Binding of Phosphorylated Sp1 Protein to Tandem Sp1 Binding Sites Regulates α2 Integrin Gene Core Promoter Activity

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 678-689 ◽  
Author(s):  
Mary M. Zutter ◽  
Ellen E. Ryan ◽  
Audrey D. Painter
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Binding Sites ◽  
Promoter Activity ◽  
Core Promoter ◽  
K562 Cells ◽  
The Core ◽  
Nuclear Extracts ◽  
Integrin Gene ◽  
Α2β1 Integrin

The α2β1 integrin, a collagen/laminin receptor, is expressed by a variety of cell types, including epithelial cells, mesenchymal cells, and hematopoietic cells. To understand the molecular mechanisms that regulate expression of the α2β1 integrin in cells with megakaryocytic differentiation, we characterized the 5′ flanking region of the α2 integrin gene and identified three distinct regulatory regions, including a core promoter, a silencer, and megakaryocyte enhancers in the distal 5′ flank (Zutter et al, Blood 96:3006, 1995 and Zutter et al, J Biol Chem 269:463, 1994). We now focus on the core promoter of the α2 integrin gene located between bp −30 and −92 that is required for transcriptional activity of the α2 integrin gene. Sequence analysis identified two Sp1 consensus sites and a potential AP2 site. Gel retardation assays showed that nuclear proteins from uninduced K562 cells and K562 cells induced to become megakaryocytic bound specifically to the core promoter region (bp −30 to bp −92) producing two DNA-protein complexes. In addition, nuclear extracts from cells induced along the megakaryocyte lineage produced a selective increase in the slower migrating complex. Site-directed mutagenesis of the 5′, the 3′, or both Sp1 binding sites suggested that both Sp1 binding sites are required for full promoter activity and for DNA-protein complex formation. DNA footprinting also showed specific protection of the 5′ Sp1 site by nuclear extracts from uninduced K562 cells and protection of both the 5′ and the 3′ Sp1 sites by nuclear extracts from induced K562 cells. Sp1 protein-DNA complex formation was dependent on Sp1 phosphorylation. The faster migrating DNA-protein complex was enhanced by dephosphorylation; the slower migrating DNA-protein complex was diminished or lost.

scholarly journals Binding of Phosphorylated Sp1 Protein to Tandem Sp1 Binding Sites Regulates α2 Integrin Gene Core Promoter Activity

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 678-689 ◽  
Author(s):  
Mary M. Zutter ◽  
Ellen E. Ryan ◽  
Audrey D. Painter
Keyword(s):  
Complex Formation ◽  
Protein Complex ◽  
Binding Sites ◽  
Promoter Activity ◽  
Core Promoter ◽  
K562 Cells ◽  
The Core ◽  
Nuclear Extracts ◽  
Integrin Gene ◽  
Α2β1 Integrin

Abstract The α2β1 integrin, a collagen/laminin receptor, is expressed by a variety of cell types, including epithelial cells, mesenchymal cells, and hematopoietic cells. To understand the molecular mechanisms that regulate expression of the α2β1 integrin in cells with megakaryocytic differentiation, we characterized the 5′ flanking region of the α2 integrin gene and identified three distinct regulatory regions, including a core promoter, a silencer, and megakaryocyte enhancers in the distal 5′ flank (Zutter et al, Blood 96:3006, 1995 and Zutter et al, J Biol Chem 269:463, 1994). We now focus on the core promoter of the α2 integrin gene located between bp −30 and −92 that is required for transcriptional activity of the α2 integrin gene. Sequence analysis identified two Sp1 consensus sites and a potential AP2 site. Gel retardation assays showed that nuclear proteins from uninduced K562 cells and K562 cells induced to become megakaryocytic bound specifically to the core promoter region (bp −30 to bp −92) producing two DNA-protein complexes. In addition, nuclear extracts from cells induced along the megakaryocyte lineage produced a selective increase in the slower migrating complex. Site-directed mutagenesis of the 5′, the 3′, or both Sp1 binding sites suggested that both Sp1 binding sites are required for full promoter activity and for DNA-protein complex formation. DNA footprinting also showed specific protection of the 5′ Sp1 site by nuclear extracts from uninduced K562 cells and protection of both the 5′ and the 3′ Sp1 sites by nuclear extracts from induced K562 cells. Sp1 protein-DNA complex formation was dependent on Sp1 phosphorylation. The faster migrating DNA-protein complex was enhanced by dephosphorylation; the slower migrating DNA-protein complex was diminished or lost.

Genomic structure and promoter characterization of the human Sprouty4 gene, a novel regulator of lung morphogenesis

2004 ◽  
Vol 287 (1) ◽  
pp. L52-L59 ◽  
Author(s):  
Wei Ding ◽  
Saverio Bellusci ◽  
Wei Shi ◽  
David Warburton
Keyword(s):  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Core Promoter ◽  
Genomic Structure ◽  
Inducible Promoter ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Significant Similarity ◽  
The Core ◽  
Flanking Region

The expression of Sprouty4 ( Spry4), an intracellular FGF receptor antagonist, shows a temporally and spatially restricted pattern in embryonic lung and is induced by ERK signaling. To clarify the molecular mechanisms regulating Spry4 transcription, the genomic structure of the human Sprouty4 ( hSpry4) gene was first determined by using the GenomeWalker kit. The hSpry4 gene spans > 14 kb and is organized in three exons and two introns. Multiple transcription start sites were subsequently mapped by 5′-rapid amplification of cDNA ends. Analysis of up to 4 kb of sequence in the 5′-flanking region of the gene showed the presence of multiple potential transcription factor binding sites but no TATA or CAAT boxes. Transient transfection using luciferase reporter gene constructs with progressive deletions of the hSpry4 5′-flanking region revealed that the core promoter activity is located within the proximal 0.4-kb region, whereas the minimal ERK-inducible promoter activity is between −69 and −31. Homology analysis further showed that the core promoter region of the hSpry4 gene exhibits significant similarity to the 5′-flanking region of the mouse gene.

scholarly journals Characterization and Regulation of the Rat and Human Ghrelin Promoters

Endocrinology ◽  
2005 ◽  
Vol 146 (3) ◽  
pp. 1611-1625 ◽  
Author(s):  
Wei Wei ◽  
Guiyun Wang ◽  
Xiang Qi ◽  
Ella W. Englander ◽  
George H. Greeley
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Core Promoter ◽  
Gh3 Cells ◽  
Ags Cells ◽  
The Core ◽  
Intron 1 ◽  
Proximal Site ◽  
Exon 1

Ghrelin is a recently discovered stomach hormone and endogenous ligand for the GH secretagogue receptor. The aim of these studies is to elucidate molecular mechanisms underlying regulation of the ghrelin gene. Distal and proximal transcription initiation sites are present. A short transcript, a product of the proximal site, showed a more widespread distribution. Two sets of 5′-upstream segments of the rat and human ghrelin genes were cloned and sequenced. Rat promoter segments upstream of the distal site showed highest activity in kidney (COS-7) and stomach (AGS) cells, whereas human promoter segments upstream of the proximal site showed highest activity in AGS and pituitary (GH3) cells in transient transfection assays. For the human, the core promoter spanned −667 to −468 bp, including the noncoding exon 1 and a short 5′ sequence of intron 1. For the rat, the core promoter spanned −581 to −469 bp, and inclusion of exon 1 and a short 5′-sequence of intron 1 reduced activity by 67%. Mutation of initiator-like elements in the rat lowered activity by 20–50%, whereas in the human, all activity was abolished. Overexpression of upstream stimulatory factors increased ghrelin core promoter activity. Fasting increases stomach ghrelin expression, glucagon-a fasting-induced hormone, increased ghrelin expression in vivo in rats, and promoter activity by approximately 25–50%. Together, these findings indicate that structural differences between the rat and human ghrelin core promoters may account in part for the differences in their transcriptional regulation. Nonetheless, upstream stimulatory factor and glucagon exert similar effects on regulation of rat and human ghrelin promoters.

The human receptor tyrosine kinase Axl gene – promoter characterization and regulation of constitutive expression by Sp1, Sp3 and CpG methylation

2008 ◽  
Vol 28 (3) ◽  
pp. 161-176 ◽  
Author(s):  
Giridhar Mudduluru ◽  
Heike Allgayer
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Hela Cells ◽  
Binding Sites ◽  
Promoter Activity ◽  
Core Promoter ◽  
Constitutive Expression ◽  
Gene Promoter ◽  
Start Site ◽  
Small Interfering Rnas

Axl is a receptor tyrosine kinase which promotes anti-apoptosis, mitogenesis, invasion, angiogenesis and metastasis, and is highly expressed in cancers. However, the transcriptional regulation of this important gene has never been characterized. The present study was initiated to characterize the promoter, cis-acting elements and promoter methylation driving expression of Axl. The 2.4 kb sequence upstream of the translational start site, and sequential 5′-deletions were cloned and revealed a minimal GC-rich region (−556 to +7) to be sufficient for basal Axl promoter activity in Rko, HCT116 and HeLa cells. Within this minimal region, five Sp (specificity protein)-binding sites were identified. Two sites (Sp a and Sp b) proximal to the translation start site were indispensable for Axl promoter activity, whereas mutation of three additional upstream motifs (Sp c, Sp d and Sp e) was of additional relevance. Gel-shift assays and chromatin immunoprecipitation identified that Sp1 and Sp3 bound to all five motifs, and mutation of all motifs abolished binding. Mithramycin, which inhibits binding of Sp factors to GC-rich sites, dramatically reduced Axl promoter activity and Axl, Sp1 and Sp3 expression. In Drosophila Schneider SL2-cells, exogenous expression of Sp1/Sp3 increased Axl promoter activity. Use of Sp1/Sp3 siRNAs (small interfering RNAs) significantly reduced Axl promoter activity and protein levels in Rko and HeLa cells. Methylation-bisulfite sequencing detected methylated CpG sites within three Sp motifs (Sp a, Sp b and Sp c) and GC-rich flanking sequences, and demethylation by 5-aza-2′-deoxycytidine up-regulated Axl and Sp3 expression in low-Axl-expressing Colo206f/WiDr cells, but not in high-Axl-expressing Rko cells. The results of the present study suggest that Axl gene expression in cancer cells is (1) constitutively driven by Sp1/Sp3 bound to five core promoter motifs, and (2) restricted by methylation within/around Sp-binding sites. This might enhance the understanding and treatment of essential mechanisms associated with cancer and other diseases.

Multiple protein-binding sites in the 5'-flanking region regulate c-fos expression

1986 ◽  
Vol 6 (12) ◽  
pp. 4305-4316
Author(s):  
M Z Gilman ◽  
R N Wilson ◽  
R A Weinberg
Keyword(s):  
Binding Sites ◽  
Mammalian Cells ◽  
Promoter Activity ◽  
Protein Complexes ◽  
Protein Binding Sites ◽  
Nuclear Extracts ◽  
Multiple Protein ◽  
Flanking Region ◽  
Fos Expression

We tested sequences flanking the mouse c-fos gene for the ability to form specific DNA-protein complexes with factors present in crude nuclear extracts prepared from mammalian cells. Three such complexes were detected. One complex formed in a region necessary for the induction of c-fos expression by serum growth factors. Two additional complexes formed at sequences that contribute to basal c-fos promoter activity in vivo. These complexes represent three novel sequence-specific DNA-binding activities which appear to participate in the regulation of c-fos transcription.

scholarly journals Regulation of Pdha-2 expression is mediated by proximal promoter sequences and CpG methylation.

1997 ◽  
Vol 17 (2) ◽  
pp. 612-619 ◽  
Author(s):  
R C Iannello ◽  
J Young ◽  
S Sumarsono ◽  
M J Tymms ◽  
H H Dahl ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Promoter Activity ◽  
Core Promoter ◽  
Pyruvate Dehydrogenase Complex ◽  
Cpg Methylation ◽  
Proximal Promoter ◽  
Specific Gene ◽  
Specific Expression ◽  
Cpg Dinucleotides ◽  
The Core

Spermatogenesis is a complex process requiring the coordinate expression of a number of testis-specific genes. One of these, Pdha-2, codes for the murine spermatogenesis-specific isoform of the E1a subunit of the pyruvate dehydrogenase complex. To begin to delineate the mechanisms regulating its expression in vivo, we have generated transgenic mice lines carrying Pdha-2 promoter deletion constructs. Here we report that transgenic mice harboring a construct containing only 187 bp of promoter and upstream sequences (core promoter) is sufficient for directing the testis-specific expression of a chloramphenicol acetyltransferase (CAT) reporter gene. Like the endogenous Pdha-2, the CAT gene is expressed in testis in a stage-specific manner. Our studies also show a correlation between CpG methylation within the core promoter and its capacity to regulate transcription. In NIH 3T3 cell lines stably transfected with the Pdha-2 core promoter-CAT construct, high levels of CAT reporter expression are observed, whereas the endogenous Pdha-2 gene is repressed. In these cells, the CpG dinucleotides residing within the transfected promoter are hypomethylated whereas those residing in the endogenous promoter are methylated. Furthermore, promoter activity can be abated by the in vitro methylation of its CpG dinucleotides. DNase I footprint analysis indicates that at least one site for the methylation-mediated repression may occur through the ATF/cyclic AMP response element binding element located within the core promoter. Mutations within this element reduces activity to approximately 50% of the wild-type promoter activity. These results suggest that tissue-specific gene expression may be modulated by other mechanisms in addition to specific transcription factor availability and cooperativity. We propose that methylation may be a mechanism by which repression of the testis-specific Pdha-2 gene is established in somatic tissue.

Predicting Polymerase II Core Promoters by Cooperating Transcription Factor Binding Sites in Eukaryotic Genes

2004 ◽  
Vol 36 (4) ◽  
pp. 250-258 ◽  
Author(s):  
Xiao-Tu Ma ◽  
Min-Ping Qian ◽  
Hai-Xu Tang
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Core Promoter ◽  
Transcription Factor Binding Sites ◽  
Transcription Factor Binding ◽  
Upstream Gene ◽  
Core Promoters ◽  
Factor Binding ◽  
The Core ◽  
Eukaryotic Genes

Abstract Several discriminate functions for predicting core promoters that based on the potential cooperation between transcription factor binding sites (TFBSs) are discussed. It is demonstrated that the promoter predicting accuracy is improved when the cooperation among TFBSs is taken into consideration. The core promoter region of a newly discovered gene CKLFSF1 is predicted to locate more than 1.5 kb far away from the 5′ end of the transcript and in the last intron of its upstream gene, which is experimentally confirmed later. The core promoters of 3402 human RefSeq sequences, obtained by extending the mRNAs in human genome sequences, are predicted by our algorithm, and there are about 60% of the predicted core promoters locating within the ± 500 bp region relative to the annotated transcription start site.

scholarly journals Functional Analysis of the Rod Photoreceptor cGMP Phosphodiesterase α-Subunit Gene Promoter

2004 ◽  
Vol 279 (19) ◽  
pp. 19800-19807 ◽  
Author(s):  
Steven J. Pittler ◽  
Youwen Zhang ◽  
Shiming Chen ◽  
Alan J. Mears ◽  
Donald J. Zack ◽  
...  
Keyword(s):  
Binding Sites ◽  
Promoter Region ◽  
Promoter Activity ◽  
Core Promoter ◽  
Dnase I ◽  
Hek293 Cells ◽  
Upstream Sequence ◽  
Rod Photoreceptor ◽  
Α Subunit ◽  
Cgmp Phosphodiesterase

To understand the factors controlling expression of the cGMP phosphodiesterase type 6 (PDE6) genes, we have characterized the promoter of the human PDE6A gene that encodes the catalytic α-subunit.In vivoDNase I hypersensitivity assays revealed two sites immediately upstream of the PDE6A core promoter region. Transient transfection assay in Y79 cells of constructs containing varying lengths of the promoter region showed a decrease in promoter activity with increasing length. The most active segment contained a 177-bp upstream sequence including apparent Crx and Nrl transcription factor binding sites. Both Crx and Nrl transactivated the PDE6A promoter in HEK293 cells and showed a >100-fold increase when coexpressed. Coexpression of a dominant negative inhibitor of Nrl abolished Nrl transactivation but had no effect on Crx. DNase I footprinting assays identified three potential Crx binding sites within a 55-bp segment beginning 29 bp upstream of the transcription start point. Mutation of two of these sites reduced reporter gene activity by as much as 69%. Gel shifts showed that all three Crx sites required a TAAT sequence for efficient binding. Consistent with a requirement for Crx and Nrl inPde6apromoter activity,Pde6amRNA is reduced by 87% in the retina of Crx–/–mice and is undetectable in Nrl–/–mice at postnatal day 10. These results establish that both Nrl and Crx are required for full transcriptional activity of the PDE6A gene.

scholarly journals A conserved tissue-specific structure at a human T-cell receptor beta-chain core promoter.

1997 ◽  
Vol 17 (8) ◽  
pp. 4220-4229 ◽  
Author(s):  
J P Halle ◽  
P Haus-Seuffert ◽  
C Woltering ◽  
G Stelzer ◽  
M Meisterernst
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Binding Sites ◽  
Core Promoter ◽  
Cell Receptor ◽  
Specific Structure ◽  
Beta Chain ◽  
Tissue Specific ◽  
The Core ◽  
Human T Cell

The T-cell receptor (TCR) beta-chain promoters have been characterized as nonstructured basal promoters that carry a single conserved ubiquitous cyclic AMP-responsive element. Our investigation of the human TCR beta gene uncovers a surprisingly complex and tissue-specific structure at the TCR Vbeta 8.1 promoter. The core of the promoter (positions -42 to +11) is recognized by the lymphoid cell-specific transcription factors Ets-1, LEF1, and AML1 as well as by CREB/ATF-1, as is demonstrated in gel shift and footprinting experiments. With the exception of LEF1, these factors activate transcription in T cells. Binding sites at the core region show little conservation with consensus sites. Nonetheless, CREB, Ets-1, and AML1 bind and activate cooperatively and very efficiently through the nonconsensus binding sites at the core promoter region. Moderate ubiquitous activation is further induced by CREB/ATF and Sp1 factors through proximal upstream elements. The tissue-specific core promoter structure is apparently conserved in other T-cell-specifically expressed genes such as the CD4 gene. Our observations suggest that both the enhancer and the promoter have a complex tissue-specific structure whose functional interplay potentiates T-cell-specific transcription.

scholarly journals Regulation of alpha 2 integrin gene expression in cells with megakaryocytic features: a common theme of three necessary elements

Blood ◽  
1995 ◽  
Vol 86 (8) ◽  
pp. 3006-3014 ◽  
Author(s):  
MM Zutter ◽  
AA Painter ◽  
WD Staatz ◽  
YL Tsung
Keyword(s):  
Transcriptional Activation ◽  
Core Promoter ◽  
K562 Cells ◽  
Integrin Subunit ◽  
Common Theme ◽  
Platelet Factor ◽  
Megakaryocytic Differentiation ◽  
Beta 1 Integrin ◽  
Integrin Gene ◽  
In Cells

The alpha 2 beta 1 integrin mediates interactions between cells and the extracellular matrix molecules, collagen and/or laminin. The alpha 2 beta 1 integrin is expressed in a variety of cell types, but in cells of hematopoietic lineage, expression is restricted to megakaryocytes and platelets. Increased expression of the alpha 2 beta 1 integrin during megakaryocytic differentiation is a consequence of transcriptional activation of the alpha 2 gene. We have begun to characterize the role of the 52 flanking region of the alpha 2 integrin gene in regulating expression during megakaryocyte differentiation. A 5-kb fragment of the 52 region directs both cell type and differentiation-dependent expression of a reporter gene in the pluripotent hematopoietic K562 cells upon megakaryocytic differentiation and in the megakaryocytic cell line, Dami. Analysis of a series of 52 deletion mutants indicates that expression of the alpha 2 integrin gene in cells with megakaryocytic features requires a core promoter region, a silencer region, and megakaryocytic enhancers in the distal 52 end. The organization of these three distinct regulatory regions of the alpha 2 promoter/enhancer suggests a common theme for megakaryocytic gene regulation shared with other megakaryocyte-specific proteins, including alpha IIb integrin subunit and platelet factor 4.

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