Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes

Hagen, Gustav; Müller, Susanne; Beato, Miguel; Suske, Guntram

doi:10.1093/nar/20.21.5519

Cited by 547 publications

(476 citation statements)

References 40 publications

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“…This result is consistent with the observation that the recognition sequence for the members of the Sp family of transcription factors is highly variable, and it deviates¯exibly from the consensus Sp1 binding site (Kadonaga et al, 1986;Sa er et al, 1990). Unlike Sp1, Sp2 and Sp3 which are ubiquitously expressed in various tissues and cell types, Sp4 is known to be brain-speci®c (Kingsley and Winoto, 1991;Fischer et al, 1993;Hagen et al, 1992). Thus Sp4 may activate HGF expression in the central nervous system since the HGF gene is also expressed in this tissue (Jung et al, 1994).…”

Section: Discussionsupporting

confidence: 85%

Transcriptional regulation of the hepatocyte growth factor (HGF) gene by the Sp family of transcription factors

et al. 1997

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In this study, we have localized an enhancer element in the 5'-¯anking region of the HGF gene promoter and have identi®ed its functional transcriptional factors. Through transient transfection of NIH3T3 ®broblast cells and gel mobility shift assays, the functional binding site was localized to a short region (7318 to 7303 bp from the transcription start site) which has a CTCCC sequence. This motif is also conserved in the human HGF promoter and acts as a binding site for the Sp family of transcription factors. In the presence of NIH3T3 nuclear protein extracts, this enhancer region formed speci®c DNA-protein complexes which were totally abrogated by excess amounts of consensus Sp1 oligonucleotide. In gel mobility supershift assays, antiSp1 and anti-Sp3 antibodies speci®cally recognized these complexes as was evident by their slower migration. Sitespeci®c mutation of this binding motif resulted in total loss of Sp1 and Sp3 binding and a concomitant loss of enhancer function within the context of the HGF promoter. Furthermore, in transient cotransfection assays, overexpression of Sp1 and/or Sp3 stimulated HGF promoter activity independently and additively through binding to the Sp1 binding site in the HGF gene promoter region. DNaseI hypersensitive analysis of freshly isolated nuclei from NIH3T3 cells revealed that ®ve hypersensitive sites (HSS) are present within the 2 kb region immediately upstream of the HGF promoter. One of these sites was mapped to position 70.3 kb from the transcription start site. These data show that both Sp1 and Sp3 transcription factors upregulate HGF promoter activity by binding to the Sp1 binding site at position 7318 to 7303 bp in the HGF gene promoter.

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Section: Discussionsupporting

confidence: 85%

Transcriptional regulation of the hepatocyte growth factor (HGF) gene by the Sp family of transcription factors

et al. 1997

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“…We found that the human MIP gene sequence -253/+42 contains an active promoter in cultured lens cells but is inactive in non-lens cells (17). In the present study we have characterized a cis regulatory element required for MIP gene (18,19), is present in lens nuclear extracts and also interacts with this Sp1 binding site. Even though Sp1 is a ubiquitous factor, it regulates tissue-specific expression of a variety of genes (20)(21)(22)(23)(24)(25).…”

Section: Introductionmentioning

confidence: 61%

Overlapping Sp1 and AP2 binding sites in a promoter element of the lens-specific MIP gene

Ohtaka-Maruyama

Wang

et al. 1998

Nucleic Acids Research

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The MIP gene, the founder of the MIP family of channel proteins, is specifically expressed in fiber cells of the ocular lens and expression is regulated temporally and spatially during development. We previously found that a DNA fragment containing 253 bp of 5′-flanking sequence and 42 bp of exon 1 of the human MIP gene contains regulatory elements responsible for lensspecific expression of the MIP gene. In this report we have analyzed the function of overlapping Sp1 and AP2 binding sites present in the MIP promoter. Using DNase I footprinting analysis we found that purified Sp1 and AP2 transcription factors interact with several domains of the human MIP promoter sequence -253/+42. Furthermore, addition of purified Sp1 to Drosophila nuclear extracts activates in vitro transcription from the MIP promoter -253/+42. This promoter activity is competed by oligonucleotides containing domains footprinted with Sp1. Using promoter-reporter gene (CAT) constructs we found that the sequence -39/-70 contains a cis regulatory element essential for promoter activity in transient assays in lens cells. EMSA analysis showed that lens nuclear extracts contain factors that bind to the MIP 5′-flanking sequence containing overlapping Sp1 and AP2 binding domains at positions -37/-65. Supershift experiments with lens nuclear extracts indicated that Sp3 is also able to interact with this regulatory element, suggesting that Sp1 and Sp3 may be involved in regulation of transcription of the MIP gene in the lens.

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“…We then decided to perform supershift assays to specifically identify which TF are producing the different retarded bands. We primarily used specific antibodies against the SP1 and SP3 TF, which are known to be able to bind with similar specificity and affinity to GC-boxes [24]. The results obtained are shown in Fig.…”

Section: Identification Of Tf By Supershift Analysismentioning

confidence: 99%

Transcription of PRDM1, the master regulator for plasma cell differentiation, depends on an SP1/SP3/EGR‐1 GC‐box

et al. 2008

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The positive regulatory domain containing 1, encoded by the PRDM1 gene, is a transcriptional repressor considered as a master regulator that is required and sufficient for plasma cell differentiation. In the present study we have performed sequence analysis of the upstream region of the human PRDM1 gene to detect the minimal promoter region necessary for PRDM1 gene transcription. This region comprises the region upstream of the initiation site, as well as the first exon. Collectively, deletion and mutation analysis in conjunction with luciferase reporter assays, EMSA and supershift assays identified a phylogenetically conserved GC-box as an essential element for PRDM1 expression. This GC-box element matches to a binding site for multiple transcription factors such as SP1 and SP3 isoforms as well as early growth response 1. Chromatin immunoprecipitation assays confirmed the in vivo binding capability of these factors to the human PRDM1 promoter. These studies together characterize for the first time the basal activity of the human PRDM1 promoter, through which several factors, including SP1, SP3 and early growth response 1, modulate its expression through a conserved GC-box.Key words: B cell differentiation Á Human Á PRDM1 Á Transcription factors Supporting Information available online IntroductionPlasma cells (PC), i.e. terminally differentiated B lymphocytes in a post-mitotic state, are immunoglobulin (Ig)-secreting cells playing a central role in the antigen-specific immune response. The plasmacytic differentiation process is regulated by the "turn off" and "turn on" of many specific transcription factors (TF), which are now starting to be elucidated [1][2][3][4]. One of these, the human positive regulatory domain containing 1 with zinc-finger domain (PRDM1), is considered as a key factor that promotes the nonproliferative and differentiated stage of PC [4, 5]. The PRDM1 factor was initially identified as a 98-kDa DNA-binding protein that contains five zinc-finger domains and acts as a repressor of interferon-b gene expression [6]. Its murine homolog, initially called B lymphocyte-induced maturation protein-1 (Blimp-1), was isolated as a mouse gene that is induced during B lymphocyte maturation [7]. It is expressed at high levels only in late B cells and PC, and the ectopic overexpression of PRDM1 is sufficient to drive B lymphocytes to differentiate into PC [7]. Moreover, its expression is required for the formation of Ig-secreting cells [8] and for the maintenance of long-lived PC in bone marrow [9].MYC Here we have defined the minimal promoter for human PRDM1 transcription enclosing a GC-rich element necessary for basal transcription. Moreover, this GC-rich element contains an SP1/early growth response 1 (EGR-1) overlapping binding site to which the regulatory factors SP1, SP3 and EGR-1 can bind, which activate the PRDM1 transcription. Results and discussionStructure of the 5 0 -flanking region and initiation sites of the human PRDM1 gene To obtain insight into the regulation of the human PRDM1 gene and b...

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Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes

Cited by 547 publications

References 40 publications

Transcriptional regulation of the hepatocyte growth factor (HGF) gene by the Sp family of transcription factors

Transcriptional regulation of the hepatocyte growth factor (HGF) gene by the Sp family of transcription factors

Overlapping Sp1 and AP2 binding sites in a promoter element of the lens-specific MIP gene

Transcription of PRDM1, the master regulator for plasma cell differentiation, depends on an SP1/SP3/EGR‐1 GC‐box

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