CpG methylation at the USF-binding site is important for the liver-specific transcription of the chipmunk HP-27 gene

The chipmunk hibernation-specific HP-27 gene is expressed specifically in the liver and has a CpG-poor promoter. To reveal how the liver-specific transcription of the HP-27 gene is regulated, we performed yeast one-hybrid screening of a chipmunk liver cDNA library. A 5′-flanking sequence of the HP-27 gene, extending from −170 to −140 and containing an E-box (5′-CACGTG-3′), is essential for the liver-specific transcription of HP-27. We used this sequence as bait and found that a ubiquitously expressed transcription factor, USF (upstream stimulatory factor), bound to the E-box. In COS-7 cells, USF activated transcription from the HP-27 gene promoter. We then used bisulphite genomic sequencing to analyse the methylation status of the four CpG dinucleotides that lie in the 5′-flanking sequence of the HP-27 gene up to −450, to investigate how the ubiquitously expressed USF activates transcription of the HP-27 gene only in the liver, while its transcription is repressed elsewhere. The only difference in methylation in the tissues tested was in the CpG dinucleotide in the USF-binding site, which was hypomethylated in the liver, but highly methylated in the kidney and heart. The specific methylation of the CpG dinucleotide at the USF-binding site impeded both the binding of USF and its transcriptional activation of the HP-27 gene. Chromatin immunoprecipitation using anti-USF antibodies revealed that USF bound to the HP-27 gene promoter in the liver, but not in the kidney or heart. Thus CpG methylation at the USF-binding site functions in establishing and maintaining tissue-specific transcription from the CpG-poor HP-27 gene promoter.

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Promoter elements in the transcriptional activation of the human stromelysin-1 gene by the inflammatory cytokine, interleukin 1

Biochemical Journal ◽

10.1042/bj3020471 ◽

1994 ◽

Vol 302 (2) ◽

pp. 471-477 ◽

Cited By ~ 42

Author(s):

S Quinones ◽

G Buttice ◽

M Kurkinen

Keyword(s):

Transcriptional Activation ◽

Inflammatory Cytokine ◽

Interleukin 1 ◽

Gene Promoter ◽

Flanking Sequence ◽

Tissue Remodelling ◽

Native Promoter ◽

Cell Extracts ◽

Extracellular Matrix Components ◽

The Absolute

Stromelysin-1, a tissue-remodelling metalloproteinase synthesized by fibroblasts, has proteolytic activity against a variety of extracellular matrix components. Stromelysin-1 gene transcription is induced by the inflammatory cytokine interleukin (IL)-1. In fibroblasts transiently transfected with constructs containing 5′-deletion mutants of the human stromelysin-1 gene promoter, IL-1-induced transcriptional activity was abolished with the removal of region -102 to -54. This region includes an AP-1 binding site at positions -70 to -64. The AP-1 site alone increased the basal activity of and conferred minimal IL-1 inducibility onto the heterologous gene promoter of thymidine kinase. Interestingly, although the removal of the AP-1 site from the native promoter (-1303 to +4) affected the absolute levels of IL-1-induced and basal promoter activity, it did not alter their ratio, indicating the involvement of regions outside the AP-1 site in the IL-1 response. Of the stromelysin-1 5′ flanking sequence examined, only the region -274 to -54 could confer IL-1 inducibility to a heterologous promoter independently of the AP-1 site. This region also bound specific nuclear factors. Further analysis revealed that the region composed of -86 to -71 and -63 to -54 could independently respond to IL-1 and bind protein of whole cell extracts. Protein binding to this region and to the AP-1 site was modestly induced by IL-1 treatment. From these results we conclude that, in fibroblasts, the AP-1 site (-70 to -64) is not necessary for the IL-1 response; however, it probably interacts through protein associations with the responsive region immediately surrounding it in the absolute transcriptional activation of the human stromelysin-1 gene by IL-1.

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Pyrosequencing Analysis of the Methylation Status of the CpG Island in the Retinoic Acid Receptor-β2 (RAR-β2) Gene Promoter.

Blood ◽

10.1182/blood.v104.11.4297.4297 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4297-4297

Author(s):

Da-Cheng Zhou ◽

David Reynolds ◽

Robert E. Gallagher

Keyword(s):

Retinoic Acid ◽

Cell Lines ◽

Cpg Island ◽

Retinoic Acid Receptor ◽

Methylation Status ◽

Cpg Islands ◽

Leukemia Cell ◽

Gene Promoter ◽

Cpg Dinucleotides ◽

Retinoic Acid Receptor Β2

Abstract CpG islands are associated with the 5′-ends of most housekeeping genes and many regulated genes. We have hypothesized that the methylation status of CpG islands in the promoter region of all-trans retinoic acid (ATRA) target genes such as retinoic acid receptor-β2 (RAR-β2) may be related to ATRA resistance and relapse of acute promyelocytic leukemia (APL). In the present study, we developed a highly quantitative method to assess the degree of DNA methylation at specific sites using PyrosequencingTM technology (Biotage, Uppsala, Sweden). This method is more quantitative than methylation-specific PCR, and is as accurate as but simpler and more robust than combined bisulfite restriction analysis (COBRA) or direct sequencing of plasmid clones of PCR products. We used this method to study 14 CpG dinucleotides in the CpG island of the RAR-β2 promoter. In reconstruction experiments in which 100% methylated and 100% unmethylated DNAs were admixed in different proportions (100:0; 80:20, 60:40, etc), a straightline graph was obtained over the entire range from 0 – 100% for each of the 14 CpG dinucleotides (r2 > 0.98). The results were highly reproducible and the variation between the results obtained from repetitive pyrosequencing of the same DNA was very low (S.D.<2%). Also the standard deviation between measurements of different PCR-amplified, bisulfite-converted DNAs prepared in separate experiments was <5%. We then used this method to measure the methylation level of the CpG island of the RAR-β2 promoter in several leukemia cell lines. Of 3 APL cell lines, the two with PML-RARα mutations, i.e., UF-1 and AP-1060, had higher overall methylation, compared to the NB4 cell line with non-mutant PML-RARα (mean ± SD = 52 ± 25% and 55 ± 21%, versus 43 ± 20%; p = 0.04 and 0.08, respectively; SD calculated from the variation across the 14 CpG dinucleotides for each source). Two myeloid leukemia cell lines with predominantly erythroid lineage characteristics, K562 and TF-1, had much lower levels of RAR-β2 methylation (2.6 ± 0.9% and 8.9 ± 3.2%, respectively). In the AP-1060 culture system, recently developed in our lab, there was little difference in methylation status between the patient bone marrow source and an intermediate, non-immortalized cell strain AP-1060S (27 ± 13% vs. 31 ± 25%). Further, there was no difference between lower and higher passage generations of AP-1060S (31 ± 25% vs. 30 ± 26%), which had markedly different replicative potential, indicating that replicative senescence at higher AP-1060 passages was not associated with altered methylation of the RAR-β2 gene promoter. However, the established, immortalized AP-1060 cell line had significantly greater methylation (52 ± 25%) than either the bone marrow source or AP-1060S (p <0.0001 and p = 0.0002, respectively), consistent with published reports of increased promoter methylation of cell lines. In conclusion, pyrosequencing is a high throughput method with great quantitative strength, and can be used for accurate and consistent analysis of methylation status in large numbers of samples.

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Forkhead Box A1 (FOXA1) and A2 (FOXA2) Oppositely Regulate Human Type 1 Iodothyronine Deiodinase Gene in Liver

Endocrinology ◽

10.1210/en.2011-1310 ◽

2012 ◽

Vol 153 (1) ◽

pp. 492-500 ◽

Cited By ~ 11

Author(s):

Naotetsu Kanamoto ◽

Tetsuya Tagami ◽

Yoriko Ueda-Sakane ◽

Masakatsu Sone ◽

Masako Miura ◽

...

Keyword(s):

Binding Site ◽

Promoter Activity ◽

Short Interfering Rna ◽

Upstream Stimulatory Factor ◽

Iodothyronine Deiodinase ◽

Forkhead Box ◽

E Box ◽

Stimulatory Factor ◽

Interfering Rna

Type 1 iodothyronine deiodinase (D1), a selenoenzyme that catalyzes the bioactivation of thyroid hormone, is expressed mainly in the liver. Its expression and activity are modulated by several factors, but the precise mechanism of its transcriptional regulation remains unclear. In the present study, we have analyzed the promoter of human D1 gene (hDIO1) to identify factors that prevalently increase D1 activity in the human liver. Deletion and mutation analyses demonstrated that a forkhead box (FOX)A binding site and an E-box site within the region between nucleotides −187 and −132 are important for hDIO1 promoter activity in the liver. EMSA demonstrated that FOXA1 and FOXA2 specifically bind to the FOXA binding site and that upstream stimulatory factor (USF) specifically binds to the E-box element. Overexpression of FOXA2 decreased hDIO1 promoter activity, and short interfering RNA-mediated knockdown of FOXA2 increased the expression of hDIO1 mRNA. In contrast, overexpression of USF1/2 increased hDIO1 promoter activity. Short interfering RNA-mediated knockdown of FOXA1 decreased the expression of hDIO1 mRNA, but knockdown of both FOXA1 and FOXA2 restored it. The response of the hDIO1 promoter to USF was greatly attenuated in the absence of FOXA1. Taken together, these results indicate that a balance of FOXA1 and FOXA2 expression modulates hDIO1 expression in the liver.

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A base-resolution panorama of the in vivo impact of cytosine methylation on transcription factor binding

10.1101/2021.08.27.457995 ◽

2021 ◽

Author(s):

Aldo Hernandez-Corchado ◽

Hamed S Najafabadi

Keyword(s):

Binding Site ◽

Cytosine Methylation ◽

Binding Activity ◽

Cpg Methylation ◽

In Vivo Models ◽

Cpg Dinucleotides ◽

Dna Accessibility ◽

Dna Binding Activity

Methylation of the cytosine base at CpG dinucleotides is traditionally considered antagonistic to the DNA-binding activity of the majority of transcription factors (TFs). Recent in vitro studies of TF-DNA interactions have revealed a more complex picture, suggesting a heterogeneous cytosine methylation impact that varies across TFs, with over a third of TFs preferring methylated sequences. Expanding these in vitro observations to in vivo TF binding preferences, however, is challenging, as the effect of methylation of individual CpG sites cannot be easily isolated from the confounding effects of DNA accessibility and regional DNA methylation. As a result, the in vivo methylation preferences of most TFs remain uncharacterized. Here, we introduce Joint Accessibility-Methylation-Sequence (JAMS) models for inferring the effect of CpG methylation on TF binding in vivo. JAMS creates quantitative models that connect the strength of the binding signal observed in ChIP-seq to the DNA accessibility of the binding site, regional methylation level, DNA sequence, and base-resolution cytosine methylation. Furthermore, by jointly modeling both the control and pull-down signal in a ChIP-seq experiment, JAMS isolates the TF-specific effects from background effects, revealing how methylation of specific CpGs within the binding site alters the TF binding affinity in vivo. We show that JAMS can quantitatively model the TF binding strength and learn the accessibility-methylation-sequence determinants of TF binding. JAMS models are reproducible and generalizable across cell lines, and can faithfully recapitulate cell type-specific TF binding. Systematic application of JAMS to 2368 ChIP-seq experiments generated high-confidence models for 260 TFs, revealing that 45% of TFs are inhibited by methylation of their potential binding sites in vivo. In contrast, only 6% prefer to bind to methylated sites, including 11 novel methyl-binding TFs. Comparison of these in vivo models to in vitro data confirmed high precision of the methyl-preferences inferred by JAMS. Finally, among the CpG-binding proteins from the ZF-KRAB family of TFs, we observed a disproportionately high preference for methylated sequences (24%), highlighting the role of CpG methylation in determining the genome-wide binding profiles of the TFs from this family.

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Regulation of the Human Secretin Gene Is Controlled by the Combined Effects of CpG Methylation, Sp1/Sp3 Ratio, and the E-Box Element

Molecular Endocrinology ◽

10.1210/me.2003-0461 ◽

2004 ◽

Vol 18 (7) ◽

pp. 1740-1755 ◽

Cited By ~ 37

Author(s):

Leo Tsz-On Lee ◽

Kian-Cheng Tan-Un ◽

Ronald Ting-Kai Pang ◽

David Tai-Wai Lam ◽

Billy Kwok-Chong Chow

Keyword(s):

Gene Expression ◽

Hepg2 Cells ◽

Cpg Island ◽

Methylation Status ◽

Gene Promoter ◽

Mobility Shift ◽

Specific Pcr ◽

E Box

Abstract To unravel the mechanisms that regulate the human secretin gene expression, in this study, we have used secretin-expressing (HuTu-80 cells, human duodenal adenocarcinoma) and non-secretin-expressing [PANC-1 (human pancreatic ductile carcinoma) and HepG2 (human hepatocellular carcinoma) cells] cell models for in vitro and in vivo analyses. By transient transfection assays, within the promoter region (−11 to −341 from ATG, relative to the ATG initiation codon), we have initially identified several functional motifs including an E-box and 2 GC-boxes. Results from gel mobility shift and chromatin immunoprecipitation assays confirmed further that NeuroD, E2A, Sp1, and Sp3 bind to these E- and GC-boxes in HuTu-80 cells in vitro and in vivo, whereas only high levels of Sp3 is observed to bind the promoter in HepG2 cells. In addition, overexpression of Sp3 resulted in a dose-dependent repression of the Sp1-mediated transactivation. Collectively, these data suggest that the Sp1/Sp3 ratio is instrumental to controlling secretin gene expression in secretin-producing and non-secretin-producing cells. The functions of GC-box and Sp proteins prompted us to investigate the possible involvement of DNA methylation in regulating this gene. Consistent with this idea, we found a putative CpG island (−336 to 262 from ATG) that overlaps with the human secretin gene promoter. By methylation-specific PCR, all the CpG dinucleo-tides (26 of them) within the CpG island in HuTu-80 cells are unmethylated, whereas all these sites are methylated in PANC-1 and HepG2 cells. The expressions of secretin in PANC-1 and HepG2 cells were subsequently found to be significantly activated by a demethylation agent, 5′-Aza-2′ deoxycytidine. Taken together, our data indicate that the human secretin gene is controlled by the in vivo Sp1/Sp3 ratio and the methylation status of the promoter.

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Sequential Action of Ets-1 and Sp1 in the Activation of the Human β-1,4-Galactosyltransferase V Gene Involved in Abnormal Glycosylation Characteristic of Cancer Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m611862200 ◽

2007 ◽

Vol 282 (38) ◽

pp. 27702-27712 ◽

Cited By ~ 28

Author(s):

Takeshi Sato ◽

Kiyoshi Furukawa

Keyword(s):

Gene Expression ◽

Binding Site ◽

Cancer Cells ◽

Transcriptional Activation ◽

Promoter Activity ◽

A549 Cells ◽

Gene Promoter ◽

Dominant Negative ◽

Mobility Shift ◽

V Gene

Malignant transformation is associated with increased gene expression of β-1,4-galactosyltransferase (β-1,4-GalT) V, which contributes to the biosynthesis of highly branched N-linked oligosaccharides characteristic of cancer cells. Our previous study showed that expression of the human β-1,4-GalT V gene is regulated by Sp1 (Sato, T., and Furukawa, K. (2004) J. Biol. Chem. 279, 39574–39583), and a subsequent study showed that the gene expression is also activated by Ets-1, a product of the oncogene (Sato, T., and Furukawa, K. (2005) Glycoconj. J. 22, 365). Herein we report the mechanism of β-1,4-GalT V gene activation by these transcription factors. The gene expression and promoter activity of β-1,4-GalT V increased when the ets-1 cDNA was transfected into A549 cells, which contain a small amount of Ets-1, but decreased dramatically when the dominant-negative ets-1 cDNA was transfected into HepG2 cells, which contain a large amount of Ets-1. Luciferase assays using deletion constructs of the β-1,4-GalT V gene promoter showed that promoter region –116 to +22 is critical for the transcriptional activation of the gene by Ets-1. Despite the presence of one Ets-1-binding site, which overlapped the Sp1-binding site, electrophoretic mobility shift assays showed that the region bound preferentially to Sp1 rather than to Ets-1. To solve this problem, we examined the transcriptional regulation of the human Sp1 gene by Ets-1 and found that the gene expression and promoter activity of Sp1 are regulated by Ets-1 in cancer cells. Functional analyses of two Ets-1-binding sites in the Sp1 gene promoter showed that only Ets-1-binding site –413 to –404 is involved in the activation of the gene by Ets-1. These results indicate that Ets-1 enhances expression of the β-1,4-GalT V gene through activation of the Sp1 gene in cancer cells.

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A ubiquitous factor (HF-1a) and a distinct muscle factor (HF-1b/MEF-2) form an E-box-independent pathway for cardiac muscle gene expression.

Molecular and Cellular Biology ◽

10.1128/mcb.12.4.1469 ◽

1992 ◽

Vol 12 (4) ◽

pp. 1469-1479 ◽

Cited By ~ 74

Author(s):

S Navankasattusas ◽

H Zhu ◽

A V Garcia ◽

S M Evans ◽

K R Chien

Keyword(s):

Cardiac Muscle ◽

Binding Site ◽

Transcriptional Activation ◽

Regulatory Element ◽

Point Mutations ◽

Muscle Cells ◽

Luciferase Reporter ◽

Transient Assays ◽

E Box ◽

Muscle Gene

Recent studies have identified a conserved 28-bp element (HF-1) within the rat cardiac MLC-2 gene which confers cardiac muscle-specific and inducible expression during myocardial cell hypertrophy. Utilizing a combination of independent experimental approaches, this study characterizes two cardiac nuclear factors which bind to HF-1, a ubiquitous factor (HF-1a), and an A + T-rich binding factor (HF-1b) which is preferentially expressed in differentiated cardiac and skeletal muscle cells. The HF-1a binding site is located in a core region of the 28-bp conserved element, immediately upstream from the A + T-rich HF-1b site, which is homologous to the MEF-2 site found in a number of muscle genes. By a number of separate criteria (gel mobility shift, competition, and mutagenesis studies), HF-1b and MEF-2 appear to be indistinguishable and thus are either identical or closely related muscle factors. Transient assays of luciferase reporter genes containing point mutations throughout the 28-bp HF-1 regulatory element document the importance of both the HF-1a and HF-1b sites in transient assays in ventricular muscle cells. In the native 250-bp MLC-2 promoter fragment, mutations in the single E box had little effect on cardiac muscle specificity, while point mutations in either the HF-1a or HF-1b binding site significantly reduced promoter activity, underscoring the importance of both the HF-1a and HF-1b sites in the transcriptional activation of this cardiac muscle gene. Thus, this study provides evidence that a novel, ubiquitous factor (HF-1a) and a muscle factor (HF-1b/MEF-2) can form a novel, E-box-independent pathway for muscle-specific expression in ventricular cardiac muscle cells.

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A ubiquitous factor (HF-1a) and a distinct muscle factor (HF-1b/MEF-2) form an E-box-independent pathway for cardiac muscle gene expression

Molecular and Cellular Biology ◽

10.1128/mcb.12.4.1469-1479.1992 ◽

1992 ◽

Vol 12 (4) ◽

pp. 1469-1479

Author(s):

S Navankasattusas ◽

H Zhu ◽

A V Garcia ◽

S M Evans ◽

K R Chien

Keyword(s):

Cardiac Muscle ◽

Binding Site ◽

Transcriptional Activation ◽

Regulatory Element ◽

Point Mutations ◽

Muscle Cells ◽

Luciferase Reporter ◽

Transient Assays ◽

E Box ◽

Muscle Gene

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Promotor Demethylation as a Mechanism of HOX11 Activation in Adult T-ALL?.

Blood ◽

10.1182/blood.v108.11.2288.2288 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2288-2288

Author(s):

Ulrike Baak ◽

Helmut Orawa ◽

Nicola Goekbuget ◽

Olaf Hopfer ◽

Thomas Burmeister ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Activation ◽

Cytosine Methylation ◽

Methylation Status ◽

Cpg Methylation ◽

Methylation Array ◽

Aberrant Expression ◽

Methylation Specific Pcr ◽

Specific Pcr ◽

Significant Difference

Abstract Promotor demethylation of oncogenes has been associated with transcriptional activation in cancer cells. The proto-oncogene HOX11/TLX1 has been found to be aberrantly expressed in up to 30% of adult T-ALL patients. In few, a translocation between the HOX11 locus at 10q24 and the T-cell receptor locus has been identified. In the majority of cases the mechanism leading to HOX11 reactivation remains unclear. It had been proposed that an epigenetical modification by demethylation of the proximal HOX11 promotor could be responsible for an aberrant expression of HOX11. To test this hypothesis we have correlated the methylation status of CpG residues in the proximal HOX11 promotor with the gene expression status of HOX11 in adult T-ALL samples from the German Multicenter ALL Study (GMALL) 5/93 and 6/99. HOX11 expression was measured in 286 pretreatment peripheral blood and bone marrow blasts by comparative real-time RT-PCR as described previously. The methylation status was then randomly analyzed after bisulphite treatment by methylation-specific PCR (MSP) in 53 T-ALL samples with HOX11 expression (HOX11 positive) and 102 samples without HOX11 expression (HOX11 negative). Of the 150 analyzed patients only 4% of HOX11 positive patients (n=53) and 10% of HOX11 negative patients were methylated (M) in the analyzed promotor area. HOX11 negative patients were significantly more common associated with an unmethylated status (U) then HOX11 positive patients (57% vs 32%, p=0.003). The most prominent methylation phenotype in HOX11 positive patients compared to HOX11 negative samples was a mixed (MU) methylation status (55% vs. 27%, p=0.001). Interestingly, remission duration was significantly higher in pt. with the MU methylation status (M=49%, U=54% vs. MU=76%, p-logrank=0.0362). This translated also into a significant difference in the overall survival (M=55%, U=49%, MU=75%, p-logrank= 0.0202). However, in a multivariate analysis the methylation status could not be confirmed as an independent prognostic factor. The promotor-associated CpG methylation status was found to be remarkably heterogenous in the analyzed adult T-ALL patients with a predominantly unmethylated status in HOX11 negative samples and a mixed methylated/unmethylated picture in HOX11 positive samples. These findings contrast with our initial hypothesis that HOX11 expression is silenced in normal tissue by a promotor-associated CpG methylation and aberrantly reexpressed in leukemia cells by demethylation. However, various CpG residues associated with the HOX11 promotor might be of variable importance for the gene expression and intrinsic limitations of methylation-specific PCR might have influenced our analysis. Bisulphite sequencing techniques as well as the newly developed genome-wide cytosine methylation array could help overcome these issues. Understanding the role of promotor-associated methylation in HOX11 expression could clarify pathways in leukemogenesis and provide a valuable tool for better risk stratification in T-ALL.

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Transcriptional activation of the minimal human Proα1(I) collagen promoter: obligatory requirement for Sp1

Biochemical Journal ◽

10.1042/bj3230225 ◽

1997 ◽

Vol 323 (1) ◽

pp. 225-231 ◽

Cited By ~ 10

Author(s):

Helen M. POPPLETON ◽

Rajendra RAGHOW

Keyword(s):

Binding Site ◽

Transcriptional Activation ◽

Gene Promoter ◽

Tata Box ◽

Collagen Gene ◽

Drosophila Line ◽

Bona Fide ◽

Necessary And Sufficient ◽

Collagen Promoter ◽

Double Stranded Oligonucleotide

A construct containing human Proα1(I) collagen gene promoter/enhancer-driven chloramphenicol acetyltransferase (CAT), pCOL-KT, failed to be expressed significantly in Sp1-deficient Schneider Drosophila line 2 (SL2) cells. However, CAT expression was induced 200-fold in SL2 cells co-transfected with pCOL-KT and pPACSp1, an Sp1-expression vector driven by the Drosophila actin 5C promoter. Elimination of the four potential Sp1-binding sites from pCOL-KT (pCOL-KTΔI), by removal of the first intron, did not abrogate Sp1-mediated induction of CAT. Even more significantly, a minimal Proα1(I) collagen promoter (-100 to +117 bp), containing a TATA box (-28 to -25 bp) and one putative Sp1-binding site (-87 to -82 bp), elicited strong Sp1-induced transactivation. Furthermore, mutation of the Sp1 motif in the minimal Proα1(I) collagen promoter-CAT construct abolished Sp1-induced expression of the reporter gene. Purified Sp1 protein bound specifically to DNA fragments of the Proα1(I) minimal promoter encompassing the putative Sp1-binding site; Sp1 binding could be competed out by a double-stranded oligonucleotide containing the wild-type Sp1 sequence, while an oligonucleotide containing a mutated Sp1 site failed to compete. Based on these results, we postulate that Sp1 plays an obligatory role in the transcriptional activation of the human Proα1(I) collagen gene. Additionally, we propose that a bona fide Sp1 motif, located most proximal to the TATA box, is necessary and sufficient for Sp1-mediated activation of the minimal Proα1(I) collagen promoter.

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