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PubMed1988

O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation.

Glycosylation is often regarded as being restricted to proteins confined to the cell surface or within the lumen of intracellular organelles. Here we show that the human RNA polymerase II transcription factor Sp1 bears multiple O-linked N-acetylglucosamine (GlcNAc) monosaccharide residues. The lectin wheat germ agglutinin specifically inhibits the transcriptional activation but not the DNA binding function of Sp1. Furthermore, many other RNA polymerase II transcription factors also bear terminal GlcNAc residues, whereas most nuclear proteins, including RNA polymerase I and III transcription factors tested, do not. In some cases, only a subset of the polypeptide species within a particular family of closely related RNA polymerase II factors appears to be glycosylated. Our findings raise the possibility that O-linked GlcNAc residues play a role in the mechanism or regulation of transcriptional activation of RNA polymerase II.

Animals↗

PubMed1994

Transcriptional induction of genes by IFN-beta in mouse cells is regulated by a transcription factor similar to human ISGF-3.

Previous studies of IFN-stimulated transcription factors in murine cells have identified a variety of trans-acting factors that bind to the IFN-stimulated response element (ISRE) whose role in gene expression remain unclear. The present investigation was undertaken to delineate the signal transduction pathway as well as to identify the transcription factors regulated by murine IFN-beta in L929 cells. Tyrosine kinase inhibitor, Genistein, abrogated gene induction and activation of transcription factors by IFN-beta. As early as 5 min after IFN-beta treatment, a transcription factor was activated in the cytoplasm which subsequently migrated into the nucleus. Anti-phosphotyrosine antibodies detected a specific transcription factor induced by mIFN-beta. Antibodies raised against human ISGF-3 subunit proteins p48, p84, p91 and p113 recognized this factor in the cytoplasm as well as in the nucleus of IFN-beta-treated L929 cells. An antibody raised against an oligopeptide of human p113 (residues 435-450) recognized the ISGF-3 complexes both in human and murine cells. However, a different antibody against the C-terminus of human p113 (residues 671-806) did not recognize the ISGF-3 like complex in mouse cells, indicating differences in the primary sequence of these proteins.

Animals↗

PubMed2005

A transcriptional regulatory element screening system reveals a novel E2F1/pRb transcription regulation pathway.

We developed a transcriptional regulatory element library which contains 160 independent known transcriptional regulatory elements linked to luciferase reporter vectors. That library proved valuable in the identification of p53 response elements and of E-box sequence preferences of several E-box binding proteins, and we used it to explore E2F1 target regulatory elements. Among those 160 elements, we found 3 E2F1 response elements, an E2F1 consensus sequence, an insulin response element which contained the E2F consensus sequence, and a basal level enhancer (BLE1) which had a nonconsensus E2F binding sequence. BLE1 functioned as multiple copy, with E2F1 in a dose-dependent manner, and had a sequence specificity for E2F1. Electrophoretic mobility shift assay revealed that BLE1 specifically interacts with E2F1 comparable to the E2F element. Interestingly, transactivation via five copies of BLE1 was not repressed but rather was stimulated by E2F1 in combination with the retinoblastoma tumor suppressor protein (pRb). The retinoblastoma control element (RCE) contains a direct repeated BLE1 in the c-fos gene promoter which also functioned like the multiple BLE1. Our data show that E2F1 has potential binding activity to the RCE and a different transcriptional regulation pathway which cooperates with pRb. Our transcriptional regulatory element screening system is useful for identifying novel transcriptional pathways.

Base Sequence↗

PubMed2005

Model of transcriptional regulation of the BRCA1-NBR2 bi-directional transcriptional unit.

In contrast to hundreds of mutations found in familial breast and/or ovarian cancers, somatic mutations of BRCA1 are very rare. However, a high percentage of sporadic breast and ovarian cancers show a reduction in BRCA1 expression, suggesting that defects in transcriptional regulation is a contributing factor. BRCA1 shares a promoter with its neighboring gene, NBR2, which is transcribed in the opposite direction. We have previously shown that the transcription of BRCA1 is negatively regulated by protein factors that interact with a 36-bp segment, located 575 bp into its first intron. We now report the localization of an 18-bp transcriptional repressor element for NBR2, which resides 948 bp into its first intron. The binding of nuclear proteins to this repressor element was detected by electrophoretic mobility shift assays (EMSAs), and it conferred an orientation-dependent functional suppression onto a heterologous thymidine kinase promoter. Combined with our previous studies, a model of transcriptional regulation of the closely aligned BRCA1-NBR2 bi-directional unit is proposed. A minimal 56-bp DNA region is functional in driving transcription in both directions, while uni-directional control is provided by distinct repressors that bind to sequences located in the first intron of the respective genes.

Base Sequence↗

PubMed2005

Transcriptional regulation of the human reduced folate carrier A1/A2 promoter: Identification of critical roles for the USF and GATA families of transcription factors.

The human reduced folate carrier (hRFC) gene has a complex regulation involving 6 alternatively spliced non-coding exons and promoters (A1/A2, A, B, C, D, and E). The hRFC-A1/A2 promoter is unique in that it transcribes a novel transcript with an in-frame AUG in non-coding exon A1/A2 that encodes a modified hRFC protein with altered transport function. In this report, we characterize the hRFC-A1/A2 promoter in HepG2 human hepatoma cells. By transfecting HepG2 cells with 5' and 3' deletion constructs, a transcriptionally important 270 bp region was identified. Gel shift assays identified transcription factor binding to three E-box elements and one GATA site within this region. These elements were verified by transfections of mutant constructs into HepG2 cells. Cotransfections in Drosophila Mel-2 cells confirmed promoter activation by USF1 and GATA1. A physical association between USF1 and GATA1 was demonstrated by their co-immunoprecipitation. By real time PCR analysis of transfected HepG2 cells, USF1 and GATA1 increased endogenous hRFC-A1/A2 transcripts. Altogether, our results demonstrate a transcriptionally important region in the hRFC-A1/A2 promoter including E-box and GATA elements, and a transactivation by USF1 and GATA1 proteins. Our results further establish the complexity of hRFC regulation, as a means of ensuring adequate folate cofactor transport for cell proliferation.

Animals↗

PubMed2006

Beta-catenin control of T-cell transcription factor 4 (Tcf4) importation from the cytoplasm to the nucleus contributes to Tcf4-mediated transcription in 293 cells.

Beta-catenin has essential roles in morphogenesis and human cancer, both as a subunit of adhesive complexes in the cell membrane and as a transcriptional coactivator in the Wnt signaling pathway. In addition, beta-catenin also has the ability to transport lymphoid enhancer binding factor-1 into the nucleus. In this study, we examined a constitutive active mutation, beta-catenin (T41A, S45A), for its potential as a nuclear import receptor for T-cell transcription factor 4 in 293 cells. Immunoblot analysis demonstrated that this constitutive active form of beta-catenin increased the amount of Tcf4 in the nucleus about 4-5-fold compared to controls. However, the overall expression of Tcf4 remained the same with or without over-expression of beta-catenin (T41A, S45A). T-cell transcription factor 4 reporter gene and electrophoretic mobility shift assay further indicated that the increase in Tcf4 in the nucleus was consistent with its accrued DNA binding capacity and transcription activity. Microscopic immunofluorescence examination showed that Tcf4 was mainly located in the cytoplasm and transported into the nucleus, without or with over-expression of beta-catenin (T41A, S45A), respectively. Our results suggest that beta-catenin might be a major factor regulating the import of Tcf4 from the cytoplasm into the nucleus, consequently controlling its transcription activity.

Active Transport, Cell Nucleus↗

PubMed2004

Transcriptional regulation of mouse alphaB- and gammaF-crystallin genes in lens: opposite promoter-specific interactions between Pax6 and large Maf transcription factors.

Mammalian alphaB-crystallin is highly expressed both in lens epithelium and lens fibers. In contrast, gammaF-crystallin is highly expressed in the lens fiber cells. Crystallin gene expression in lens is regulated at the level of transcription by a sparse number of specific DNA-binding transcription factors. Here, we report studies on transcriptional regulation of mouse alphaB- and gammaF-crystallin promoters by specific combinations of Pax6/Pax6(5a), large Mafs (MafA, MafB, c-Maf, and NRL), Sox1, Sox2, Six3, and RARbeta/RXRbeta. Two sets of these factors, co-expressed both in lens epithelium and in lens fibers, were tested in co-transfection assays using cultured lens and non-lens cells. Regulation of alphaB-crystallin was studied in the presence of lens epithelial-factors Pax6, MafB, and RARbeta/RXRbeta, and lens fiber-factors Pax6, MafA, c-Maf, and NRL. Pax6 proteins activated the alphaB-crystallin promoter (-162 to +45) with any combination of Mafs. Addition of RARbeta/RXRbeta further increased its promoter activity. Gel shift assays using lens nuclear extracts demonstrated interactions of Pax6, Maf, and retinoic acid nuclear receptor proteins with two lens-specific regions, the distal LSR1 (-147/-118) and proximal LSR2 (-78/-40), of the alphaB-crystallin promoter. In contrast, Pax6 proteins acted as repressors of gammaF-crystallin promoter activity elicited by a combination of large Mafs, Sox, and RARbeta/RXRbeta proteins in transiently transfected lens and non-lens cells. The results show that Pax6 conversely regulates these two lens crystallin promoters. We propose that the opposite roles of Pax6 in crystallin gene regulation are results of different promoter architectures of the alphaB- and gammaF-crystallin genes, developmentally regulated association of transcription factors with the corresponding cis-regulatory sites, and specific recruitment of transcriptional co-activators and co-repressors by Pax6.

Animals↗

PubMed2026

Acidic transcription factors position the genome at nuclear speckles through transcription-dependent and -independent mechanisms.

A small fraction of the genome reproducibly positions near nuclear speckles (NSs), increasing the expression and/or splicing efficiency of NS-associated genes. How specific genomic regions in mammalian cells are targeted to NSs remains unclear. Here, we demonstrate the establishment of genome-wide NS association without active transcription. We show that DNA sequences derived from NS-associated regions, when integrated as transgenes, are autonomously targeted to NSs. By systematically dissecting one such genomic locus, the COL1A1-SGCA locus, we identified redundant NS-targeting cis-regulatory elements, including an ∼600-bp fragment with 17 binding motifs for 8 transcription factors (TFs). Four NS-targeting TFs within this fragment contain acidic activation domains (AADs) that provide both chromatin-context and transcription-dependent NS targeting, properties that appear to be common among several other tested AADs. A subset of acidic activator TFs contains an additional, transcription-independent NS-targeting activity. Our findings establish diverse and partially redundant NS-targeting activities, which may facilitate dynamic gene positioning at the NS periphery for context-specific transcriptional responses.

Transcription, Genetic↗

PubMed2000

Site A of the MCP-1 distal regulatory region functions as a transcriptional modulator through the transcription factor NF1.

The monocyte chemoattractant protein-1 (MCP-1) functions to recruit monocytes and macrophages to areas of inflammation and is a prototypic chemokine subjected to coordinate regulation by immunomodulatory agents. TNF mediated regulation of MCP-1 occurs through a distal regulatory region located 2.5 kb upstream of the transcriptional start site. Within this region are two NF-kB motifs that are each critical for function. Site A, located within the distal regulatory region and upstream of the kappaB elements is required for maximal induction by TNF. However, unlike the kappaB elements and other MCP-1 regulatory elements, Site A is constitutively occupied by factors in vivo. To better understand the nature of Site A function, this report identified a Site A binding protein and provides a functional analysis of the element in driving transcription. The results showed that the transcription factor NF1/CTF binds to Site A both in vitro and in vivo. While Site A has no transcriptional activity on its own, it was found to augment the transcriptional activity of a GAL4-VP16 reporter system in an orientation and position independent manner. Because NF1 is known to interact with factors that modify nucleosomes, these results suggest a unique role for Site A in regulating MCP-1 expression.

3T3 Cells↗

PubMed2002

RE-1 silencing transcription factor-4 (REST4) is neither a transcriptional repressor nor a de-repressor.

The zinc finger protein RE-1 silencing transcription factor (REST) is a transcriptional repressor that represses neuronal genes in non-neuronal tissues. A neuronal splice form of REST, termed REST4, has been described in the rat. It encompasses the N-terminus of REST, including the N-terminal repressor domain and five of the eight zinc fingers of the DNA-binding domain. The biological function of REST4 is controversial. Transcriptional repression as well as transcriptional de-repression activity has been attributed to the REST4 protein of rat. Here, we have expressed a 'humanized' version of REST4 (hREST4) to facilitate a comparison of the biological functions of hREST4 and REST. The biological activity the human REST protein has been extensively studied in the past. Additionally, hREST4 has a high degree of homology with the REST4 protein of rat. An immunofluorescence analysis showed that hREST4 is expressed in the nucleus, indicating that the protein may have a potential impact on gene regulation. We analyzed the biological function of hREST4 in NS20Y neuroblastoma cells using human synapsin I promoter/reporter gene constructs. The human synapsin I gene is negatively regulated by REST. The results show that hREST4, in contrast to the full-length human REST protein, does not impair human synapsin I promoter activity. Moreover, co-transfection experiments with expression vectors encoding REST and hREST4 did not reveal any evidence that REST4 blocks the transcriptional repression activity of REST.

Amino Acid Sequence↗

PubMed2006

Combinatorial control of Arabidopsis proline dehydrogenase transcription by specific heterodimerisation of bZIP transcription factors.

Proline metabolism has been implicated in plant responses to abiotic stresses. The Arabidopsis thaliana proline dehydrogenase (ProDH) is catalysing the first step in proline degradation. Transcriptional activation of ProDH by hypo-osmolarity is mediated by an ACTCAT cis element, a typical binding site of basic leucine zipper (bZIP) transcription factors. In this study, we demonstrate by gain-of-function and loss-of-function approaches, as well as chromatin immunoprecipitation (ChIP), that ProDH is a direct target gene of the group-S bZIP factor AtbZIP53. Dimerisation studies making use of yeast and Arabidopsis protoplast-based two-hybrid systems, as well as bimolecular fluorescence complementation (BiFC) reveal that AtbZIP53 does not preferentially form dimers with group-S bZIPs but strongly interacts with members of group-C. In particular, a synergistic interplay of AtbZIP53 and group-C AtbZIP10 was demonstrated by colocalisation studies, strong enhancement of ACTCAT-mediated transcription as well as complementation studies in atbzip53 atbzip10 T-DNA insertion lines. Heterodimer mediated activation of transcription has been found to operate independent of the DNA-binding properties and is described as a crucial mechanism to modulate transcription factor activity and function.

Arabidopsis↗

PubMed2006

Interactions between hairy/enhancer of split-related proteins and the pancreatic transcription factor Ptf1-p48 modulate function of the PTF1 transcriptional complex.

In the developing pancreas, the onset of exocrine differentiation is driven by the activity of the PTF1 (pancreas transcription factor 1) transcriptional complex, which is comprised of the class II bHLH (basic helix-loop-helix) protein, Ptf1-p48 [also known as Ptf1a (pancreas specific transcription factor 1a)], and a class I E-box binding partner. Activity of the PTF1 complex is normally inhibited by the Notch signalling pathway, a process mediated by Notch effector proteins in the HES (Hairy/Enhancer of Split) family of bHLH transcriptional repressors. In the present study, we show that this inhibitory effect occurs through direct interaction between HES family members and Ptf1-p48. The HES family members Hey1 (hairy/enhancer-of-split related with YRPW motif 1) and Hey2 co-immunoprecipitate with Ptf1-p48, and Ptf1-p48 binding by Hes1 is also evident in yeast two-hybrid and GST (glutathione S-transferase) pull-down assays. The ability of Hes1 to interact with Ptf1-p48 resides within a fragment comprised of the bHLH, Orange and C-terminal domains, and does not require the N-terminal or WRPW elements. The ability of truncated versions of Hes1 to bind Ptf1-p48 correlates with their ability to down-regulate the activity of the PTF1 transcriptional complex, defining Ptf1-p48 binding as the most likely mechanism by which Notch effector proteins delay exocrine pancreatic differentiation.

Animals↗

PubMed1998

Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes.

Mechanisms controlling transcription and its regulation are fundamental to our understanding of molecular biology and, ultimately, cellular biology. Our knowledge of transcription initiation and integral factors such as RNA polymerase is considerable, and more recently our understanding of the involvement of enhancers and complexes such as holoenzyme and mediator has increased dramatically. However, an understanding of transcriptional repression is also essential for a complete understanding of promoter structure and the regulation of gene expression. Transcriptional repression in eukaryotes is achieved through 'silencers', of which there are two types, namely 'silencer elements' and 'negative regulatory elements' (NREs). Silencer elements are classical, position-independent elements that direct an active repression mechanism, and NREs are position-dependent elements that direct a passive repression mechanism. In addition, 'repressors' are DNA-binding trasncription factors that interact directly with silencers. A review of the recent literature reveals that it is the silencer itself and its context within a given promoter, rather than the interacting repressor, that determines the mechanism of repression. Silencers form an intrinsic part of many eukaryotic promoters and, consequently, knowledge of their interactive role with enchancers and other transcriptional elements is essential for our understanding of gene regulation in eukaryotes.

Animals↗

PubMed1998

The bZip transcription factor vitellogenin-binding protein is post transcriptional down regulated in chicken liver.

The vitellogenin-binding protein (VBP) is a member of the proline and acidic-region rich (PAR) family of bZip transcription factors. PAR is located N-terminally to the DNA-binding domain. VBP binds to specific sites within the 300-bp 5'-flanking region of the chicken-liver-specific estrogen-dependent very-low-density apolipoprotein gene (apoVLDL II). One of these binding sites (site D) resembles the albumin site D and is positioned in close proximity of the major estrogen-responsive element. Previous studies showed that VBP can bind simultaneously with the estrogen receptor to the putative complex regulatory element E1D. To investigate whether VBP is involved in apoVLDL II gene expression, we examined its capacity to enhance apoVLDL II transcription and its presence in liver. We show that VBP is capable of enhancing transcription in transfection experiments. However, VBP could not be detected in liver by Western-blots or immuno-electro mobility shift assays (EMSAs) using antibodies against different moieties of the protein. We examined the possible reduction in translation efficiencies due to a small upstream open reading frame in the VBP leader sequence, but did not find any. Although VBP binds to the proximal apoVLDL II promoter region and enhances transcription in co-transfection experiments, the protein is unlikely to be involved in apoVLDL II gene transcription because of its undetectable low level in liver nuclei.

Animals↗

PubMed1990

Cloned yeast and mammalian transcription factor TFIID gene products support basal but not activated metallothionein gene transcription.

Transcription factor IID (TFIID), the "TATA binding factor," is thought to play a key role in the regulation of eukaryotic transcriptional initiation. We have studied the role of TFIID in the transcription of the yeast metallothionein gene, which is regulated by the copper-dependent activator protein ACE1. Both basal and induced transcription of the metallothionein gene require TFIID and a functional TATA binding site. Crude human and mouse TFIID fractions, prepared from mammalian cells, respond to stimulation by ACE1. In contrast, human and yeast TFIID proteins expressed from the cloned genes do not respond to ACE1, except in the presence of wheat germ or yeast total cell extracts. These results indicate that the cloned TFIID gene products lack a component(s) or modification(s) that is required for regulated as compared to basal transcription.

Animals↗

PubMed1991

The initiator directs the assembly of a transcription factor IID-dependent transcription complex.

Highly purified RNA polymerase II was found to be able to weakly recognize the initiator (Inr) present in the adenovirus IVa2 and major late promoters. The association of RNA polymerase II with the Inr was enhanced by the general transcription factors. The Inr was capable of directing the formation of a DNA-protein complex. Transcription competent complexes on the adenovirus major late and IVa2 promoters appear to be formed by alternative pathways mediated through the Inr and/or "TATA" motif. The presence of both motifs, however, is required for efficient transcription utilizing a discrete start site. Complexes formed at either site required transcription factor TFIID, the TATA binding protein. Consistent with this observation, a TFIID requirement was demonstrated for transcription from a mutant adenovirus major late promoter construct lacking a functional TATA motif.

Adenoviridae↗

PubMed1995

Transcription factor TFIID is a direct functional target of the adenovirus E1A transcription-repression domain.

The 243-amino acid adenovirus E1A oncoprotein both positively and negatively modulates the expression of cellular genes involved in the regulation of cell growth. The E1A transcription repression function appears to be linked with its ability to induce cellular DNA synthesis, cell proliferation, and cell transformation, as well as to inhibit cell differentiation. The mechanism by which E1A represses the transcription of various promoters has proven enigmatic. Here we provide several lines of evidence that the "TATA-box" binding protein (TBP) component of transcription factor TFIID is a cellular target of the E1A repression function encoded within the E1A N-terminal 80 amino acids. (i) The E1A N-terminal 80 amino acids [E1A-(1-80)protein] efficiently represses basal transcription from TATA-containing core promoters in vitro. (ii) TBP reverses completely E1A repression in vitro. (iii) TBP restores transcriptional activity to E1A-(1-80) protein affinity-depleted nuclear extracts. (iv) The N-terminal repression domain of E1A interacts directly and specifically with TBP in vitro. These results may help explain how E1A represses a set of genes that lack common upstream promoter elements.

Adenovirus E1A Proteins↗

PubMed1996

Separation of the transcriptional coactivator and antirepression functions of transcription factor IIA.

Human transcription factor IIA (TFIIA) is composed of three subunits (alpha, beta, and gamma). TFIIA interacts with the TATA-box binding protein and can overcome repression of transcription. TFIIA was found to be necessary for VP16-mediated transcriptional activation through a coactivator function. We have separated the coactivator and antirepression activities of TFIIA. A TFIIA lacking the alpha subunit was isolated from HeLa cells. This "mini-TFIIA" interacts with the TATA-box binding protein and can overcome repression of transcription, but it is defective in transcriptional coactivator function.

DNA-Binding Proteins↗