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PubMed1994

Temperature-sensitive mutations in the gene encoding the small subunit of the vaccinia virus early transcription factor impair promoter binding, transcription activation, and packaging of multiple virion components.

The vaccinia virus D6R open reading frame encodes the small subunit of the heterodimeric vaccinia virus early transcription factor (VETF) that activates transcription of early genes in vitro. VETF binds early gene promoters and has a DNA-dependent ATPase activity that is essential for activation of transcription. To examine the relationship between the structure and function of VETF, we have localized the mutations in two temperature-sensitive viruses whose lesions previously were mapped to the D6R gene. For both mutants, a single G-to-A nucleotide change that would alter protein coding potential was identified. In mutant E93, the codon for alanine 25 was changed to that of threonine, and in mutant S4 the codon for valine 278 was replaced with that for methionine. The molecular phenotype of each mutant was assessed by expressing mutant transcription factors in HeLa cells by using a vaccinia virus-T7 system and characterizing the proteins' activities in vitro. The A25T mutant activated transcription to a lesser extent than wild-type VETF, and the V278M mutant had no demonstrable transcription factor activity. Both mutant proteins were shown to be defective for promoter binding, accounting for their impairment in transcription activation. The functional defects for both mutants were observed at permissive as well as nonpermissive temperatures. The mutant proteins retained ATPase activity but required higher DNA concentrations to activate the ATPase. These results indicate that the small subunit of VETF is essential for its promoter binding activity and likely contacts the promoter DNA. Immunoblotting experiments showed that the virion particles from the two mutant viruses contained about half the VETF of wild-type virus, suggesting that promoter binding may contribute to packaging of VETF into the virion particle. RNA polymerase, mRNA capping enzyme, and nucleoside triphosphate phosphohydrolase I were found at similarly reduced levels in the virion, indicating that packaging of some virion core enzymes may be interdependent.

Adenosine Triphosphatases↗

PubMed1997

Antioxidant-induced changes of the AP-1 transcription complex are paralleled by a selective suppression of human papillomavirus transcription.

Considering the involvement of a redox-regulatory pathway in the expression of human papillomaviruses (HPVs), HPV type 16 (HPV-16)-immortalized human keratinocytes were treated with the antioxidant pyrrolidine-dithiocarbamate (PDTC). PDTC induces elevated binding of the transcription factor AP-1 to its cognate recognition site within the viral regulatory region. Despite of increased AP-1 binding, normally indispensable for efficient HPV-16 transcription, viral gene expression was selectively suppressed at the level of initiation of transcription. Electrophoretic mobility supershift assays showed that the composition of the AP-1 complex, predominantly consisting of Jun homodimers in untreated cells, was altered. Irrespective of enhanced c-fos expression, c-jun was phosphorylated and became primarily heterodimerized with fra-1, which was also induced after PDTC incubation. Additionally, there was also an increased complex formation between c-jun and junB. Because both fra-1 and junB overexpression negatively interferes with c-jun/c-fos trans-activation of AP-1-responsive genes, our results suggest that the observed block in viral transcription is mainly the consequence of an antioxidant-induced reconstitution of the AP-1 transcription complex. Since expression of the c-jun/c-fos gene family is tightly regulated during cellular differentiation, defined reorganization of a central viral transcription factor may represent a novel mechanism controlling the transcription of pathogenic HPVs during keratinocyte differentiation and in the progression to cervical cancer.

Antioxidants↗

PubMed1999

Cell-type-dependent activity of the ubiquitous transcription factor USF in cellular proliferation and transcriptional activation.

USF1 and USF2 are basic helix-loop-helix transcription factors implicated in the control of cellular proliferation. In HeLa cells, the USF proteins are transcriptionally active and their overexpression causes marked growth inhibition. In contrast, USF overexpression had essentially no effect on the proliferation of the Saos-2 osteosarcoma cell line. USF1 and USF2 also lacked transcriptional activity in Saos-2 cells when assayed by transient cotransfection with USF-dependent reporter genes. Yet, there was no difference in the expression, subcellular localization, or DNA-binding activity of the USF proteins in HeLa and Saos-2 cells. Furthermore, Gal4-USF1 and Gal4-USF2 fusion proteins activated transcription similarly in both cell lines. Mutational analysis and domain swapping experiments revealed that the small, highly conserved USF-specific region (USR) was responsible for the inactivity of USF in Saos-2 cells. In HeLa, the USR serves a dual function. It acts as an autonomous transcriptional activation domain at promoters containing an initiator element and also induces a conformational change that is required for USF activity at promoters lacking an initiator. Taken together, these results suggest a model in which the transcriptional activity of the USF proteins, and consequently their antiproliferative activity, is tightly controlled by interaction with a specialized coactivator that recognizes the conserved USR domain and, in contrast to USF, is not ubiquitous. The activity of USF is therefore context dependent, and evidence for USF DNA-binding activity in particular cells is insufficient to indicate USF function in transcriptional activation and growth control.

Amino Acid Sequence↗

PubMed2000

Architectural transcription factors and the SAGA complex function in parallel pathways to activate transcription.

Recent work has shown that transcription of the yeast HO gene involves the sequential recruitment of a series of transcription factors. We have performed a functional analysis of HO regulation by determining the ability of mutations in SIN1, SIN3, RPD3, and SIN4 negative regulators to permit HO expression in the absence of certain activators. Mutations in the SIN1 (=SPT2) gene do not affect HO regulation, in contrast to results of other studies using an HO:lacZ reporter, and our data show that the regulatory properties of an HO:lacZ reporter differ from that of the native HO gene. Mutations in SIN3 and RPD3, which encode components of a histone deacetylase complex, show the same pattern of genetic suppression, and this suppression pattern differs from that seen in a sin4 mutant. The Sin4 protein is present in two transcriptional regulatory complexes, the RNA polymerase II holoenzyme/mediator and the SAGA histone acetylase complex. Our genetic analysis allows us to conclude that Swi/Snf chromatin remodeling complex has multiple roles in HO activation, and the data suggest that the ability of the SBF transcription factor to bind to the HO promoter may be affected by the acetylation state of the HO promoter. We also demonstrate that the Nhp6 architectural transcription factor, encoded by the redundant NHP6A and NHP6B genes, is required for HO expression. Suppression analysis with sin3, rpd3, and sin4 mutations suggests that Nhp6 and Gcn5 have similar functions. A gcn5 nhp6a nhp6b triple mutant is extremely sick, suggesting that the SAGA complex and the Nhp6 architectural transcription factors function in parallel pathways to activate transcription. We find that disruption of SIN4 allows this strain to grow at a reasonable rate, indicating a critical role for Sin4 in detecting structural changes in chromatin mediated by Gcn5 and Nhp6. These studies underscore the critical role of chromatin structure in regulating HO gene expression.

Acetyltransferases↗

PubMed2002

DNA-dependent adenosine triphosphatase (helicaselike transcription factor) activates beta-globin transcription in K562 cells.

Correct developmental regulation of beta-like globin gene expression is achieved by preferential transcription of a gene at a given developmental stage, silencing of other beta-like gene promoters, and competition among these promoters for interaction with the locus control region (LCR). Several evolutionarily conserved DNA elements in the promoters of the beta-like genes and LCR have been studied in detail, and the role of their binding factors has been investigated. However, the beta-globin promoter includes additional evolutionarily conserved sequences of unknown function. The present study examined the properties of a 21-base pair (bp) promoter-conserved sequence (PCS) located at positions -115 to -136 bp relative to the transcription start site of the beta-globin gene. A helicaselike transcription factor (HLTF) belonging to the SWI2/SNF2 family of proteins binds to the PCS and a partly homologous sequence in the enhancer region of the LCR hypersensitive site 2 (HS2). Elevation of the level of HLTF in K562 erythroleukemic cells increases beta-promoter activity in transient transfection experiments, and mutations in the PCS that remove HLTF-binding regions abolish this effect, suggesting that HLTF is an activator of beta-globin transcription. Overexpression of HLTF in K562 cells does not affect the endogenous levels of gamma- and epsilon-globin message, but it markedly activates beta-globin transcription. In conclusion, this study reports a transcription factor belonging to the SWI2/SNF2 family, which preferentially activates chromosomal beta-globin gene transcription and which has not previously been implicated in globin gene regulation.

Animals↗

PubMed2001

Bacterial peptidoglycan-induced tnf-alpha transcription is mediated through the transcription factors Egr-1, Elk-1, and NF-kappaB.

Bacteria and their ubiquitous cell wall component peptidoglycan (PGN) activate the innate immune system of the host and induce the release of inflammatory molecules. TNF-alpha is one of the highest induced cytokines in macrophages stimulated with PGN; however, the regulation of tnf-alpha expression in PGN-activated cells is poorly understood. This study was done to identify some of the transcription factors that regulate the expression of the tnf-alpha gene in macrophages stimulated with PGN. Our results demonstrated that PGN-induced expression of human tnf-alpha gene is regulated by sequences proximal to -182 bp of the promoter. Mutations within the binding sites for cAMP response element, early growth response (Egr)-1, and kappaB3 significantly reduced this induction. The transcription factor c-Jun bound the cAMP response element site, Egr-1 bound the Egr-1 motif, and NF-kappaB p50 and p65 bound to the kappaB3 site on the tnf-alpha promoter. PGN rapidly induced transcription of egr-1 gene and this induction was significantly reduced by specific mutations within the serum response element-1 domain of the egr-1 promoter. PGN also induced phosphorylation and activation of Elk-1, a member of the Ets family of transcription factors. Elk-1 and serum response factor proteins bound the serum response element-1 domain on the egr-1 promoter, and PGN-induced expression of the egr-1 was inhibited by dominant-negative Elk-1. These results indicate that PGN induces activation of the transcription factors Egr-1 and Elk-1, and that PGN-induced expression of tnf-alpha is directly mediated through the transcription factors c-Jun, Egr-1, and NF-kappaB, and indirectly through the transcription factor Elk-1.

Animals↗

PubMed2001

Combinatorial transcriptional regulation: the interaction of transcription factors and cell signaling molecules with homeodomain proteins in Drosophila development.

Patterning and cell fate specification during development require complex interplay among multiple families of transcription factors to establish, maintain, and coordinate transcriptional cascades. During these processes, homeodomain proteins and cell signaling proteins cooperate to generate tissue-and stage-specific responses. This review of physical and genetic interactions in Drosophila melanogaster development highlights the cross-talk among these protein families. Protein-protein association can modulate regulation by both signal transduction-regulated transcription factors and homeodomain proteins, as observed in Drosophila and other organisms. Enhancers or genes regulated by multiple transcription factors provide opportunities for protein-protein binding to modulate transcription factor function. Combinatorial regulation of several enhancers by homeodomain proteins and cell signaling-regulated transcription factors is discussed; detailed maps of the genetic interactions that pattern the embryonic midgut and the larval wing imaginal disc are used to illustrate the multiplicity of potential protein-protein interactions. These interactions potentially provide direct mechanisms for communication between transcription factors as well as for generating the requisite functional specificity.

Animals↗

PubMed1991

Post-transcriptional and transcriptional control of collagen gene expression in normal and modulated rabbit corneal endothelial cells.

In a previous report, collagen synthesis did not correlate with steady-state collagen RNA levels; substantial amounts of type I collagen RNAs in endothelial cells were not translated into the respective protein. The current investigation was extended to study the level of the control mechanism in collagen gene expression in normal corneal endothelial cells or those modulated by corneal endothelium modulation factor released by polymorphonuclear leukocytes. Northern-blot analysis using cloned rabbit types I and IV cDNA probes (same species as RNA sources) demonstrated specific mRNA transcripts for collagen types I and IV in the endothelial cells, although the steady-state level of these mRNAs in modulated endothelial cells was low. The turnover rate of collagen RNAs was determined; normal cells contain very stable alpha 2(I) and alpha 2(IV) mRNAs whose half-lives exceed 24 hr. The same messages decayed rapidly in the modulated cells, where they had an apparent half-life of approximately 8 hr. Using nuclear run-off transcription, the rate of transcription in normal cells was found to be slightly lower than that in modulated cells. When the relative rate of collagen gene transcription was compared, that of alpha 2(I) was the lowest and of alpha 2(IV), the highest in both cells. The relative transcriptional rates of individual collagen chains did not account for the steady-state levels, suggesting that transcriptional regulation in corneal endothelial cells was less than was translational regulation. On the other hand, during early stages of corneal endothelial cell modulation induced by factors released by polymorphonuclear leukocytes there was a differential effect on both transcriptional rate and the steady-state level of collagen RNAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Transcriptional and post-transcriptional control of apolipoprotein E gene expression in differentiating human monocytes.

The present studies examined the mechanisms responsible for the regulation of apolipoprotein (apo) E gene expression during human monocytic differentiation. Levels of apoE mRNA were low in undifferentiated THP1 cells, a human monocytic cell line. Addition of 12-O-tetradecanylphorbol-13-acetate (PMA) induced differentiation of these cells to a macrophage-like phenotype and was associated with increased apoE mRNA abundance in a time-dependent fashion, up to 10-11-fold within 32 h. Results of nuclear run-on transcription assays demonstrated that the apoE gene was transcriptionally active in undifferentiated THP1 cells and that differentiation of monocytes with PMA was associated with a maximal increase of apoE gene transcription rate of only 2-3-fold at 6-12 h. Using actinomycin D as an inhibitor of new transcription, we could demonstrate a more rapid degradation of mature apoE mRNA in undifferentiated compared to differentiated cells, suggesting that the apoE mRNA species was more stable in differentiated THP1 cells. Primer extension assays performed using RNA extracts from undifferentiated and differentiated THP1 cells confirmed the increase of apoE mRNA abundance in the latter but failed to disclose heterogeneity in apoE gene transcription start site between these two phenotypes. These findings indicate that apoE gene expression is controlled at both transcriptional and post-transcriptional loci during human monocyte-macrophage differentiation.

Apolipoproteins E↗

PubMed1987

Multihormonal regulation of transcription of the tryptophan 2,3-dioxygenase gene in primary cultures of adult rat hepatocytes with special reference to the presence of a transcriptional protein mediating the action of glucocorticoids.

For study of hormonal regulation of gene expression of tryptophan 2,3-dioxygenase (EC 1. 13. 11. 11, TO), a DNA clone containing a sequence complementary to TO mRNA was prepared with TO mRNA from rat liver enriched 62-fold by immunoadsorption. Primary cultures of adult rat hepatocytes were treated with dexamethasone, and the amount of TO mRNA was measured by RNA dot-blot hybridization with this TO cDNA. Dexamethasone induced this TO mRNA 7-fold, while their treatments with dexamethasone plus glucagon induced the TO mRNA 18-fold. This induction of TO mRNA by dexamethasone plus glucagon was inhibited by insulin or epinephrine. Studies on transcription in isolated nuclei showed that these hormonal changes in the level of TO mRNA were caused by changes in the rate of transcription of the TO gene. Thus, expression of TO in the liver is regulated multihormonally at the transcriptional step. There was a long lag period before stimulation of transcription of the TO gene by dexamethasone in hepatocytes cultured for 20 h: the maximal rate was attained after 6-8 h. The lag time depended on the culture time without dexamethasone and was shorter after shorter culture of the cells. This finding suggested that a transcriptional factor that was lost during culture mediated the action of glucocorticoids. Consistent with this idea, cycloheximide or puromycin almost completely blocked enhanced transcription of the TO gene by dexamethasone after a 20-h culture, but not after a 2-h culture. These findings indicate that a short-lived transcriptional protein, which is also regulated by glucocorticoids, mediates their effect on expression of the TO gene.

Animals↗

PubMed1994

Mechanism of differential regulation of IL-2 in murine Th1 and Th2 T cell subsets. 1. Induction of IL-2 transcription in Th2 cells by up-regulation of transcription factors with the protein synthesis initiation factor 4E.

Regulation of IL-2 gene expression in response to receptor-mediated stimuli is known to be mediated primarily by the IL-2 transcriptional enhancer and multiple transcription factors. However, the mechanism that controls the differential expression of the IL-2 gene in both human and murine CD4+ Th cell subsets (Th1-IL-2+ and Th2-IL-2-) is not clearly understood. Differential IL-2 gene expression was assessed in murine Th1 and Th2 subsets by analyzing the expression of a Escherichia coli lacZ reporter gene under control of the human IL-2 enhancer (IL2ZH) transfected in both T cell subsets. Stimulation of transfected T cells with the mitogen Con A, anti-CD3 Ab, or PMA plus ionomycin activated the IL2ZH construct in Th1 but not Th2 cells. However, IL2ZH was activated in stimulated Th2 cells that were co-transfected with a vector that overexpressed the eukaryotic initiation factor 4E (eIF-4E). It has been shown that eIF-4E is rate limiting for protein synthesis and its overexpression leads to increased rates of protein synthesis. Hence, eIF-4E overexpression could have overcome a deficiency in transcriptionally active levels of IL-2 regulatory factors in Th2 cells leading to IL-2 enhancer activation. This possibility was supported by demonstrating that transcriptionally active levels of the critical IL-2 transcription factor, nuclear factor of activated T cells (NF-AT), occurred only in Th2 cells overexpressing eIF-4E but not in normal Th2 cells, thus indicating that the inability of Th2 cells to express IL-2 was associated with inadequate levels of at least one transcription factor, NF-AT. Moreover, these results were confirmed by the observation that eIF-4E overexpression augmented NF-AT binding activity in Th2 cells. These data suggest that concentrations of inducible transcription factors are a major component of the regulatory mechanisms dictating IL-2 expression and may be under translational control in Th1/Th2 T cell subsets.

Animals↗

PubMed1993

Transcriptional activation of the mouse mdr3 gene coincides with the appearance of novel transcription initiation sites in multidrug-resistant P388 tumor cells.

In independently derived drug-resistant sublines of the mouse lymphoid tumor P388, multidrug resistance is associated with the exclusive overexpression of the mdr3 gene. In P388/VCR cells, mdr3 overexpression occurs in the absence of gene amplification, while in P388/ADM-2 cells overexpression is associated with mdr3 gene amplification. The mechanism underlying mdr3 overexpression in these cells was investigated. Measurement of the rate of transcription by nuclear "run-on" assays showed that increased mdr3 expression in P388/VCR cells was caused by transcriptional activation of the gene. Analysis of the 5' end of mdr3 mRNA transcripts by primer extension indicated that in P388/VCR cells, these mRNAs extended approximately 200 nucleotides upstream exon 2, about 60 nucleotides longer than their counterparts expressed in normal tissues from the known transcription start site of the gene (TS1). Northern blotting experiments using discrete exon and intron probes derived from the 5' end of the gene near TS1, together with ribonuclease protection using a complementary RNA probe from the same region, demonstrated that transcriptional activation in P388/VCR cells occurred from a novel transcription start site named TS3, located either upstream of TS1 or within intron 1 at a site immediately upstream a novel exon. In P388/ADM-2 cells, Northern blotting and ribonuclease protection identified overexpressed mdr3 mRNAs initiating near TS1 and a large partially spliced mdr3 mRNA species initiating upstream of TS1 at a novel initiation site designated TS2. Therefore, mdr3 overexpression in independently derived multidrug-resistant isolates of P388 cells is associated with the appearance of novel transcription start sites in the gene and novel sequences at the 5' end of the overexpressed mRNAs.

Animals↗

PubMed2003

Nuclear distribution of Oct-4 transcription factor in transcriptionally active and inactive mouse oocytes and its relation to RNA polymerase II and splicing factors.

The intranuclear distribution of the transcription factor Oct-4, which is specifically expressed in totipotent mice stem and germ line cells, was studied in mouse oocytes using immunogold labeling/electron microscopy and immunofluorescence/confocal laser scanning microcopy. The localization of Oct-4 was studied in transcriptionally active (uni/bilaminar follicles) and inactive (antral follicles) oocytes. Additionally, the Oct-4 distribution was examined relative to that of the unphosphorylated form of RNA polymerase II (Pol II) and splicing factor (SC 35) in the intranuclear entities such as perichromatin fibrils (PFs), perichromatin granules (PGs), interchromatin granule clusters (IGCs), Cajal bodies (CBs), and nucleolus-like bodies (NLBs). It was shown that: (i) Oct-4 is localized in PFs, IGCs, and in the dense fibrillar component (DFC) of the nucleolus at the transcriptionally active stage of the oocyte nucleus; (ii) Oct-4 present in PFs and IGCs colocalizes with Pol II and SC 35 at the transcriptionally active stage; (iii) Oct-4 accumulates in NLBs, CBs, and PGs at the inert stage of the oocyte. The results confirm the previous suggestion that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcription/processing. The colocalization of Oct-4 with Pol II in both IGCs and PFs in active oocytes (uni/bilaminar follicles) suggests that Oct-4 is intimately associated with the Pol II holoenzyme before and during transcription. The colocalization of Oct-4, Pol II, and SC 35 with coilin-containing structures such as NLBs and CBs at the inert stage (antral follicles) suggests that the latter may represent storage sites for the transcription/splicing machinery during the decline of transcription.

Animals↗

PubMed1992

The herpes simplex virus immediate-early protein ICP27 stimulates the transcription of cellular Alu repeated sequences by increasing the activity of transcription factor TFIIIC.

Infection with herpes simplex virus (HSV) results in an increase in the transcription of the endogenous Alu repeated sequence by RNA polymerase III. This effect is also observed in uninfected cells stably transformed with a plasmid expressing the HSV immediate-early protein ICP27 or in cells transfected with the gene encoding this protein. Both uninfected cells expressing ICP27 and cells infected with virus producing functional ICP27 display increased activity of the cellular transcription factor TFIIIC when compared with untreated cells. This increase is not observed, however, in cells infected with a mutant strain of virus which does not produce ICP27. Hence ICP27 induces elevated Alu transcription by activating transcription factor TFIIIC, which is the limiting factor for such transcription. This is the first report of increased activity of a cellular transcription factor during HSV infection, when most cellular gene activity is inhibited.

Animals↗

PubMed1996

A role for the Msx-1 homeodomain in transcriptional regulation: residues in the N-terminal arm mediate TATA binding protein interaction and transcriptional repression.

In a previous study we showed that the murine homeodomain protein Msx-1 is a potent transcriptional repressor and that this activity is independent of its DNA binding function. The implication of these findings is that repression by Msx-1 is mediated through its association with certain protein factors rather than through its interaction with DNA recognition sites, which prompted investigation of the relevant protein factors. Here we show that Msx-1 interacts directly with the TATA binding protein (TBP) but not with several other general transcription factors. This interaction is mediated by the Msx-1 homeodomain, specifically through residues in the N-terminal arm. These same N-terminal arm residues are required for repression by Msx-1, suggesting a functional relationship between TBP association and transcriptional repression. This is further supported by the observation that addition of excess TBP blocks the repressor action of Msx-1 in in vitro transcription assays. Finally, DNA binding activity is separable from both TBP interaction and repression, which further shows that these other activities of the Msx-1 homeodomain are distinct. Therefore, these findings define a role for the Msx-1 homeodomain, particularly the N-terminal arm residues in protein-protein interaction and transcriptional repression, and implicate a more complex role overall for homeodomains in transcriptional regulation.

Amino Acid Sequence↗

PubMed1995

Members of the Sp transcription factor family control transcription from the uteroglobin promoter.

Previous analyses of the uteroglobin promoter revealed seven distinct regions, which contribute to its overall activity in epithelial cells from endometrium and lung. Most significantly, a mutation of the promoter sequence around 65 base pairs upstream of the transcriptional start site severely impairs promoter activity. The transcription factor acting through this sequence has not been identified yet. Here, we report that members of the Sp transcription factor family specifically recognize this non-classical GC box, in addition to another functional motif located 230 base pairs upstream of the transcriptional start site. We have characterized in detail the interaction of recombinant Sp3 with both motifs by DNase I footprinting and methylation protection using the wild-type uteroglobin promoter and various linker scanning mutants as templates. Electrophoretic mobility shift analyses show that Sp1 and Sp3 both bind with similar affinity to these elements. We demonstrate that the DNA-binding proteins in the endometrial cell line Ishikawa which recognize these motifs are also Sp1 and Sp3. Gene transfer experiments into Drosophila Schneider cells that do not contain endogenous Sp factors revealed that both DNA motifs respond to transiently expressed Sp1 and Sp3. Our results show thus that the level of transcription from the uteroglobin promoter is controlled by members of the Sp transcription factor family through unusual Sp binding sites.

Animals↗

PubMed1999

The interaction between the forkhead thyroid transcription factor TTF-2 and the constitutive factor CTF/NF-1 is required for efficient hormonal regulation of the thyroperoxidase gene transcription.

The forkhead thyroid-specific transcription factor TTF-2 is the main mediator of thyrotropin and insulin regulation of thyroperoxidase (TPO) gene expression. This function depends on multimerization and specific orientation of its DNA-binding site, suggesting that TTF-2 is part of a complex interaction network within the TPO promoter. This was confirmed by transfection experiments and by protein-DNA interaction studies, which demonstrated that CTF/NF1 proteins bind 10 base pairs upstream of the TTF-2-binding site to enhance its action in hormone-induced expression of the TPO gene. GST pull-down assays showed that TTF-2 physically interacts with CTF/NF1 proteins. In addition, we demonstrate that increasing the distance between both transcription factors binding sites by base pair insertion results in loss of promoter activity and in a drastic decrease on the ability of the promoter to respond to the hormones. CTF/NF1 is a family of transcription factors that contributes to constitutive and cell-type specific gene expression. Originally identified as factors implicated in the replication of adenovirus, this group of proteins (CTF/NF1-A, -B, -C, and -X) is now known to be involved in the regulation of several genes. In contrast to other reports regarding the involvement of these proteins in inducible gene expression, we show here that members of this family of transcription factors are regulated by hormones. With the use of specific CTF/NF1 DNA probes and antibodies we demonstrate that CTF/NF1-C is a thyrotropin-, cAMP-, and insulin-inducible protein. Thus CTF/NF1 proteins do not only mediate hormone-induced gene expression cooperating with TTF-2, but are themselves hormonally regulated. All these findings are clearly of important value in understanding the mechanisms governing the transcription regulation of RNA polymerase II promoters, which often contain binding sites for multiple transcription factors.

Base Sequence↗

PubMed1999

GATA-6 activates transcription of thyroid transcription factor-1.

Thyroid transcription factor-1 (TTF-1) is expressed in respiratory epithelial cells, where it regulates the transcription of target genes expressed in a cell-selective manner. GATA-5 and -6, members of the zinc finger family of transcription factors, are also expressed in various cell types within in the developing lung. In the present work, GATA-6 mRNA was detected in adult mouse lung, purified mouse type II epithelial cells, and differentiated mouse pulmonary adenocarcinoma cells (MLE-15 cells), being co-expressed with TTF-1 mRNA. In order to test whether GATA factors regulated TTF-1 gene transcription, GATA-5 and -6 expression vectors were co-transfected with TTF-1 luciferase expression vector. GATA-6, but not GATA-5, markedly activated TTF-1 gene transcription in HeLa cells. EMSA and supershift analysis with GATA-6 antiserum demonstrated that GATA-6 in MLE-15 cell nuclear extracts bound to an element located 96-101 base pairs from major start of TTF-1 gene transcription. Site directed mutagenesis of the GATA element in the TTF-1 promoter region inhibited transactivation by GATA-6 in HeLa cells. GATA-6 is co-expressed with TTF-1 in the respiratory epithelium in vivo and respiratory epithelial cells in vitro. GATA-6 strongly enhanced activity of the human TTF-1 gene promoter in vitro. These findings support the concept that GATA-6 may play an important role in lung cell differentiation and gene expression, at least in part by altering the expression of TTF-1 and its potential targets.

Animals↗