Binding Of Transcription Factor Sp1 Research Articles

Chromatin modifications induced by the AML1-ETO fusion protein reversibly silence its genomic targets through AML1 and Sp1 binding motifs

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.

Histone Deacetylase Inhibitors and Mithramycin A Impact a Similar Neuroprotective Pathway at a Crossroad between Cancer and Neurodegeneration

Mithramycin A (MTM) and histone deacetylase inhibitors (HDACi) are effective therapeutic agents for cancer and neurodegenerative diseases. MTM is a FDA approved aureolic acid-type antibiotic that binds to GC-rich DNA sequences and interferes with Sp1 transcription factor binding to its target sites (GC box). HDACi, on the other hand, modulate the activity of class I and II histone deacetylases. They mediate their protective function, in part, by regulating the acetylation status of histones or transcription factors, including Sp1, and in turn chromatin accessibility to the transcriptional machinery. Because these two classes of structurally and functionally diverse compounds mediate similar therapeutic functions, we investigated whether they act on redundant or synergistic pathways to protect neurons from oxidative death. Non-protective doses of each of the drugs do not synergize to create resistance to oxidative death suggesting that these distinct agents act via a similar pathway. Accordingly, we found that protection by MTM and HDACi is associated with diminished expression of the oncogene, Myc and enhanced expression of a tumor suppressor, p21waf1/cip1. We also find that neuroprotection by MTM or Myc knockdown is associated with downregulation of class I HDAC levels. Our results support a model in which the established antitumor drug MTM or canonical HDACi act via distinct mechanisms to converge on the downregulation of HDAC levels or activity respectively. These findings support the conclusion that an imbalance in histone acetylase and HDAC activity in favor of HDACs is key not only for oncogenic transformation, but also neurodegeneration.

Impact of the MDM2 promoter polymorphisms SNP285 and SNP309 on endometrial cancer risk among caucasians.

5092 Background: MDM2 is a key regulator of p53 and, thus, of cell cycle arrest and apoptosis. The MDM2 intronic promoter polymorphism SNP309T>G increases MDM2 expression through enhanced Sp1 transcription factor binding. Conflicting evidence has linked 309G to elevated risk and early diagnosis of different cancer forms. Recently we reported a second MDM2 promoter polymorphism SNP285G>C, located on the 309G allele (Knappskog et al, Cancer Cell 2011; doi:10.1016/j.ccr.2010.12.019). SNP285C/309G accounts for 12% of the 309G alleles in Western Europeans but is non-existent among Asians. It antagonizes the effect of SNP309G by reducing Sp1 binding and reduces the risk of breast and ovarian cancer. Here, we explored the effect of SNP309 and SNP285 on risk of endometrial cancer in a European population. Methods: We analysed SNP285 and SNP309 status in 915 Norwegian endometrial cancer patients and 2.465 healthy controls. Our primary hypothesis was to examine the effect of the SNP285C/309G haplotype on endometrial cancer risk among individuals carrying the SNP309G allele. Our secondary hypothesis was to explore the effect of SNP309G on endometrial cancer risk and explore the effect of excluding individuals harbouring the SNP285C/309G haplotype from this analysis. Results: The SNP285C/309G haplotype strongly reduces the risk of endometrial cancer among carriers of the SNP309TG genotype (OR: 0.62; CI 0.42-0.91) but not among SNP309GG homozygotes (OR: 1.47; CI 0.89-2.35). SNP309TG and GG genotype carriers revealed a moderately increased risk of endometrial cancer compared to SNP309TT homozygotes (TG: OR 1.17; CI 0.99-1.37; GG: OR 1.21; CI 0.95-1.58; TG + GG combined: OR: 1.18; CI 1.01-1.37). Removing individuals harbouring the SNP309G-counteracting SNP285C from the analysis strengthened this association (TG + GG: OR: 1.20; CI 1.03-1.41). Conclusions: MDM2 promoter haplotypes influence risk of endometrial cancer, mirroring their effect on ovarian cancer. The finding that the SNP285C/309G haplotype reduces the risk of endometrial cancer by 38% among individuals expressing SNP309TG heterozygosity (about 40% of the Caucasian population) reveals ethnic diversity with respect to endometrial cancer risk.

NFATc1 Regulation of TRAIL Expression in Human Intestinal Cells

TNF-related apoptosis-inducing ligand (TRAIL; Apo2) has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT) participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187 (Io) increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA), an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter.

A protein-based electrochemical method for label-free characterization of sequence-specific protein–DNA interactions

Interactions between DNA binding proteins and specific DNA elements are the fundamental basis of many biological pathways during gene expression and regulation. The sequence diversity of DNA elements leads to affinity variation, which could play important roles in regulatory and/or pathogenic processes. Therefore detection and analysis of such interactions, particularly the interactions between essential protein factors and their various DNA targets, are crucial for unveiling the molecular mechanisms behind these processes. For this purpose, a simple electrochemical method based on protein-modified electrode was developed. Transcription factor Sp1, an important and well-studied DNA binding protein, was used as a proof example and immobilized onto a glass carbon electrode (GCE) surface, with the wild type Sp1 consensus binding sequence (wtDNA) and a mutant sequence carrying two point mutations (mutDNA) within the Sp1 binding site served as testing samples. Binding of DNA samples to GCE-immobilized Sp1 was analyzed by electrochemical impedance spectroscopy (EIS), and the wtDNA showed a 3-fold higher change of the charge-transfer resistance ( R ct) value than that of the mutDNA, correlating well with the known high affinity of wtDNA to Sp1. The reversible disassociation and re-association of Sp1-DNA complexes were also achieved and monitored closely by EIS. Further detection of a series of concentrations of wtDNA using difference pulse voltammetry (DPV) showed that as low as 1 × 10 −10 mol/L wtDNA can be selectively detected in a DNA pool containing 0.05 g/L DNA fragments derived from salmon sperm. Exhibiting high affinity resolution, excellent selectivity, and low detection limit, this functionalized and reusable electrode provides a promising and convenient electrochemical approach for label-free, rapid monitoring, and comprehensive study of the specific interactions and/or affinity changes of DNA binding proteins with their various target DNAs under different conditions.

Histone deacetylases (HDACs) in XPC gene silencing and bladder cancer

Bladder cancer is one of the most common malignancies and causes hundreds of thousands of deaths worldwide each year. Bladder cancer is strongly associated with exposure to environmental carcinogens. It is believed that DNA damage generated by environmental carcinogens and their metabolites causes development of bladder cancer. Nucleotide excision repair (NER) is the major DNA repair pathway for repairing bulk DNA damage generated by most environmental carcinogens, and XPC is a DNA damage recognition protein required for initiation of the NER process. Recent studies demonstrate reduced levels of XPC protein in tumors for a majority of bladder cancer patients. In this work we investigated the role of histone deacetylases (HDACs) in XPC gene silencing and bladder cancer development. The results of our HDAC inhibition study revealed that the treatment of HTB4 and HTB9 bladder cancer cells with the HDAC inhibitor valproic acid (VPA) caused an increase in transcription of the XPC gene in these cells. The results of our chromatin immunoprecipitation (ChIP) studies indicated that the VPA treatment caused increased binding of both CREB1 and Sp1 transcription factors at the promoter region of the XPC gene for both HTB4 and HTB9 cells. The results of our immunohistochemistry (IHC) staining studies further revealed a strong correlation between the over-expression of HDAC4 and increased bladder cancer occurrence (p < 0.001) as well as a marginal significance of increasing incidence of HDAC4 positivity seen with an increase in severity of bladder cancer (p = 0.08). In addition, the results of our caspase 3 activation studies demonstrated that prior treatment with VPA increased the anticancer drug cisplatin-induced activation of caspase 3 in both HTB4 and HTB9 cells. All of these results suggest that the HDACs negatively regulate transcription of the XPC gene in bladder cancer cells and contribute to the severity of bladder tumors.

Hepatitis C virus core protein promotes the migration and invasion of hepatocyte via activating transcription of extracellular matrix metalloproteinase inducer

The chronic infection of hepatitis C virus (HCV) becomes a main factor evoking hepatocellular carcinoma, where the HCV core protein plays a central role in hepatocarcinogenesis. Whether the core protein directly contributes to metastasis of hepatocytes still remains to be reported in literature. Transwell chamber migration assay, Boyden chamber invasion assays and scanning electron microscopy observations were performed to determine the prometastatic ability of HCV core protein when expressed in human hepatocyte L02 cells. In addition, western blots, dual-luciferase assays, and chromatin immunoprecipitation assays were used to elucidate HCV core protein dependent pathways that promote metastasis in hepatocytes. Our investigation suggests that HCV core protein markedly enhances the capability of migration and invasion in L02 clones expressing HCV core proteins. The metastasis-promoting effect of the core protein is, in part, highly dependent on its effect on promoting the binding of transcription factor Sp1 to the extracellular matrix metalloproteinase inducer promoter. The effect of Sp1 binding resulted in an increase in extracellular matrix metalloproteinase inducer expression and progression of metastasis. Thus, we report that the expression of HCV core protein contributes to the metastasis of hepatocyte cells through activating transcription of extracellular metalloproteinase inducer.

MDM2promoter SNP285 and SNP309; phylogeny and impact on cancer risk

MDM2 plays a key role to physiological processes like growth arrest, senescence and apoptosis. It binds to and inhibits key proteins like p53 and the RB protein, and MDM2 amplification as well as protein overexpression without amplification is seen in many solid tumors. An MDM2 promoter polymorphism (SNP309T>G) has been found associated with enhanced Sp1 transcription factor binding and elevated MDM2 transcription. While 309G has been found associated with elevated cancer risk and young age at diagnosis of different cancers, results in Caucasians have been at variance. Recently, we reported a second polymorphism (SNP285G>C) located on the 309G allele. The 285C/309G haplotype accounts for about 12% of all 309G alleles among Norwegians, Dutch and British habitants. Assessing Sp1 binding to the MDM2 promoter using surface plasmon resonance technology, we found SNP309G to enhance Sp1 binding by 22% while SNP285C reduced Sp1 binding by 51%. SNP285C reduced the risk of breast cancer and ovarian cancer among 309TG/309GG carriers by 21 and 26%, respectively, but in particular the risk of ovarian cancer among 309TG heterozygotes (reduction by 37%). The fact that the 285C/309G haplotype accounted for only 1.9% of all 309G alleles among Finns and was absent in Chinese indicate 285C to be a young polymorphism.

Human ZNF312b oncogene is regulated by Sp1 binding to its promoter region through DNA demethylation and histone acetylation in gastric cancer

In a previous study, human ZNF312b was identified as a cell proliferation-associated oncogene via the K-ras/extracellular signal-regulated kinase cascade in gastric cancer. However, the mechanism concerning its transcriptional activation remains unknown. Here, we show that DNA methylation and histone acetylation of the ZNF312b promoter function as a switch for ZNF312b transcriptional activation in gastric cancer. The transcription level of ZNF312b was increased by treatment with a demethylating agent, 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor sodium butyrate, in several human cancer cell lines including gastric cancer. Consistent with these results, epigenetic analysis, such as pyrosequencing, bisulfate sequencing and methyl-specific polymerase chain reaction (MSP), showed that the expression level of ZNF312b is highly dependent on the degree of DNA methylation in gastric cancer cell lines. In addition, by ChIP assay using anti-acetyl/methyl H3K9 antibodies, histone acetylation was shown to mediate the expression of the ZNF312b gene. Interestingly, ChIP assay using the Sp1 antibody revealed that the binding of transcription factor Sp1 to the ZNF312b promoter for its transcriptional activation requires DNA demethylation and histone acetylation. Moreover, a knockdown of Sp1 resulted in a decrease in ERK-mediated proliferation via downregulation of the ZNF312b gene in gastric cancer cells. Taken together, these results suggest that the aberrant expression of ZNF312b promotes gastric tumorigenesis through epigenetic modification of its promoter region and provides a molecular mechanism for ZNF312b expression to contribute to the progression of gastric cancer.

The MDM2 Promoter SNP285C/309G Haplotype Diminishes Sp1 Transcription Factor Binding and Reduces Risk for Breast and Ovarian Cancer in Caucasians

MDM2 plays a key role in modulating p53 function. The MDM2 SNP309T > G promoter polymorphism enhances Sp1 binding and has been linked to cancer risk and young age at diagnosis although with conflicting evidence. We report a second MDM2 promoter polymorphism, SNP285G > C, residing on the SNP309G allele. SNP285C occurs in Caucasians only, where 7.7% (95% CI 7.6%-7.8%) of healthy individuals carry the SNP285C/309G haplotype. Invitro analyses reveals that SNP309G enhances but SNP285C strongly reduces Sp1 promoter binding. Comparing MDM2 promoter status among different cohorts of ovarian (n= 1993) and breast (n= 1973) cancer patients versus healthy controls (n= 3646), SNP285C reduced the risk of both ovarian (OR 0.74; CI 0.58-0.94) and breast cancer (OR 0.79; CI 0.62-1.00) among SNP309G carriers.

β-Ionone Enhances TRAIL-Induced Apoptosis in Hepatocellular Carcinoma Cells through Sp1-Dependent Upregulation of DR5 and Downregulation of NF-κB Activity

beta-Ionone (ION), an end-ring analogue of beta-carotenoid, has been known to inhibit tumor cell growth and induce apoptosis in various types of cancer cells. Nevertheless, its apoptosis-related molecular mechanisms remain unclear. Here, we first investigated the molecular mechanisms by which ION sensitizes cancer cells to the therapeutic potential of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, treatment with subtoxic concentrations of ION and TRAIL effectively inhibited cell viability in the hepatocellular carcinoma cell line Hep3B and other cancer cell lines such as colon carcinoma cell line HCT116 and leukemia cell line U937. Combined treatment with ION and TRAIL was also more effective in inducing DR5 expression, caspase activities, and apoptosis than treatment with either agent alone. ION-mediated sensitization to TRAIL was efficiently reduced by treatment with a chimeric blocking antibody or small interfering RNA specific for DR5. Electrophoretic mobility shift assay and a chromatin immunoprecipitation assay confirmed that ION treatment upregulates the binding of transcription factor Sp1 to its putative site within the DR5 promoter region, suggesting that Sp1 is an ION-responsive transcription factor. In addition, ION significantly increased hepatocellular carcinoma cell sensitivity to TRAIL by abrogating TRAIL-induced NF-kappaB activation and decreasing the expression of antiapoptotic proteins such as XIAP and IAP-1/2. Taken together, these data suggest that ION is a useful agent for TRAIL-based cancer treatments. Mol Cancer Ther; 9(4); 833-43. (c)2010 AACR.

Left ventricle transcriptome in aldosterone‐mediated cardiac injury

Primary hyperaldosteronism patients develop cardiac injury and dysfunction. In animal experimental models, excess aldosterone (ALDO) only causes cardiac injury in the presence of high salt (HS) intake. To study which molecular mechanisms are involved ALDO/HS‐mediated cardiac injury and its dependence on HS intake, we studied cardiac whole genome gene expression (GE). Male Sprague Dawley rats (n=6 per group) were uninephrectomized, and treated with ALDO (0.75 μg/h delivered by miniosmotic pumps), 0.9 % NaCl in the drinking water or a combination of both of them for 2 days, or 1, 2, 4 or 8 wks. Whole genome GE was performed using Illumina Beadarrays and GE was analyzed using GeneSpring software. ALDO/HS caused severe left ventricle GE changes after only 2 wks of treatment. Although either ALDO or HS alone changed GE (209 and 289 genes, respectively) the combination synergistically modified the expression of 1046 genes (493 UP and 553 DOWN). Bioinformatics analysis indicate that the Gene Ontology set of inflammatory response genes (n=17) and genes with the Sp1 transcription factor binding site in their promoter are significantly enriched. Similar GE results were observed after 4 and 8 wks of treatment. In summary, we report a left ventricle time series GE analysis in an animal experimental model of ALDO‐mediated cardiac injury highlighting many new genes involved in this pathological condition.

Histone Acetylation Up‐regulates Phosphatidylinositol‐3,4,5‐triphosphate‐dependent Rac Exchange Factor 1 (P‐Rex1) Expression to Promote Prostate Cancer Metastasis

We have reported that P‐Rex1 up‐regulation promotes metastasis, the major cause of prostate cancer death (Oncogene, 28: 1853–1863). The objective of this study was to combine biochemical, molecular and cellular techniques to define mechanisms underlying P‐Rex1 up‐regulation. Quantitative real‐time PCR and Western blot analysis indicated that histonedeacetylase (HDAC) inhibitors but not DNA methylation inhibitors increased P‐Rex1 expression in non‐metastatic prostate cancer cells to levels similar to that in metastatic prostate cancer PC‐3 cells. HDAC inhibitors enhanced P‐Rex1 promoter activity and a transcription factor Sp1 binding site in this promoter is responsible for the effect of HDAC inhibitors. Chromatin immunoprecipitation assays demonstrated that Sp1 association with the P‐Rex1 promoter is essential for P‐Rex1 expression. HDAC inhibitors caused dissociation of HDAC from Sp1, leading to histone acetylation and P‐Rex1 up‐regulation. Interestingly, HDAC1 over‐expression in PC‐3 cells caused histone deacetylation and P‐Rex1 down‐regulation, which inhibited metastatic activity in Transwell chamber assays. Our results demonstrate that histone acetylation stimulates P‐Rex1 expression in a Sp1‐dependent manner and suggest that targeting HDAC and Sp1 may provide a novel strategy for suppressing prostate cancer metastasis. Supported by NIH 1R011CA125661 and Department of Defense.

Polymorphisms in MMP-2 and MMP-9 promoter regions are associated with endometriosis

In this case-control study, we investigated the potential associations of MMP-2 and MMP-9 gene promoter region polymorphisms as well as MMP-2 promoter haplotypes with susceptibility to endometriosis in women of caucasian origin. The results demonstrated that polymorphisms in MMP-2 (-735 C/T) and MMP-9 (-1562 C/T) were associated with elevated risk of endometriosis and that certain MMP-2 promoter haplotypes were more common in control group.

Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts

IntroductionExtracellular matrix (ECM) turnover is controlled by the synthetic rate of matrix proteins, including type I collagen, and their enzymatic degradation by matrix metalloproteinases (MMPs). Fibrosis is characterized by an unbalanced accumulation of ECM leading to organ dysfunction as observed in systemic sclerosis. We previously reported that proteasome inhibition (PI) in vitro decreases type I collagen and enhances MMP-1 production by human fibroblasts, thus favoring an antifibrotic fibroblast phenotype. These effects were dominant over the pro-fibrotic phenotype induced by transforming growth factor (TGF)-β. Here we investigate the molecular events responsible for the anti-fibrotic phenotype induced in fibroblasts by the proteasome inhibitor bortezomib.MethodsThe steady-state mRNA levels of COL1A1, COL1A2, TIMP-1, MMP-1, and MMP-2 were assessed by quantitative PCR in human dermal fibroblasts cultured in the presence of TGF-β, bortezomib, or both. Transient fibroblast transfection was performed with wild-type and mutated COL1A1 and MMP-1 promoters. Chromatin immunoprecipitation, electrophoretic mobility shift assay (EMSA), and DNA pull-down assays were used to assess the binding of c-Jun, SP1, AP2, and Smad2 transcription factors. Immunoblotting and immunofluorescent microscopy were performed for identifying phosphorylated transcription factors and their cellular localization.ResultsBortezomib decreased the steady-state mRNA levels of COL1A1 and COL1A2, and abrogated SP1 binding to the promoter of COL1A2 in both untreated and TGF-β-activated fibroblasts. Reduced COL1A2 expression was not due to altered TGF-β-induced Smad2 phosphorylation, nuclear translocation, or binding to the COL1A2 promoter. In contrast to collagen, bortezomib specifically increased the steady-state mRNA levels of MMP-1 and enhanced the binding of c-Jun to the promoter of MMP-1. Furthermore, disruption of the proximal AP-1-binding site in the promoter of MMP-1 severely impaired MMP-1 transcription in response to bortezomib.ConclusionsBy altering the binding of at least two transcription factors, c-Jun and SP1, proteasome inhibition results in increased production of MMP-1 and decreased synthesis of type I collagen in human dermal fibroblasts. Thus, the antifibrotic phenotype observed in fibroblasts submitted to proteasome inhibition results from profound modifications in the binding of key transcription factors. This provides a novel rationale for assessing the potential of drugs targeting the proteasome for their anti-fibrotic properties.

Molecular Basis for A Thrombophilic Patient's Combined Deficiencies in Proteins C and S.

Abstract 4198 Background/objectivesProteins C and S (PC and PS) are vitamin K-dependent plasma proteins with anticoagulant properties. Protein S functions as a non-enzymatic cofactor for activated protein C (APC). APC proteolytically degrades coagulation factors Va and VIIIa, thereby diminishing the activities of the prothrombinase and tenase complexes, respectively. The human PC gene (PROC) is located on the long arm of chromosome 2 (2q13-q14) and contains 9 exons which code for 461 amino acid residues. The human PS (PROS1) gene resides on chromosome 3 (3p11.1-q11.2) and contains 15 exons coding for 636 amino acid residues. Hereditary PS and PC deficiencies are both autosomal dominant disorders in which patients have diminished functional levels of the respective protein (usually ∼50% relative to normal controls). Clinically, this results in increased propensity toward thromboembolic disease, also known as thrombophilia. In this study, we describe a unique thrombophilic patient who has combined deficiencies in both proteins C and S. The objective of the study was to elucidate the precise genetic defect(s) causing these deficiencies. MethodsFollowing purification of the patient's DNA from peripheral blood leukocytes, PCR amplifications were performed using oligonucleotide primers flanking all exons of the PROS1 and PROC genes. In addition, the 400bp region upstream of the first exon of PROS1 (corresponding to the promoter region) was also amplified by PCR. The PCR products were purified and their DNA sequences determined in both forward and reverse directions using the dye-terminator method. The resultant nucleotide sequences were compared with the PROS1 and PROC reference sequences [web address]. ResultsThe patient was found to be heterozygous for a novel missense PROC gene mutation in exon 8, which codes in part for the proteolytic domain of protein C. The resulting ValàGly substitution of residue 221 is in close proximity to the catalytic triad, which could abrogate its enzymatic activity. Although no mutations were present in any of the patient's PROS1 exons, there was a novel heterozygous CàG nucleotide substitution in the PROS1 promoter region. This substitution is present within an Sp1 transcription factor binding site that is highly conserved among mammals. Therefore, this mutation could significantly diminish expression of the otherwise normal PROS1 gene, leading to the observed decreased protein S level. Both mutations are unreported in the literature and are not listed in the PROC and PROS1 mutation databases. ConclusionsWe have successfully identified two novel genetic defects leading to deficiencies of the anticoagulant proteins C and S in a patient with thrombophilia. Further studies are underway to confirm the suggested biochemical effects of the mutation on protein C and protein S gene expression. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Rac1 GTPase downregulates vascular endothelial growth factor receptor‐2 expression by suppressing Sp1‐dependent DNA binding in human endothelial cells

RhoA, Rac1 and CDC42 are small GTP-binding proteins of the Rho family that play a crucial role in regulation of the actin-based cytoskeleton. In addition to cell growth regulation, they are implicated in transcriptional activation, oncogenic transformation and angiogenesis. The small Rho-GTPases have been linked to vascular endothelial growth factor (VEGF)-induced signalling pathways, but their role has not yet been elucidated. As signalling via the VEGF receptor-2 (VEGFR2) pathway is critical for angiogenic responses in cancer, wound repair and ischaemic and inflammatory diseases, we investigated whether the small Rho-GTPase Rac1 influences VEGFR2 expression in human endothelial cells. In this study, we show that a dominant negative Rac1 expression vector led to a pronounced decrease in VEGFR2 mRNA and protein expression. To identify minimal promoter requirements and potential applications of the small Rho-GTPases, we used VEGFR2 promoter-reporter gene constructs containing various deletions. The inhibitory effects of dominant negative Rac1 on the transcriptional activity of the VEGFR2 promoter localized to an element between -77 and -60 that contains an Sp1 transcription factor binding site. Electrophoretic mobility shift assays demonstrated that constitutive Sp1-dependent DNA binding decreased with Rac1 inhibition. Hence, repression of the small Rho GTPase Rac1 seems to be an additional critical molecular mechanism in the regulation of VEGFR2 expression.

Overexpression of Protease-Activated Receptor-1 Contributes to Melanoma Metastasis via Regulation of Connexin 43

Protease-activated receptor-1 (PAR-1) is a key player in melanoma metastasis with higher expression seen in metastatic melanoma cell lines and tissue specimens. cDNA microarray and Western blot analyses reveal that the gap junctional intracellular communication molecule connexin 43 (Cx-43), known to be involved in tumor cell diapedesis and attachment to endothelial cells, is significantly decreased after PAR-1 silencing in metastatic melanoma cell lines. Furthermore, Cx-43 promoter activity was significantly inhibited in PAR-1-silenced cells, suggesting that PAR-1 regulates Cx-43 at the transcriptional level. Chromatin immunoprecipitation studies showed a reduction in the binding of SP-1 and AP-1 transcription factors to the promoter of Cx-43. Both transcription factors have been shown previously to be required for maximal Cx-43 promoter activity. These results were corroborated by mutating the AP-1 and SP-1 binding sites resulting in decreased Cx-43 promoter activity in PAR-1-positive cells. Moreover, as Cx-43 has been shown to facilitate arrest of circulating tumor cells at the vascular endothelium, melanoma cell attachment to endothelial cells was significantly decreased in PAR-1-silenced cells, with this effect being abrogated after PAR-1 rescue. Herein, we report that up-regulation of PAR-1 expression, seen in melanoma progression, mediates high levels of Cx-43 expression. As both SP-1 and AP-1 transcription factors act as positive regulators of Cx-43, our data provide a novel mechanism for the regulation of Cx-43 expression by PAR-1. Indeed, Cx-43 expression was restored following PAR-1 rescue in PAR-1-silenced cells. Taken together, our data support the tumor promoting function of Cx-43 in melanoma.

133 O-linked N-Acetylglucosaminylation Represses HIV-1 Replication and Sp1-Mediated Trans-Activation of the HIV-1-LTR

The gene expression and replication of the human immunodeficiency virus-1 (HIV-1) is regulated by the promoter/enhancer located in the U3 region of the proviral 5'-long terminal repeat (LTR). Binding of the cellular transcription factor Sp1 to specific regulatory sites in the 5'-LTR is a key event in the replication cycle of HIV-1. O-linked N-acetylglucosaminylation (OGlcNAcylation) is a metabolism-associated highly dynamic and inducible posttranslational modification found on cytoplasmic and nuclear proteins in eukaryotes, catalyzed by O-GlcNAc transferase (OGT) and reversed by OGlcNAc hexosaminidase (O-GlcNAcase). Since the activity of Sp1 has been described to be regulated by O-GlcNAcylation, we evaluated whether increased O-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the OGlcNAcylation- enhancing agent glucosamine (GlcN) repressed viral replication in a dose dependent manner, and that this effect is due to a decreased activity of the LTR promoter. Overexpression of OGT specifically and dose dependently inhibited the activation of the LTR promoter containing the Sp1-binding sites and strongly augmented the O-GlcNAcylation of Sp1. Knockdown of Sp1 by short hairpin RNA blocked OGT repression of the HIV-1-LTR promoter, and depletion of OGT by RNA interference completely restored the promoter activity. In contrast to the LTR promoter, OGT had no inhibitory effect on the Sp1-dependent control promoter EF1-alpha. Finally, gel shift assays demonstrated that O-GlcNAcylation does not alter the DNA-binding affinity of Sp1. From this study, we conclude that OGlcNAcylation of Sp1 represses trans-activation of the HIV-1-LTR promoter. OGT does not act as a general repressor of Sp1-triggered gene expression, but selectively inhibits the Sp1-mediated HIV-1-LTR promoter activity. Thus, modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation, and may link viral replication to the metabolic state of the host cell.

Sodium butyrate upregulates expression of NKG2D ligand MICA/B in HeLa and HepG2 cell lines and increases their susceptibility to NK lysis.

Natural killer (NK) cells are important effectors in the immune response to tumors. A number of cell-surface inhibitory and activating receptors on NK cells tightly regulate their interaction with target cell ligands. In particular, the strength of an anti-tumor immune response appears to depend critically on surface levels of one activating receptor, NKG2D. Correspondingly, expression of NKG2D ligands on target cells is a requirement for effective tumor immunosurveillance and the elimination of pathogen-infected cells. Sodium butyrate, a potent repressor of histone deacetylase (HDAC), has recently been proposed as a potential agent in cancer treatment based on its ability to modify, in several cancer cell types, the expression of a variety of genes related to cell cycle regulation and apoptosis. Here we report that, in the HeLa and HepG2 tumor cell lines, sodium butyrate upregulated the expression of the MHC class I-related chain molecules A and B (MICA and MICB) at both the mRNA and protein levels, resulting in an enhanced susceptibility of cells in both lines to NK lysis. It also led to an elevated expression of heat shock protein 70 (HSP70) and transcription factor Sp1, and increased the binding of transcription factors Sp1 and heat shock transcription factor 1 (HSF1) to the MICA/B promoter, resulting in increased expression of MICA and MICB. siRNA targeting Sp1 significantly attenuate the enhancement of MICA expression by sodium butyrate. These results suggest that sodium butyrate and other HDAC inhibitors may have therapeutic potential by enhancing the immune response to cancer.