Transcription Factor Sp1 Promotes the Expression of Porcine ROCK1 Gene.
ABSTRACT: Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) gene plays a crucial role in maintaining genomic stability, tumorigenesis and myogenesis. However, little is known about the regulatory elements governing the transcription of porcine ROCK1 gene. In the current study, the transcription start site (TSS) was identified by 5'-RACE, and was found to differ from the predicted one. The region in ROCK1 promoter which is critical for promoter activity was investigated via progressive deletions. Site-directed mutagenesis indicated that the region from -604 to -554 bp contains responsive elements for Sp1. Subsequent experiments showed that ROCK1 promoter activity is enhanced by Sp1 in a dose-dependent manner, whereas treatment with specific siRNA repressed ROCK1 promoter activity. Electrophoretic mobility shift assay (EMSA), DNA pull down and chromatin immunoprecipitation (ChIP) assays revealed Sp1 can bind to this region. qRT-PCR and Western blotting research followed by overexpression or inhibition of Sp1 indicate that Sp1 can affect endogenous ROCK1 expression at both mRNA and protein levels. Overexpression of Sp1 can promote the expression of myogenic differentiation 1(MyoD), myogenin (MyoG), myosin heavy chain (MyHC). Taken together, we conclude that Sp1 positively regulates ROCK1 transcription by directly binding to the ROCK1 promoter region (from -604 to -532 bp) and may affect the process of myogenesis.
Project description: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.
Project description:The functional 5' flanking region of the human 12-lipoxygenase in epidermoid carcinoma A431 cells was characterized. By a primer extension method, the transcription initiation sites were mapped at -47 adenosine, -48 guanosine and -55 guanosine upstream of the ATG translation start codon. Transient transfection with a series of 5' and 3' deletion constructs showed that the 5' flanking region spanning from -224 to -100 bp was important for the basal expression of 12-lipoxygenase gene. Gel mobility shift assays with antibodies of transcription factors showed that both Sp1 and Sp3 required highly GC-rich Sp1 sites within this region for binding. Disruption of two Sp1 recognition motifs residing at -158 to -150 bp and -123 to -114 bp by site-directed mutagenesis markedly reduced the basal 12-lipoxygenase promoter activity and abolished the retarded bands in a gel-shift assay, indicating that these two Sp1-binding sites were essential for gene expression. The same two Sp1-binding sites in this promoter region were also responsible for epidermal growth factor (EGF)-induced expression of 12-lipoxygenase gene. Moreover, EGF also induced the transcriptional activation of luciferase driven by SV40 early promoter, which contained rich Sp1-binding sites. Taken together, the results suggest that two specific Sp1 consensus sites are involved in the mediation of the basal promoter activity as well as EGF induction of the 12-lipoxygenase gene and that Sp1 and Sp3 transcription factors might have a role in their regulation.
Project description:Cytoplasmic long non-coding RNAs have been shown to act at many different levels to control post-transcriptional gene expression, although their role in translational control is poorly understood. Here, we show that lnc-31, a non-coding RNA required for myoblast proliferation, promotes ROCK1 protein synthesis by stabilizing its translational activator, YB-1. We find that lnc-31 binds to the Rock1 mRNA as well as to the YB-1 protein and that translational activation requires physical interaction between the two RNA species. These results suggest a localized effect of YB-1 stabilization on the Rock1 mRNA. ROCK1 upregulation by lnc-31, in proliferative conditions, correlates well with the differentiation-repressing activity of ROCK1. We also show that, upon induction of differentiation, the downregulation of lnc-31, in conjunction with miR-152 targeting of Rock1, establishes a regulatory loop that reinforces ROCK1 repression and promotes myogenesis.
Project description:Human trefoil factor 3 (hTFF3) is a small-molecule peptide with potential medicinal value. Its main pharmacological function is to alleviate gastrointestinal mucosal injuries caused by various factors and promote the repair of damaged mucosa. However, how its transcription is regulated is not yet known. The aim of this study was to clone the hTFF3 gene promoter region, identify the core promoter and any transcription factors that bind to the promoter, and begin to clarify the regulation of its expression. The 5' flanking sequence of the hTFF3 gene was cloned from human whole blood genomic DNA by PCR. Truncated promoter fragments with different were cloned and inserted into the pGL3-Basic vector to determine the position of the core hTFF3 promoter. Transcription element maintaining basic transcriptional activity was assessed by mutation techniques. Protein-DNA interactions were analyzed by chromatin immunoprecipitation (ChIP). RNA interference and gene over-expression were performed to assay the effect of transcription factor on the hTFF3 expression. The results showed that approximately 1,826 bp of the fragment upstream of hTFF3 was successfully amplified, and its core promoter region was determined to be from -300 bp to -280 bp through analysis of truncated mutants. Mutation analysis confirmed that the sequence required to maintain basic transcriptional activity was accurately positioned from -300 bp to -296 bp. Bioinformatic analysis indicated that this area contained a Sp1 binding site. Sp1 binding to the hTFF3 promoter was confirmed by ChIP experiments. Sp1 over-expression and interference experiments showed that Sp1 enhanced the transcriptional activity of the hTFF3 promoter and increased hTFF3 expression. This study demonstrated that Sp1 plays an important role in maintaining the transcription of hTFF3.
Project description:BACKGROUND: LRP5, a member of the low density lipoprotein receptor superfamily, regulates diverse developmental processes in embryogenesis and maintains physiological homeostasis in adult organisms. However, how the expression of human LRP5 gene is regulated remains unclear. RESULTS: In order to characterize the transcriptional regulation of human LRP5 gene, we cloned the 5' flanking region and evaluated its transcriptional activity in a luciferase reporter system. We demonstrated that both KLF15 and Sp1 binding sites between -72 bp and -53 bp contribute to the transcriptional activation of human LRP5 promoter. Chromatin immunoprecipitation assay demonstrated that the ubiquitous transcription factors KLF15 and Sp1 bind to this region. Using Drosophila SL2 cells, we showed that KLF15 and Sp1 trans-activated the LRP5 promoter in a manner dependent on the presence of Sp1-binding and KLF15-binding motifs. CONCLUSIONS: Both KLF15 and Sp1 binding sites contribute to the basal activity of human LRP5 promoter. This study provides the first insight into the mechanisms by which transcription of human LRP5 gene is regulated.
Project description:Arylamine N-acetyltransferase 1 (NAT1) plays an important role in the biotransformation of xenobiotics, and genetic variants have been implicated in susceptibility to cancer and birth defects. A specific and quantitative reverse transcription-polymerase chain reaction assay for transcription from the major NAT1 promoter detected high expression with limited variability in human tissues. A 213-base pair (bp) minimal promoter was identified by transfection of luciferase reporter constructs into MCF-7 and HepG2 cell lines. Alignment of the 213-bp region with paralogous and orthologous promoters revealed two conserved region segments, one of which overlaps a 16-bp perfect palindrome. Transfection of luciferase constructs with artificial mutations in the minimal promoter defined two sites important for promoter function. One of these sites included a close match to the Sp1 transcription factor binding consensus sequence. Electrophoretic mobility shift assays (EMSAs), followed by competitive and supershift analyses, confirmed the Sp1 binding. Mutation of the highly conserved portion of the 16-bp palindrome reduced promoter activity more than 3-fold, and an EMSA shift was detected with an oligonucleotide, 200L29, which spans this segment. The 200L29 EMSA shift could not be competed by consensus Sp1 or AP-2 oligonucleotides, and may represent binding of a transcription factor that is common to N-acetyltransferase genes in humans and other species.
Project description:Neat1 is widely expressed in many tissues and cells and exerts pro-proliferation effects on many cancer cells. However, little is known about the function of Neat1 in myogenesis. Here we characterized the roles of Neat1 in muscle cell formation and muscle regeneration. Gain- or loss-of-function studies in C2C12 cells demonstrated that Neat1 accelerates myoblast proliferation but suppresses myoblast differentiation and fusion. Further, knockdown of Neat1 in vivo increased the cross-sectional area of muscle fibers but impaired muscle regeneration. Mechanically, Neat1 physically interacted with Ezh2 mainly through the core binding region (1001-1540 bp) and recruited Ezh2 to target gene promoters. Neat1 promoted myoblast proliferation mainly by decreasing the expression of the cyclin-dependent kinase inhibitor P21 gene but inhibited myoblast differentiation by suppressing the transcription of myogenic marker genes, such as Myog, Myh4, and Tnni2. Altogether, we uncover a previously unknown function of Neat1 in muscle development and the molecular mechanism by which Neat1 regulates myogenesis.
Project description:DHX15 plays a role in leukaemogenesis and leukaemia relapse. However, the mechanism underlying the transcriptional regulation of DHX15 in ALL has not been elucidated. Our present study aimed to explore the functional promoter region of DHX15 and to investigate the transcription factors controlling the transcription of this gene. A luciferase assay performed with several truncated constructs identified a 501-bp region as the core promoter region of DHX15. Site-directed mutagenesis, electrophoretic mobility shift and chromatin immunoprecipitation assays showed that ETS1 and SP1 occupied the DHX15 promoter. Furthermore, knockdown of ETS1 and SP1 resulted in suppression of DHX15, whereas the overexpression of these genes led to up-regulation of DHX15. Interestingly, in samples obtained from patients with ALL at diagnosis, both ETS1 and SP1 correlated positively with DHX15 expression. Additionally, differences in methylation of the DHX15 core promoter region were not observed between the patients and controls. In conclusion, we identified the core promoter region of DHX15 and demonstrated that ETS1 and SP1 regulated DHX15 expression in ALL.
Project description:Postnatal growth and regeneration of skeletal muscle are carried out mainly by satellite cells, which, upon stimulation, begin to express myogenin (Myog), the critical determinant of myogenic differentiation. DNA methylation status has been associated with the expression of Myog, but the causative mechanism remains almost unknown. Here, we report that the level of CIBZ, a methyl-CpG-binding protein, decreases upon myogenic differentiation of satellite-derived C2C12 cells, and during skeletal muscle regeneration in mice. We present data showing that the loss of CIBZ promotes myogenic differentiation, whereas exogenous expression of CIBZ impairs it, in cultured cells. CIBZ binds to a Myog promoter-proximal region and inhibits Myog transcription in a methylation-dependent manner. These data suggest that the suppression of myogenic differentiation by CIBZ is dependent, at least in part, on the regulation of Myog. Our data show that the methylation status of this proximal Myog promoter inversely correlates with Myog transcription in cells and tissues, and during postnatal growth of skeletal muscle. Notably, induction of Myog transcription by CIBZ suppression is independent of the demethylation of CpG sites in the Myog promoter. These observations provide the first reported molecular mechanism illustrating how Myog transcription is coordinately regulated by a methyl-CpG-binding protein and the methylation status of the proximal Myog promoter.
Project description:The rat lactate dehydrogenase (LDH) A subunit gene promoter contains a putative AP-1 binding site at -295/-289 bp, two consensus Sp1 binding sites at -141/-136 bp and -103/-98 bp, and a single copy of a consensus cyclic AMP-responsive element (CRE) at -48 to -41 bp upstream of the transcription initiation site. Additionally, an as yet unidentified silencer element is located within the -1173/-830 bp 5'-flanking region. Transient transfection analyses of a -1173/+25 bp LDH A-chLoramphenicol acetyltransferase fusion gene has indicated a complete inability of the promoter fragment to direct basal or forskolin-induced transcription. Deletion of the -1173/-830 bp sequence restored basal and cyclic AMP (cAMP)-inducible activity. Point mutations in the Sp1 binding sites of a -830/+25 bp promoter fragment reduced basal but not the relative degree of cAMP-inducible activity. cAMP-regulated transcriptional activity was dependent upon an 8 bp CRE, -TGACGTCA-, located at the -48/-41 bp upstream region. Mutations in the CRE abolished cAMP-mediated induction and reduced basal activity by about 65%. The CRE binds a 47 kDa protein which has previously been identified as CRE binding protein (CREB)-327, an isoform of the activating transcription factor/CREB transcription factor gene family. Co-transfection of a vector that expresses the catalytic subunit of cAMP-dependent protein kinase stimulates LDH A subunit promoter activity suggesting that cAMP induces LDH A subunit gene expression through phosphorylative modification of CREB-327. This study emphasizes a fundamental role of several modules including Sp1 and CREB binding sites in regulating basal and cAMP-mediated transcriptional activity of the LDH A gene.