Sp1 Binding Sites Research Articles

The HIV-Tat protein interacts with Sp3 transcription factor and inhibits its binding to a distal site of the sod2 promoter in human pulmonary artery endothelial cells

Redox imbalance results in damage to cellular macromolecules and interferes with signaling pathways, leading to an inflammatory cellular and tissue environment. As such, the cellular oxidative environment is tightly regulated by several redox-modulating pathways. Many viruses have evolved intricate mechanisms to manipulate these pathways for their benefit, including HIV-1, which requires a pro-oxidant cellular environment for optimal replication. One such virulence factor responsible for modulating the redox environment is the HIV Transactivator of transcription (Tat). Tat is of particular interest as it is actively secreted by infected cells and internalized by uninfected bystander cells where it can elicit pro-oxidant effects resulting in inflammation and damage. Previously, we demonstrated that Tat regulates basal expression of Superoxide Dismutase 2 (sod2) by altering the binding of the Sp-transcription factors at regions relatively near (approx. −210 nucleotides) upstream of the transcriptional start site. Now, using in silico analysis and a series of sod2 promoter reporter constructs, we have identified putative clusters of Sp-binding sites located further upstream of the proximal sod2 promoter, between nucleotides −3400 to −210, and tested their effect on basal transcription and for their sensitivity to HIV-1 Tat. In this report, we demonstrate that under basal conditions, maximal transcription requires a cluster of Sp-binding sites in the −584 nucleotide region, which is extremely sensitive to Tat. Using chromatin immunoprecipitation (ChIP) we demonstrate that Tat results in altered occupancy of Sp1 and Sp3 at this distal Tat-sensitive regulatory element and strongly stimulated endogenous expression of SOD2 in human pulmonary artery endothelial cells (HPAEC). We also report altered expression of Sp1 and Sp3 in Tat-expressing HPAEC as well as in the lungs of HIV-1 infected humanized mice. Lastly, Tat co-immunoprecipitated with endogenous Sp3 but not Sp1 and did not alter the acetylation state of Sp3. Thus, here, we have defined a novel and important cis-acting factor in HIV-1 Tat-mediated regulation of SOD2, demonstrated that modulation of Sp1 and Sp3 activity by Tat promotes SOD2 expression in primary human pulmonary artery endothelial cells and determined that pulmonary levels of Sp3 as well as SOD2 are increased in the lungs of a mouse model of HIV infection.

Stearoyl-CoA desaturase (scd1a) is epigenetically regulated by broodstock nutrition in gilthead sea bream (Sparus aurata)

ABSTRACTThe aim of this study was to generate new knowledge on fish epigenetics, assessing the effects of linolenic acid (ALA) conditioning of broodstock in the offspring of the marine fish Sparus aurata. Attention was focused on gene organization, methylation signatures and gene expression patterns of fatty acid desaturase 2 (fads2) and stearoyl-CoA desaturase 1a (scd1a). Blat searches in the genomic IATS-CSIC database (www.nutrigroup-iats.org/seabreamdb) highlighted a conserved exon-intron organization, a conserved PUFA response region, and CG islands at the promoter regions of each gene. The analysed CpG positions in the fads2 promoter were mostly hypomethylated and refractory to broodstock nutrition. The same response was achieved after conditioning of juvenile fish to low water oxygen concentrations, thus methylation susceptibility at individual CpG sites seems to be stringently regulated in fish of different origin and growth trajectories. Conversely, the scd1a promoter was responsive to broodstock nutrition and the offspring of parents fed the ALA-rich diet shared an increased DNA-methylation, mainly in CpG sites neighbouring SP1 and HNF4α binding sites. Cytosine methylation at these sites correlated inversely with the hepatic scd1a expression of the offspring. Co-expression analyses supported that the HNF4α-dependent regulation of scd1a is affected by DNA methylation. The phenotypic output is a regulated liver fat deposition through changes in scd1 expression, which would also allow the preservation of fatty acid unsaturation levels in fish fed reduced levels of n-3 LC-PUFA. Collectively, these findings reveal a reliable mechanism by which parent’s nutrition can shape scd1a gene expression in the fish offspring.

Sp1 Mediates the Constitutive Expression and Repression of the PDSS2 Gene in Lung Cancer Cells.

Prenyl diphosphate synthase subunit 2 (PDSS2) is the first key enzyme in the CoQ10 biosynthesis pathway, and contributes to various metabolic and nephritic diseases. It has been reported that PDSS2 is downregulated in several types of tumors and acts as a potential tumor suppressor gene to inhibit the proliferation and migration of cancer cells. However, the regulatory mechanism of PDSS2 expression remains elusive. In the present study, we first identified and characterized the PDSS2 promoter region. We established four different luciferase reporter constructs which mainly cover the 2 kb region upstream of the PDSS2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all four constructs have prominent promoter activity, and the core promoter of PDSS2 is mainly located within the 202 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the PDSS2 promoter contains binding sites for canonical transcription factors such as Sp1 and GATA-1. Overexpression of Sp1 significantly inhibited PDSS2 promoter activity, as well as its endogenous expression, at both mRNA and protein levels in lung cancer cells. Site-directed mutagenesis assay further confirmed that the Sp1 binding sites are essential for proximal prompter activity of PDSS2. Consistently, a selective Sp1 inhibitor, mithramycin A, treatment repressed the PDSS2 promoter activity, as well as its endogenous expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binds to the PDSS2 promoter in vivo. Of note, the expression of Sp1 and PDSS2 are negatively correlated, and higher Sp1 expression with low PDSS2 expression is significantly associated with poor prognosis in lung cancer. Taken together, our results strongly suggest the essential role of Sp1 in maintaining the basic constitutive expression of PDSS2, and the pathogenic implication of Sp1-mediated PDSS2 transcriptional repression in lung cancer cells.

CSIG-14. PROTEIN KINASE CK2 REGULATES THE EXPRESSION OF NERVE/GLIAL ANTIGEN (NG)2 IN GLIOBLASTOMA

Abstract Nerve/glial antigen (NG)2 enhances cell proliferation, migration as well as chemoresistance and is associated with a poor clinical outcome in glioblastoma. Since regulatory mechanisms of NG2 expression are still largely unknown, we herein investigated the impact of protein kinase CK2 inhibition on NG2 expression in human glioblastoma cells. CK2 was inhibited in NG2-positive human glioblastoma cell lines (A1207, U87) and primary human glioblastoma cells by pharmacological treatment (CX-4945, TBB) or siRNA. NG2 expression was analyzed by flow cytometry, Western Blot and qRT-PCR. Cytotoxicity and viability were assessed by WST-1, LDH and BrdU assays. Scratch, sprouting and BrdU assays as well as growth curves were performed to examine cell migration and proliferation. Truncated fragments of the human NG2 promotor were generated and their transcriptional activity was assessed using reporter gene assays. The effect of CK2 inhibition on tumor growth was investigated in xenografts within the flanks of NOD/SCID mice. Finally, effects of CK2 inhibition were analyzed in primary human glioblastoma cells. We found that inhibition of CK2 significantly reduces NG2 protein levels in A1207 (19%±6.2; Mean±SD), U87 (35%±11.3) and primary human glioblastoma cells (41%) when compared to controls. Further analyses revealed that this is due to a decreased NG2 mRNA level. Of note, we identified a 200 base pair fragment, including a binding site for the CK2-dependent transcription factor SP-1. Functional assays showed no cytotoxic effects of the decreased NG2 expression after CK2 inhibition, whereas cell migration and proliferation were markedly reduced. Moreover, we found that CX-4945 treatment decreases tumor growth, which is associated with a diminished NG2 expression (56%±13.0) when compared to controls. In conclusion, we identified CK2 as a novel regulator of NG2 gene expression in human glioblastoma cells. Hence, pharmacological inhibition of CK2 may represent a novel strategy in the therapy of NG2-positive glioblastoma.

8-OxoG in GC-rich Sp1 binding sites enhances gene transcription in adipose tissue of juvenile mice

The oxidation of guanine to 8-oxoguanine (8-oxoG) is the most common type of oxidative DNA lesion. There is a growing body of evidence indicating that 8-oxoG is not only pre-mutagenic, but also plays an essential role in modulating gene expression along with its cognate repair proteins. In this study, we investigated the relationship between 8-oxoG formed under intrinsic oxidative stress conditions and gene expression in adipose and lung tissues of juvenile mice. We observed that transcriptional activity and the number of active genes were significantly correlated with the distribution of 8-oxoG in gene promoter regions, as determined by reverse-phase liquid chromatography/mass spectrometry (RP-LC/MS), and 8-oxoG and RNA sequencing. Gene regulation by 8-oxoG was not associated with the degree of 8-oxoG formation. Instead, genes with GC-rich transcription factor binding sites in their promoters became more active with increasing 8-oxoG abundance as also demonstrated by specificity protein 1 (Sp1)- and estrogen response element (ERE)-luciferase assays in human embryonic kidney (HEK293T) cells. These results indicate that the occurrence of 8-oxoG in GC-rich Sp1 binding sites is important for gene regulation during adipose tissue development.

Characteristics of the BMP7 Promoter in Hu Sheep.

Simple SummaryBone morphogenetic protein 7 (BMP7) is one of the largest secretory signal conductive molecules and is in the TGF-β superfamily. It plays an important role in the growth and development of hair follicles. We cloned the proximal promoter of the BMP7 gene for bioinformatics analysis. Dual-luciferase reporter system and overexpression were used to analyze the key regions and transcription factor binding sites. There was high activity between −758 bp and −545 bp in the core region of the gene and a possible binding site for transcription factors SP1 and EGR1.The BMP7 gene is involved in the growth and development of hair follicles but its regulation mechanism is unclear. We studied the regulation mechanism of the BMP7 promoter by cloning the proximal promoter of BMP7 for bioinformatics analysis. A series of missing vectors was then constructed for dual-fluorescein activity detection based on the bioinformatics analysis results. We tested transcription-factor binding-site mutations and transcription factor over-expression to analyze the transcriptional regulation principle of the BMP7 promoter region. The upstream transcriptional regulatory region of the BMP7 gene proximal promoter was predicted by bioinformatics. There were −1216 bp to −1166 bp and −632 bp to −582 bp transcription initiation sites in the upstream transcriptional regulatory region of the BMP7 gene proximal promoter. The CpG islands’ distribution showed that there were many CpG islands at −549 bp to 1 bp. A dual-luciferase assay revealed high activity between −758 bp and −545 bp in the core region and a possible binding site for transcription factors SP1 and EGR1. The transcriptional activity of BMP7 was significantly decreased in the transcriptional regulatory region of the BMP7 after EGR1 and SP1 mutation. Transcription was significantly enhanced by over expression of the EGR1 transcription factor, which strongly suggests that EGR1 and SP1 play important roles in BMP7 regulation.

Loss of TIMP3 by promoter methylation of Sp1 binding site promotes oral cancer metastasis

The tissue inhibitor of metalloproteinase-3 (TIMP3) is the only member of the TIMP family that binds to the extracellular matrix and suppresses cancer cell growth, angiogenesis, migration, and invasion. However, whether the abnormal expression and promoter methylation of TIMP3 facilitates oral cancer metastasis remain unclear. In this study, the DNA methylation levels of TIMP3 CpG islands were assessed through pyrosequencing. Artificial modulation of TIMP3 was performed to explore the role of TIMP3 in tumor metastasis in vitro and in vivo. Our results showed that the suppression of TIMP3 transcription by DNA methylation involves the inhibition of the binding of the transcription factor Sp1 to the TIMP3 promoter as well as the upregulation of DNMT1 and DNMT3B. Functional analyses revealed that TIMP3 overexpression reduced migration and invasion abilities in oral cancer cells and inhibited lymph node metastasis in vivo. Moreover, TIMP3 regulated epithelial–mesenchymal transition by increasing the expression of the epithelial markers and reducing the expression of the mesenchymal markers. In conclusion, our findings suggested that the suppression of TIMP3 by DNA methylation contributes to oral cancer metastasis.

Sp1 is Involved in Vertebrate LC-PUFA Biosynthesis by Upregulating the Expression of Liver Desaturase and Elongase Genes.

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability for the biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all the catalytic enzymes including two fatty acyl desaturase 2 (Δ4 Fads2 and Δ6/Δ5 Fads2) and two elongases (Elovl4 and Elovl5) have been identified, providing a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in fish. Stimulatory protein 1 (Sp1) has been speculated to be a vital transcription factor in determining the promoter activity of Fads-like genes in fish, however its regulatory effects on gene expression and LC-PUFA biosynthesis have not been demonstrated. Bioinformatic analysis predicted potential Sp1 binding sites in the promoters of the rabbitfish Δ6/Δ5 fads2 and elovl5, but not in Δ4 fads2 promoter. Here we cloned full-length cDNA of the rabbitfish sp1 gene, which encoded a putative protein of 701 amino acids, and was expressed in all tissues studied with highest levels in gill and eyes. The dual luciferase reporter assay in HepG2 line cells demonstrated the importance of the Sp1 binding site for the promoter activities of both Δ6/Δ5 fads2 and elovl5. Moreover, the electrophoretic mobility shift assay confirmed the direct interaction of Sp1 with the two promoters. Insertion of the Sp1 binding site of Δ6/Δ5 fads2 promoter into the corresponding region of the Δ4 fads2 promoter significantly increased activity of the latter. In the Siganus canaliculatus hepatocyte line (SCHL) cells, mRNA levels of Δ6/Δ5 fads2 and elovl5 were positively correlated with the expression of sp1 when sp1 was overexpressed or knocked-down by RNAi or antagonist (mithramycin) treatment. Moreover, overexpression of sp1 also led to a higher conversion of 18:2n−6 to 18:3n−6, 18:2n−6 to 20:2n−6, and 18:3n−3 to 20:3n−3, which related to the functions of Δ6/Δ5 Fads2 and Elovl5, respectively. These results indicated that Sp1 is involved in the transcriptional regulation of LC-PUFA biosynthesis by directly targeting Δ6/Δ5 fads2 and elovl5 in rabbitfish, which is the first report of Sp1 involvement in the regulation of LC-PUFA biosynthesis in vertebrates.

Sp1-regulated expression of p11 contributes to motor neuron degeneration by membrane insertion of TASK1

Disruption in membrane excitability contributes to malfunction and differential vulnerability of specific neuronal subpopulations in a number of neurological diseases. The adaptor protein p11, and background potassium channel TASK1, have overlapping distributions in the CNS. Here, we report that the transcription factor Sp1 controls p11 expression, which impacts on excitability by hampering functional expression of TASK1. In the SOD1-G93A mouse model of ALS, Sp1-p11-TASK1 dysregulation contributes to increased excitability and vulnerability of motor neurons. Interference with either Sp1 or p11 is neuroprotective, delaying neuron loss and prolonging lifespan in this model. Nitrosative stress, a potential factor in human neurodegeneration, stimulated Sp1 expression and human p11 promoter activity, at least in part, through a Sp1-binding site. Disruption of Sp1 or p11 also has neuroprotective effects in a traumatic model of motor neuron degeneration. Together our work suggests the Sp1-p11-TASK1 pathway is a potential target for treatment of degeneration of motor neurons.

Consulting prostate cancer cohort data uncovers transcriptional control: Regulation of the MARCH6 gene

Cholesterol accumulation is a hallmark of prostate cancer (PCa) enabled by the upregulation of its synthesis, which presents a potential therapeutic target. This pathway is suppressed by the E3 ubiquitin ligase membrane-associated RING-CH-type finger 6 (MARCH6); however, little is known of MARCH6 regulation, particularly at the transcriptional level. Here, we consulted large transcriptomic PCa datasets to investigate transcription factors and DNA sequence elements that regulate the MARCH6 gene. Amongst 498 primary PCa tissues of The Cancer Genome Atlas, we identified a striking positive correlation between MARCH6 and androgen receptor (AR) gene expression (r = 0.81, p < 1 × 10−117) that held in other primary tumour datasets. Two putative androgen response elements were identified in the MARCH6 gene using motif prediction and mining of publicly accessible chromatin immunoprecipitation-sequencing data. However, MARCH6 expression was not androgen-responsive in luciferase reporter and qRT-PCR assays. Instead, we established that the MARCH6–AR correlation in primary PCa is due to common regulation by the transcription factor Sp1. We located a region 100 bp downstream of the MARCH6 transcriptional start site that contains three Sp1 binding sites and strongly upregulates promoter activity. The functionality of this region, and Sp1-mediated upregulation of MARCH6, was confirmed using pharmacological and genetic inhibition of Sp1. Moreover, modulation of Sp1 activity affected the stability of squalene monooxygenase, a cholesterol biosynthesis enzyme and MARCH6 substrate. We thus establish Sp1 as the first known regulator of the MARCH6 gene and demonstrate that interrogation of transcriptomic datasets can assist in the de novo inference of transcriptional regulation.

5-Aza-2-deoxycytidine Enhances the Sensitivity of 5-Fluorouracil by Demethylation of the Thymidine Phosphorylase Promoter

5-Aza-2-deoxycytidine (5-Aza-CdR) enhances the sensitivity to 5-fluorouracil (5-FU), but the molecular mechanism is not fully understood. The aim of this study was to investigate the molecular mechanism that enhances the sensitivity to 5-FU treated with 5-Aza-CdR via thymidine phosphorylase (TP). The sensitivity to drugs was determined on several cancer cell lines by the MTT assay. Protein and mRNA levels were examined by immunoblot and RT-PCR, respectively. Gene silencing, binding of Sp1 to DNA and methylation of DNA was performed by siRNA, ChIP assay and sodium bisulfate genomic sequencing, respectively. Sp1-binding sites in the TP promoter were methylated in epidermoid carcinoma. 5-Aza-CdR demethylated Sp1-binding sites and enhanced sensitivity to 5-FU. Demethylation of Sp1-binding sites by 5-Aza-CdR was a key factor enhancing 5-FU sensitivity, which may enable more effective treatments for cancer patients with the combination of 5-Aza-CdR and 5-FU.

Transcriptional regulation of human cyclic GMP-AMP synthase gene

Cyclic GMP-AMP synthase (cGAS, cGAMP synthase) plays crucial roles in autoimmune disease, anti-tumor response, anti-senescence and anti-inflammatory response. Many studies have focused on cGAS-mediated signaling pathway. However, transcriptional mechanisms of cGAS gene have remained largely unknown. Here, we cloned the cGAS promoter region and characterized the molecular mechanisms controlling the cGAS transcriptional activity. By a series of 5′ deletion and promoter constructions, we showed that the region (−414 to +76 relatives to the transcription start site) was sufficient for promoter activity. Mutation of Sp1 and CREB binding sites in this promoter region led to an apparent reduction of the cGAS promoter activity. Overexpression of Sp1 and CREB could obviously enhance promoter activity, whereas knocking-down of endogenous Sp1 and CREB markedly restrained the cGAS promoter activity. Sp1 and CREB binding to the cGAS promoter region in vivo was verified by Chromatin immunoprecipitation assay. These results pointed out that transcription factors Sp1 and CREB regulate the transcription of the cGAS gene.

Sp1 promotes cell migration and invasion in oral squamous cell carcinoma by upregulating Annexin A2 transcription

Oral squamous cell carcinoma (OSCC) is a common malignant tumor with high metastatic potential in head and neck. Revealing the mechanism of OSCC metastasis will benefit the prognosis and prevention of OSCC. Sp1 is a transcription factor involved in the progression of several tumors. Annexin A2 functions as an oncogene, and there are three putative Sp1 binding sites in the Annexin A2 promoter region. Therefore, we hypothesized that Sp1 could regulate OSCC metastasis by regulating Annexin A2 expression. Quantitative real-time PCR (qRT-PCR) and Western blot were used to evaluate Sp1 or Annexin A2 expression. Transwell assays were used to evaluate the migration and invasion capacity of OSCC cells. Luciferase assays and Chromatin immunoprecipitation assays were used to verify whether Sp1 regulate Annexin A2 at the transcriptional level. We found that the expression of Sp1 increased in OSCC tissues compared to paired adjacent normal tissues, and the overexpression of Sp1 was associated with tumor metastasis. Furthermore, Sp1 promoted cell migration and invasion through Annexin A2. In addition, we verified that Sp1 controls Annexin A2 expression at the transcriptional level and identified the binding sites involved. Our study suggests that Sp1/Annexin A2 expression could be a promising prognostic biomarker and therapeutic target for OSCC metastasis.

Sp1 contributes to overexpression of stanniocalcin 2 through regulation of promoter activity in colon adenocarcinoma.

BACKGROUNDAberrant expression of stanniocalcin 2 (STC2) is implicated in colon adenocarcinoma (COAD). A previous study identified that STC2 functions as a tumor promoter to drive development of some cancers, but the role of its overexpression in the development of COAD remains unclear.AIMTo evaluate the regulation mechanism of STC2 overexpression in COAD.METHODSThe expression of STC2 in COAD was assessed by TCGA COAD database and GEO (GSE50760). Methylation level of the STC2 promoter was evaluated with beta value in UALCAN platform, and the correlation between STC2 expression and survival rate was investigated with TCGA COAD. Transcription binding site prediction was conducted by TRANSFAC and LASAGNA, and a luciferase reporter system was used to identify STC2 promoter activity in several cell lines, including HEK293T, NCM460, HT29, SW480, and HCT116. Western blotting was performed to evaluate the role of Sp1 on the expression of STC2.RESULTSThe central finding of this work is that STC2 is overexpressed in COAD tissues and positively correlated with poor prognosis. Importantly, the binding site of the transcription factor Sp1 is widely located in the promoter region of STC2. A luciferase reporter system was successfully constructed to analyze the transcription activity of STC2, and knocking down the expression of Sp1 significantly inhibited the transcription activity of STC2. Furthermore, inhibition of Sp1 remarkably decreased protein levels of STC2.CONCLUSIONOur data provide evidence that the transcription factor Sp1 is essential for the overexpression of STC2 in COAD through activation of promoter activity. Taken together, our finding provides new insights into the mechanism of oncogenic function of COAD by STC2.

Robust hematopoietic specification requires the ubiquitous Sp1 and Sp3 transcription factors

BackgroundBoth tissue-specific and ubiquitously expressed transcription factors, such as Sp-family members, are required for correct development. However, the molecular details of how ubiquitous factors are involved in programming tissue-specific chromatin and thus participate in developmental processes are still unclear. We previously showed that embryonic stem cells lacking Sp1 DNA-binding activity (Sp1ΔDBD/ΔDBD cells) are able to differentiate into early blood progenitors despite the inability of Sp1 to bind chromatin without its DNA-binding domain. However, gene expression during differentiation becomes progressively deregulated, and terminal differentiation is severely compromised.ResultsHere, we studied the cooperation of Sp1 with its closest paralogue Sp3 in hematopoietic development and demonstrate that Sp1 and Sp3 binding sites largely overlap. The complete absence of either Sp1 or Sp3 or the presence of the Sp1 DNA-binding mutant has only a minor effect on the pattern of distal accessible chromatin sites and their transcription factor binding motif content, suggesting that these mutations do not affect tissue-specific chromatin programming. Sp3 cooperates with Sp1ΔDBD/ΔDBD to enable hematopoiesis, but is unable to do so in the complete absence of Sp1. Using single-cell gene expression analysis, we show that the lack of Sp1 DNA binding leads to a distortion of cell fate decision timing, indicating that stable chromatin binding of Sp1 is required to maintain robust differentiation trajectories.ConclusionsOur findings highlight the essential contribution of ubiquitous factors such as Sp1 to blood cell development. In contrast to tissue-specific transcription factors which are required to direct specific cell fates, loss of Sp1 leads to a widespread deregulation in timing and coordination of differentiation trajectories during hematopoietic specification.

DNA methylation status of TBX20 in patients with tetralogy of Fallot

BackgroundTBX20 plays an important role in heart development; however, its epigenetic regulation in the pathogenesis of tetralogy of Fallot (TOF) remains unclear.MethodsThe methylation levels of the TBX20 promoter region in the right ventricular myocardial tissues of TOF and control samples were measured by the Sequenom MassARRAY platform. Bisulphite-sequencing PCR (BSP) was used to confirm the TBX20 methylation of CpG sites in cells. Dual-luciferase reporter assays were performed to detect the influence of TBX20 methylation and Sp1 transcription factors on gene activity. An electrophoretic mobility shift assay (EMSA) was used to explore the binding of the Sp1 transcription factor to the TBX20 promoter.ResultsTOF cases had a significantly lower TBX20_M1 methylation level than controls (median methylation: 20.40% vs. 38.73%; p = 0.0047). The Sp1 transcription factor, which binds to Sp1 binding sites in the TBX20_M1 region and promotes TBX20 gene activity, was blocked by the methylation of Sp1 binding sites in normal controls. With decreasing methylation in the TOF cases, the Sp1 transcription factor can bind to its binding site within the TBX20 promoter M1 region and promote TBX20 gene expression.ConclusionsHypomethylation of the TBX20 promoter region was observed in the TOF cases, and the high expression of the TBX20 gene may be caused by activated Sp1 transcription factor binding because of the decreasing methylation at the Sp1 transcription factor binding sites within TBX20_M1.

Amlodipine induces vasodilation via Akt2/Sp1-activated miR-21 in smooth muscle cells.

The calcium antagonist amlodipine exerts important cardioprotective effects by modulating smooth muscle and endothelial functions. However, the mechanisms underlying these effects are incompletely understood. Western blotting was used to compare the expression of key genes involved in vascular smooth muscle cell (VSMC) phenotype conversion. Recombinant adeno-associated virus system was used to regulate miRNA expression in rats via tail vein. Bioinformatics was used to predict the transcriptional regulation of miR-21 upstream followed by biochemical validation using quantitative real-time polymerase chain reaction, ChIP-qPCR and EMSA assays. Only the calcium antagonist amlodipine, and no other type of anti-hypertensive drug, induced miR-21 overexpression in plasma and aortic vessels in the animal model. Real-time PCR and luciferase assays showed that amlodipine induced miR-21 overexpression in vascular smooth muscle cells. Western blot and immunofluorescence assays demonstrated that amlodipine activated Akt2, rather than Akt1, followed by activation of transcription factor Sp1, which regulated VSMC phenotype conversion via binding to the miR-21 promoter. Furthermore, bioinformatic analyses and luciferase assays demonstrated that amlodipine activated miR-21 transcription at the -2034/-2027 Sp1-binding site, which was further demonstrated by ChIP-qPCR and EMSA assays. Consistently, small-interfering RNA-mediated knockdown of Akt2 and Sp1 significantly attenuated the effects of amlodipine on miR-21 expression in smooth muscle cells. These results indicate that amlodipine induces smooth muscle cell differentiation via miR-21, which is regulated by p-Akt2 and Sp1 nuclear translocation, thereby providing a novel target for cardiovascular diseases.

SP1 regulates KLF4 via SP1 binding motif governed by DNA methylation during odontoblastic differentiation of human dental pulp cells.

DNA methylation is a critical epigenetic modulation in regulating gene expression in cell differentiation process, however, its detailed molecular mechanism during odontoblastic differentiation remains elusive. We aimed to study the global effect of DNA methylation on odontoblastic differentiation and how DNA methylation affects the transactivation of transcription factor (TF) on its target gene. DNA methyltransferase (DNMTs) inhibition assay and following odontoblastic differentiation assay were performed to evaluate the effect of DNA methylation inhibition on odontoblastic differentiation. Promoter DNA methylation microarray and motif enrichment assay were performed to predict the most DNA-methylation-affected TF motifs during odontoblastic differentiation. The enriched target sites and motifs were further analyzed by methylation-specific polymerase chain reaction (MS-PCR) and sequencing. The functional target sites were validated in vitro with Luciferase assay. The regulatory effect of DNA methylation on the enriched target sites in primary human dental pulp cells and motifs were confirmed by in vitro methylation assay. Inhibition of DNMTs in preodontoblast cells increased the expression level of Klf4 as well as marker genes of odontoblastic differentiation including Dmp1 and Dspp, and enhanced the efficiency of odontoblastic differentiation. SP1/KLF4 binding motifs were found to be highly enriched in the promoter regions and showed demethylation during odontoblastic differentiation. Mutation of SP1 binding site at -75 within KLF4's promoter region significantly decreased the luciferase activity. The in vitro methylation of KLF4's promoter decreased the transactivation of SP1 on KLF4. We confirmed that SP1 regulates KLF4 through binding site lying in a CpG island in KLF4's promoter region which demethylated during odontoblastic differentiation thus enhancing the efficiency of SP1's binding and transcriptional regulation on KLF4.

Effect of CREB1 promoter non-CpG island methylation on its differential expression profile on sheep ovaries associated with prolificacy

This study aimed to investigate the effect of different methylated regions of cyclic-AMP response element binding protein 1 (CREB1) by comparing the high prolificacy (HP) group and low prolificacy (LP) group, which was detected in our previous study. The expression level of CREB1 mRNA in the ovaries of the HP group was higher than in the LP group (P < 0.05). The differential methylated region (DMR) had 4 methylated CG dinucleotides(CGs): −1546, −1544, −1494 and −1464. The DNA methylation levels of -1546 CGs and −1464 CGs were significantly higher in the HP group than in the LP group (P < 0.05). The activity from −1296 to +26 (without DMR) was significantly higher than the activity from −1598 to +26 (with DMR) (P < 0.05). The result of 5-aza-2′-deoxycytidine treatment indicated that the inhibition DNA methylation of DMR reduced the transcription of CREB1. The bioinformatics predictive analysis were found that the −1546 CG site was located in the CCAAT/enhancer-binding protein alpha (CEBPA) binding site and the -1464 CG site was located in the Sp1 binding site. Finally, this study revealed the relationship between the methylation of non-CpG sites of the promoter and transcription of CREB1. This study will provide a theoretical basis of the Hu sheep ovaries associated with DNA methylation.

Three novel point mutations in the keratinocyte transglutaminase (TGK) gene in lamellar ichthyosis: significance for mutant transcript level, TGK immunodetection and activity.

We have investigated 8 patients from 7 unrelated families with lamellar ichthyosis (LI) for defects in the keratinocyte transglutaminase (TGK) gene. We have characterized three novel homozygous mutations and a previously reported splice acceptor site mutation. One patient showed a C-to-T change in the binding site for the transcription factor Sp1 within the promoter region. Another patient had a Gly 143-to-Glu mutation in exon 3 and a third patient, affected with a particular form of LI sparing the four limbs, demonstrated a Val382-to-Met mutation within exon 7. These three patients exhibited drastically reduced transglutaminase activity and an absence of detectable TGK polypeptide, as assessed by immunofluorescence and immunoblotting. Northern blot analysis showed that the Sp1 site mutation was associated with profound reduction of TGK transcript levels whereas normal transcript levels were observed for the two missense mutations. We hypothesize that the Sp1 site mutation impairs transcription of the TGK gene, whereas the two missense mutations induce structural changes leading to protein instability. Linkage to TGK was excluded in another family and no evidence for TGK defect was found in 3 other patients. These results further support the involvement of TGK in some patients with LI. They identify a TGK mutation as a cause for non-generalized LI and further delineate the molecular mechanisms underlying TGK deficiency in LI.