Isolation of genes encoding novel transcription factors which interact with the Hap complex from Aspergillus species. (65/416)

The Saccharomyces cerevisiae CCAAT-binding factor is composed of four subunits Hap2p, Hap3p, Hap4p and Hap5p. Three subunits, Hap2/3/5p, are required for DNA-binding and Hap4p is involved in transcriptional activation. Although homologues of Hap2/3/5p (in the case of Aspergillus nidulans; HapB/C/E, respectively) were found in many eukaryotes, no Hap4p homologues have been found except for the other yeast, Kluyveromyces lactis. With the lexA-hap2, -hapB, -hapC, or -hapE fusion gene, we evaluated the ability of interaction between Aspergillus Hap subunits and S. cerevisiae Hap4p subunit in S. cerevisiae. Using the system with lexA-hapB, a gene encoding a novel transcriptional activator, which interacted with the Hap complex, was isolated from A. nidulans and designated hapX.  (+info)

p38 MAPK-mediated transcriptional activation of inducible nitric-oxide synthase in glial cells. Roles of nuclear factors, nuclear factor kappa B, cAMP response element-binding protein, CCAAT/enhancer-binding protein-beta, and activating transcription factor-2. (66/416)

Previous studies have shown that mitogen-activated protein kinase (MAPK) cascades signal the induction of inducible nitric-oxide synthase (iNOS) in glial cells (Bhat, N. R., Zhang, P., Lee, J. C., and Hogan E. L. (1998) J. Neurosci. 18, 1633-1641; Bhat, N. R., Zhang, P., and Bhat, A. N. (1999) J. Neurochem. 72, 472-478). This study further investigates the role of p38 MAPK in the transcriptional activation of the iNOS gene by transient transfection with constitutively active upstream kinases in the pathway (i.e. MAPK kinase 3 (MKK3b(E)) and MAPK kinase 6 (MKK6b(E)). Expression in C-6 glial cells of either MKK3b(E) or MKK6b(E) resulted in an induction of the activity of a cotransfected rat iNOS promoter-reporter (iNOS-luciferase (Luc)) gene and an enhancement of cytokine-induced expression of iNOS mRNA, both of which were inhibitable by the p38 MAPK inhibitor SB203580. The MKK constructs also induced cAMP response element-mediated (CRE-Luc) and nuclear factor kappa B-dependent (nuclear factor kappa B-Luc) transcriptional activities. Transfection with dominant negative (dn) forms of CRE-binding protein (CREB) and CCAAT/enhancer-binding protein (C/EBP), the two CRE-binding transcription factors targeted by the p38 MAPK pathway, resulted in opposite effects; dnCREB enhanced and dnC/EBP inhibited iNOS-Luc parallel to their effects on CRE-Luc. In addition, the induction, by MKK3b(E) and MKK6b(E), of iNOS promoter activity was enhanced by a wild-type activating transcription factor (ATF-2), whereas a phosphorylation-defective form of ATF-2 had a suppressive effect. The results of these molecular studies provide evidence for an important role for the p38 MAPK pathway in the transcriptional activation of the iNOS gene in rat glial cells involving the transcription factors nuclear factor kappa B, C/EBP, and ATF-2.  (+info)

Ecteinascidin-743 inhibits activated but not constitutive transcription. (67/416)

Ecteinascidin-743 (ET-743) is a promising chemotherapeutic agent currently in Phase III clinical trials. Previous studies indicated a novel spectrum of activity for this agent, including transcriptional inhibition. Initially hypothesized to target a single transcription factor (NF-Y), we now show that ET-743 is a more general inhibitor of activated transcription. Induction of the Sp1-regulated p21 gene by Trichostatin A (TSA) was blocked by ET-743 at concentrations that had minimal effect on uninduced (constitutive) expression. Moreover, ET-743 blocked induction of Gal4 fusion proteins by TSA without affecting activation mediated by the fusion proteins in the absence of the inducer. Finally, microarray analysis of SW620 cells treated with TSA and/or ET-743 indicated that activation of TSA-responsive promoters was blocked by ET-743 with little affect on nonresponsive promoters. These results, taken together with previous reports, leads us to suggest a mechanism whereby ET-743 is a novel, potent, and general inhibitor of activated but not uninduced transcription.  (+info)

Transcriptional regulation of myeloid differentiation primary response (MyD) genes during myeloid differentiation is mediated by nuclear factor Y. (68/416)

To understand the molecular mechanism by which interleukin-6 (IL-6) regulates myeloid differentiation primary response (MyD) genes at the onset of M1 myeloid differentiation, we used JunB as a representative MyD gene to isolate and characterize IL-6 responsive elements. An IL-6 responsive element was localized between -65 and -52 of the JunB promoter (-65/-52 IL-6RE). By using antibody and oligonucleotide competition assays in electrophoretic mobility shift assay experiments, we have shown that the heterotrimeric transcription nuclear factor Y (NF-Y) complex binds to this element. A dominant-negative form of NF-YA, ectopically expressed in M1 cells, blocked NF-Y binding to the -65/-52 IL-6RE and reduced induction of JunB by IL-6. Furthermore, inhibition of NF-Y binding also reduced MyD gene induction by IL-6 and dampened the IL-6-induced M1 differentiation program. These findings are consistent with the observation that most MyD genes contain intact NF-Y binding motifs in their promoter regions. In contrast to M1 cells, during myeloid differentiation of bone marrow (BM), there was induction of NF-Y binding to the -65/-52 IL-6RE. This induced binding can be attributed to the observed induction of NF-YA protein expression and may reflect the molecular mechanism that couples proliferation to terminal differentiation of normal myeloblasts. Similar to M1 cells, blocking NF-Y binding in BM resulted in a reduction in mature macrophages. It can be concluded that NF-Y plays a role in the transcriptional regulation of MyD genes and is required for optimum myeloid differentiation.  (+info)

AML1 stimulates G1 to S progression via its transactivation domain. (69/416)

Inhibition of AML1-mediated transactivation potently slows G1 to S cell cycle progression. In Ba/F3 cells, activation of exogenous AML1 (RUNX1)-ER with 4-hydroxytamoxifen prevents inhibition of G1 progression mediated by CBFbeta-SMMHC, a CBF oncoprotein. We expressed three AML1-ER variants with CBFbeta-SMMHC in Ba/F3 cells. In these lines, CBFbeta-SMMHC expression is regulated by the zinc-responsive metallothionein promoter. Deletion of 72 AML1 C-terminal residues, which includes a transrepression domain, did not alter the activity of AML1-ER, whereas further deletion of 98 residues, removing the most potent AML1 transactivation domain (TAD), prevented rescue of cell cycle inhibition. Notably, the two variants which did not stimulate G1 exacerbated CBFbeta-SMMHC-mediated cell cycle arrest, suggesting that they dominantly inhibit AML1 activities. In addition, the two variants which stimulated G1 also induced apoptosis in 5-15% of the cells, an effect consistent with excessive G1 stimulation. These observations indicate that AML1 activates transcription of one or more genes critical for the G1 to S transition via its C-terminal transactivation domain. Inactivation of AML in acute leukemia is expected to slow proliferation unless additional genetic alterations co-exist which accelerate G1.  (+info)

Major histocompatibility complex class II transcriptional platform: assembly of nuclear factor Y and regulatory factor X (RFX) on DNA requires RFX5 dimers. (70/416)

Major histocompatibility complex class II (MHC-II) genes are regulated in a B-cell-specific and gamma interferon-inducible manner. Conserved upstream sequences (CUS) in their compact promoters bind nuclear factor Y (NFY) and regulatory factor X (RFX) complexes. These DNA-bound proteins form a platform that attracts the class II transactivator, which initiates and elongates MHC-II transcription. In this report, we analyzed the complex assembly of these DNA-bound proteins. First, we found that NFY can interact with RFX in cells. In particular, NFYA and NFYC bound RFXANK/B in vitro. Next, RFX5 formed dimers in vivo and in vitro. Within a leucine-rich stretch N-terminal to the DNA-binding domain in RFX5, the leucine at position 66 was found to be critical for this self-association. Mutant RFX5 proteins that could not form dimers also did not support the formation of higher-order DNA-protein complexes on CUS in vitro or MHC-II transcription in vivo. We conclude that the MHC-II transcriptional platform begins to assemble off CUS and then binds DNA via multiple, spatially constrained interactions. These findings offer one explanation of why in the Bare Lymphocyte Syndrome, which is a congenital severe combined immunodeficiency, MHC-II promoters are bare when any subunit of RFX is mutated or missing.  (+info)

Spatially specific expression of Hoxb4 is dependent on the ubiquitous transcription factor NFY. (71/416)

Understanding how boundaries and domains of Hox gene expression are determined is critical to elucidating the means by which the embryo is patterned along the anteroposterior axis. We have performed a detailed analysis of the mouse Hoxb4 intron enhancer to identify upstream transcriptional regulators. In the context of an heterologous promoter, this enhancer can establish the appropriate anterior boundary of mesodermal expression but is unable to maintain it, showing that a specific interaction with its own promoter is important for maintenance. Enhancer function depends on a motif that contains overlapping binding sites for the transcription factors NFY and YY1. Specific mutations that either abolish or reduce NFY binding show that it is crucial for enhancer activity. The NFY/YY1 motif is reiterated in the Hoxb4 promoter and is known to be required for its activity. As these two factors are able to mediate opposing transcriptional effects by reorganizing the local chromatin environment, the relative levels of NFY and YY1 binding could represent a mechanism for balancing activation and repression of Hoxb4 through the same site.  (+info)

CCAAT binding factor (CBF) binding mediates cell cycle activation of topoisomerase IIalpha. Conventional CBF activation domains are not required. (72/416)

To understand the role of the CCAAT binding factor (CBF) in transcription during the cell cycle, we studied the mouse topoisomerase II alpha (topo II alpha) promoter, which is activated during the late S and G(2)/M phases of the cell cycle and contains multiple CBF binding sites. Mutational analysis of the promoter shows that CBF binding to an inverted orientation of the CCAAT motif in the topo II alpha promoter, but not to a direct orientation, is required for transcription activation during the cell cycle. In contrast, analysis of the promoter in an in vitro reconstituted transcription system shows that CBF activates transcription of the topo II alpha promoter irrespective of the orientation of the CBF binding sites. This analysis demonstrates that only one of the three transcription start sites of the topo II alpha promoter is stimulated by CBF, indicating that transcription activation by CBF is dependent on basal promoter structure. Interestingly, mutations of the start site that abolish CBF-dependent transcription activation in vitro do not inhibit activation of the promoter during the cell cycle. Consistent with this observation, expression of a truncated CBF-B subunit lacking a transcription activation domain, which inhibits activity of a collagen promoter, does not affect activity of the topo II alpha promoter in fibroblast cells. In contrast, expression of an allele-specific CBF-B mutant that binds high affinity to a mutant CBF binding site containing a CCAAC motif revives transcription activation of an inactive mutant topo II alpha promoter containing CCAAC during the cell cycle. Altogether, this study indicates that CBF binding, but not conventional CBF activation domains, are required for activation of the topo II alpha promoter during the cell cycle. Considering these results together with results of another recent study, we hypothesize that binding of CBF that disrupts the nucleosomal structure in the topo II alpha promoter is a major function of CBF by which it regulates the cell cycle-dependent transcription of the topo II alpha promoter and possibly many other cell cycle-regulated promoters containing CBF binding sites.  (+info)