Nuclear factor I-mediated repression of the mouse mammary tumor virus promoter is abrogated by the coactivators p300/CBP and SRC-1. (1/466)

To better understand the function of nuclear factor I (NFI) proteins in transcription, we have used transient transfection assays to assess transcriptional modulation by NFI proteins on the NFI-dependent mouse mammary tumor virus (MMTV) promoter. Expression of NFI-C or NFI-X, but not NFI-A or NFI-B proteins, represses glucocorticoid induction of the MMTV promoter in HeLa cells. Repression is DNA binding-independent as a deletion construct expressing the NH2-terminal 160 residues of NFI-C represses but does not bind DNA. Repression by NFI-C is cell type-dependent and occurs in HeLa and COS-1 cells but not 293 or JEG-3 cells. NFI-C does not repress progesterone induction of the MMTV promoter in HeLa cells, suggesting that progesterone induction of the promoter differs mechanistically from glucocorticoid induction. NFI-C-mediated repression is alleviated by overexpression of glucocorticoid receptor (GR), suggesting that NFI-C represses the MMTV promoter by preventing GR function. However, repression by NFI-C occurs with only a subset of glucocorticoid-responsive promoters, as the chimeric NFIGREbeta-gal promoter that is activated by GR is not repressed by NFI-C. Since the coactivator proteins p300/CBP, SRC-1A, and RAC3 had previously been shown to function at steroid hormone-responsive promoters, we asked whether they could influence NFI-C-mediated repression of MMTV expression. Expression of p300/CBP or SRC-1A alleviates repression by NFI-C, whereas RAC3 has no effect. This abrogation of NFI-C-mediated repression by p300/CBP and SRC-1A suggests that repression by NFI-C may occur by interference with coactivator function at the MMTV promoter.  (+info)

Reciprocal interaction between two cellular proteins, Puralpha and YB-1, modulates transcriptional activity of JCVCY in glial cells. (2/466)

Cross communication between regulatory proteins is an important event in the control of eukaryotic gene transcription. Here we have examined the structural and functional interaction between two cellular regulatory proteins, YB-1 and Puralpha, on the 23-bp sequence element derived from the enhancer-promoter of the human polyomavirus JCV. YB-1 and Puralpha are single-stranded DNA binding proteins which recognize C/T- and GC/GA-rich sequences, respectively. Results from band shift studies demonstrated that while both proteins interact directly with their DNA target sequences within the 23-bp motif, each protein can regulate the association of the other one with the DNA. Affinity chromatography and coimmunoprecipitation provide evidence for a direct interaction between Puralpha and YB-1 in the absence of the DNA sequence. Ectopic expression of YB-1 and Puralpha in glial cells synergistically stimulated viral promoter activity via the 23-bp sequence element. Results from mutational studies revealed that residues between amino acids 75 and 203 of YB-1 and between amino acids 85 and 215 of Puralpha are important for the interaction between these two proteins. Functional studies with glial cells indicated that the region within Puralpha which mediates its association with YB-1 and binding to the 23-bp sequence is important for the observed activation of the JCV promoter by the Puralpha and YB-1 proteins. The results of this study suggest that the cooperative interaction between YB-1 and Puralpha mediates the synergistic activation of the human polyomavirus JCV genome by these cellular proteins. The importance of these findings for cellular and viral genes which are regulated by Puralpha and YB-1 is discussed.  (+info)

CREB binding protein coordinates the function of multiple transcription factors including nuclear factor I to regulate phosphoenolpyruvate carboxykinase (GTP) gene transcription. (3/466)

Nuclear factor I (NFI) binds to a region of the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene promoter adjacent to the cAMP regulatory element (CRE) and inhibits the induction of transcription from the gene promoter caused by the catalytic subunit of protein kinase A. In vivo footprinting studies demonstrated that both the CRE and the NFI-binding site are occupied by transcription factors, regardless of the presence of factors that stimulate (dibutyryl cAMP or dexamethasone) or inhibit (insulin) transcription from the PEPCK gene promoter. The NFI effects on transcription from the PEPCK gene promoter were observed even in the absence of the NFI binding site, suggesting the possibility of other weaker binding sites on the promoter or an interaction of NFI with a transcriptional co-activator. A mammalian two-hybrid system was used to demonstrate direct interaction between the transactivation domain of NFI-C and the CREB binding domain of the CREB-binding protein (CBP). Overexpression of a gene fragment encoding the CREB binding domain of CBP stimulates transcription from the PEPCK gene promoter. The inhibitory effect of NFI on transcription of the PEPCK gene induced by the catalytic subunit of protein kinase A appears to be the result of an interaction between NFI and the CREB-binding protein in which NFI competes with CREB for binding to the CREB-binding site on CBP. In contrast, glucocorticoids and thyroid hormone use the steroid hormone receptor binding domain of CBP to stimulate transcription from the PEPCK gene promoter. NFI-A combines with dexamethasone or thyroid hormone in an additive manner to stimulate PEPCK gene transcription. We conclude that CBP coordinates the action of the multiple factors known to control transcription of the PEPCK gene.  (+info)

The transcription factor nuclear factor I mediates repression of the GLUT4 promoter by insulin. (4/466)

Insulin represses GLUT4 expression in 3T3-L1 adipocytes through an insulin response element located at bases -706 to -676 in the 5'-flanking sequence. Nuclear proteins related to the nuclear factor I (NF1) family of transcription factors bind to this insulin response element. Mutations that disrupt binding of NF1 proteins to the insulin response element impair the insulin response in reporter gene assays. Insulin treatment of 3T3-L1 adipocytes induces a rapid change in the level of phosphorylation of NF1 proteins, providing a potential mechanism for insulin's ability to regulate gene expression through NF1. Another as yet unidentified protein, not related to NF1, also binds to the GLUT4 insulin response element and is able to mediate partial repression of the GLUT4 promoter in reporter gene assays.  (+info)

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. (5/466)

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.  (+info)

The Wilms' tumor suppressor gene (wt1) product represses different functional classes of transcriptional activation domains. (6/466)

We have studied the ability of the wt1 tumor suppressor gene product to repress different classes of activation domains previously shown to stimulate the initiation and elongation steps of RNA polymerase II transcription in vivo. Repression assays revealed that WT1 represses all three classes of activation domains: Sp1 and CTF, which stimulate initiation (type I), human immunodeficiency virus type I Tat fused to a DNA-binding domain, which stimulates predominantly elongation (type IIA), and VP16, p53 and E2F1, which stimulate both initiation and elongation (type IIB). WT1 is capable of exerting its repression effect over a significant distance when positioned approximately 1700 bp from the core promoter. Deletion analysis of WT1 indicates that the responsible domain resides within the first 180 N-terminal amino acids of the protein. Nuclear run-ons analyzing the effects of WT1 on initiation of transcription demonstrate inhibition of this process. Our observations imply that WT1 can repress activators that stimulate initiation and/or elongation.  (+info)

Activation of the thymidine kinase promoter by herpes simplex virus type 1 immediate early proteins. (7/466)

The herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene promoter contains binding sites for the cellular transcription factors such as Spl, CTF, and TFIID, each of which affects basal level expression of the TK gene. The transcription of the TK gene was induced by viral immediate early proteins, ICP0 and ICP4 in an additive manner, but was repressed by ICP22 and ICP27. To gain further insights into the role of ICP0 and ICP4 for expression of the TK gene during virus infection, several mutants with deletions or point mutations in each of the transcriptional regulatory elements were generated starting at -109 and progressing toward +1. According to the CAT assay involving these mutants, the cellular transcription factor (CTF) binding site was necessary for efficient expression in the presence of transfected ICP0 and ICP4 or during virus infection, whereas the Sp1 binding site had a minor effect on ICP0-mediated TK expression. These results indicate that the immediate early proteins of HSV-1 regulate expression of the TK gene during virus infection by modulating activities of cellular transcription factors such as CTF.  (+info)

Expression, DNA-binding specificity and transcriptional regulation of nuclear factor 1 family proteins from rat. (8/466)

Nuclear factor 1 (NF1) family proteins, which are encoded by four different genes (NF1-A, NF1-B, NF1-C and NF1-X), bind to the palindromic sequence and regulate the expression of many viral and cellular genes. We have previously purified NF1-A and NF1-B from rat liver as factors that bind to the silencer in the glutathione transferase P gene, and have also reported the repression domain of NF1-A. In the present study we cloned five cDNA species (NF1-B1, NF1-B2, NF1-B3, NF1-C2 and NF1-X1) and compared their expression profiles and the affinity and specificity of the DNA binding of these NF1 family members. By Northern blot analysis, we found that the expression profiles of the NF1s are indistinguishable in the various tissues of the rat. The DNA-binding affinities of NF1-A and NF1-X are higher than those of NF1-B and NF1-C, whereas all four NF1 proteins showed the same DNA-binding specificity. Transfection analyses revealed that the function of NF1-B on the transcriptional regulation differed between NF1-B isoforms and was affected by the factor(s) that bind to the promoter regions. In addition, we identified the transcriptional regulatory domain of NF1-B, which is enriched with proline and serine residues.  (+info)