxia-induced VEGF secretion.