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At least 577 records · Page 32Linked to original sources
PubMed2001

Repressor element silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) can act as an enhancer as well as a repressor of corticotropin-releasing hormone gene transcription.

The repressor element-1/neuron-restrictive silencing element (RE-1/NRSE) mediates transcriptional repression by the repressor element silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF) in many neuron-specific genes. REST/NRSF is expressed most highly in non-neural tissues, where it is thought to repress gene transcription, but is also found in developing neurons and at low levels in the brain. Its null mutation in vivo results in embryonic lethality in mice. While the RE-1/NRSE-mediated repressive influence of REST/NRSF is well established, results in transgenic studies have suggested that the action of the system is more complex. Here, we report that transcription of the corticotropin releasing hormone (CRH) gene is regulated by REST/NRSF, in part through the RE-1/NRSE. Expression of transfected Crh-luciferase constructs was down-regulated by REST/NRSF in a RE-1/NRSE-dependent fashion in both muscle-derived L6 and REST/NRSF co-transfected neuronal PC12 cells. Treatment of L6 cells with trichostatin A revealed that REST/NRSF repression depends, in part, on histone deacetylase activity in these cells. In another neuronal cell line, NG108, REST/NRSF also repressed expression from constructs containing an intact RE-1/NRSE. However, unexpectedly, REST/NRSF up-regulated expression levels of constructs lacking an intact RE-1/NRSE. These results suggest that REST/NRSF can act as both a repressor of Crh transcription, via the Crh RE-1/NRSE, and an enhancer of Crh transcription, via a mechanism independent of the Crh RE-1/NRSE.

Animals↗
PubMed2001

The DNA glycosylase T:G mismatch-specific thymine DNA glycosylase represses thyroid transcription factor-1-activated transcription.

The transcription factor thyroid transcription factor-1 (TTF-1) is a homeodomain-containing protein that belongs to the NK2 family of genes involved in organogenesis. TTF-1 is required for normal development of the forebrain, lung, and thyroid. In a search for factors that regulate TTF-1 transcriptional activity, we isolated three genes (T:G mismatch-specific thymine DNA glycosylase (TDG), homeodomain-interacting protein kinase 2 (HIPK2), and Ajuba), whose products can interact with TTF-1 in yeast and in mammalian cells. TDG is an enzyme involved in base excision repair. In the present paper, we show that TDG acts as a strong repressor of TTF-1 transcriptional activity in a dose-dependent manner, while HIPK2 and Ajuba display no effect on TTF-1 activity, at least under the tested conditions. TDG-mediated inhibition occurs specifically on TTF-1-responsive promoters in thyroid and non thyroid cells. TDG associates with TTF-1 in mammalian cells through the TTF-1 carboxyl-terminal activation domain and is independent of the homeodomain. These findings reveal a previously unsuspected role for the repair enzyme TDG as a transcriptional repressor and open new routes toward the understanding of the regulation of TTF-1 transcriptional activity.

Animals↗
PubMed2002

Coordination of ATF6-mediated transcription and ATF6 degradation by a domain that is shared with the viral transcription factor, VP16.

ATF6 is a 670-amino acid endoplasmic reticulum (ER) transmembrane protein that is cleaved in response to ER stress. The resulting N-terminal fragment of approximately 400 amino acids translocates to the nucleus and activates selected ER stress-inducible genes, such as GRP-78 and sarco/endoplasmic reticulum ATPase, which are required for cell survival. In studying the mechanism of ATF6-activated transcription, we found that when HeLa cells were transfected with a plasmid encoding ATF6-(1-373), ER stress-inducible reporter gene activation was high, but ATF6-(1-373) expression was low, unless a proteasome inhibitor was added. In contrast, transfection with a plasmid encoding ATF6-(94-373) resulted in low reporter activation and high expression of ATF6-(94-373), which was independent of the proteasome inhibitor. Thus, the information responsible for transcriptional activation and proteasomal degradation must lie within the N-terminal 93 amino acids of ATF6. This portion of ATF6 was found to be homologous to the herpes simplex viral protein, VP16. One 8-amino acid domain of particular interest in this region of ATF6 is 75% identical to the VN8 region in VP16. VN8 is required for VP16-mediated transcription, as well as rapid degradation of VP16 by proteasomes. Point mutations in the VN8-like region of ATF6 caused a loss of transcription, increased expression levels, and an increase in half-life. Thus, the potent transcriptional activities and rapid degradation of ATF6 and VP16 require the VN8 domains in each protein. Homology searches indicate that ATF6 is the only eukaryotic protein known that possesses an active VN8 domain, raising questions about how this domain evolved and the functional importance underlying its appearance in only these two transcription factors.

Activating Transcription Factor 6↗
PubMed2002

Transforming growth factor-beta inhibits pulmonary surfactant protein B gene transcription through SMAD3 interactions with NKX2.1 and HNF-3 transcription factors.

Transforming growth factor-beta (TGF-beta) represses surfactant protein B (Sp-B) gene transcription through a mechanism that remains unknown. A homeodomain and a forkhead transcription factor, NKX2.1 and HNF-3, respectively, are known activators of Sp-B transcription. Because SMADs are the effectors of TGF-beta-induced gene activation, we examined the possibility that gene repression by TGF-beta may also occur through interactions of SMADs with NKX2.1 and HNF-3. We found that lung epithelial carcinoma H441 cells contain SMAD2/3 and -4, which localize to the nucleus in response to TGF-beta treatment. The activity of a transfected Sp-B promoter/reporter construct was reduced in a dose-dependent manner by TGF-beta. Cotransfection with a mutant, constitutively activated form of the Tgf-beta type I receptor repressed Sp-B promoter activity in the absence of TGF-beta ligand. Dominant negative mutants of Smads blocked the repressor activity of TGF-beta. SMAD3, but not SMAD2, mediated the repressor activity of TGF-beta on the Sp-B promoter. Mutations within a 70-base pair domain that includes binding sites for NKX2.1, hepatocyte nuclear factor 3 (HNF-3), or cAMP response element-binding protein (CREB) eliminated SMAD3-dependent repression of Sp-B transcription. Electrophoretic mobility shift analysis showed no evidence for direct binding of SMAD3 to the Sp-B promoter, and a DNA binding mutant of SMAD3 also repressed Sp-B, suggesting that direct DNA binding of SMAD3 may not be required. Using a mammalian two hybrid assay, we found physical and functional interactions between SMAD3 and both NKX2.1 and HNF-3. Also, a glutathione S-transferase-fused SMAD3 directly binds to in vitro synthesized NKX2.1 or HNF-3, demonstrating protein-protein interactions between SMAD3 and the two transcriptional factors. The DNA binding of NKX2.1 to Sp-B promoter was reduced in response to TGF-beta treatment, although expression of Nkx2.1 was not affected. We conclude that SMAD3 interactions with the positive regulators NKX2.1 and HNF-3 underlie the molecular basis for TGF-beta-induced repression of Sp-B gene transcription.

Activin Receptors, Type I↗
PubMed2002

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.

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.

Activating Transcription Factor 2↗
PubMed2003

Human PLU-1 Has transcriptional repression properties and interacts with the developmental transcription factors BF-1 and PAX9.

PLU-1 is a large (1544 amino acids) nuclear protein that is highly expressed in breast cancers and is proposed to function as a regulator of gene expression. A yeast two-hybrid screen using PLU-1 as bait has identified two unrelated PLU-1 interacting proteins, namely brain factor-1 (BF-1) and paired box 9 (PAX9), both of which are developmental transcription factors. BF-1 and PAX9 interact with PLU-1 via a novel conserved sequence motif (Ala-X-Ala-Ala-X-Val-Pro-X4-Val-Pro-X8-Pro, termed the VP motif), because deletion or site-directed mutagenesis of this motif in either protein abolishes PLU-1 interaction in vivo. In a reporter assay system, PLU-1 has potent transcriptional repression activity. BF-1 and PAX9 also represses transcription in the same assay, but co-expression of PLU-1 with BF-1 or PAX9 significantly enhances this repression. Mutation of the PLU-1 binding motifs in BF-1 and PAX9 abolishes the observed PLU-1 co-repression activity. These data support a role for PLU-1 acting as a transcriptional co-repressor of two unrelated developmental transcription factors. Because both BF-1 and PAX proteins interact with members of the groucho co-repressor family, it is plausible that PLU-1 has a role in groucho-mediated transcriptional repression.

Amino Acid Sequence↗
PubMed2004

TSG101 interacts with apoptosis-antagonizing transcription factor and enhances androgen receptor-mediated transcription by promoting its monoubiquitination.

Apoptosis-antagonizing transcription factor (AATF), also termed Che-1, was identified as interacting protein of Dlk/ZIP kinase and RNA polymerase II, respectively. Che-1 has additionally been shown to bind Rb, thereby activating transcription factor E2F and promoting cell cycle progression. Moreover, AATF enhances steroid receptor-mediated transactivation in a hormone- and dose-dependent manner (Leister, P., Burgdorf, S., and Scheidtmann, K. H., (2003) Signal Transduction 3, 18-25). These data suggest that AATF exerts its functions through interaction with different transcription factors. In search of novel interaction partners of AATF, we identified the tumor susceptibility gene product TSG101, which had also been recognized as a co-regulator of nuclear hormone receptors. Interestingly, TSG101 and AATF functioned as cooperative coactivators in androgen receptor-mediated transcription. Because TSG101 was also shown to play a role in regulation of ubiquitin conjugation, we asked whether its coactivating function might be linked to ubiquitination. Indeed, TSG101 enhanced monoubiquitination of the androgen receptor in a ligand-dependent manner, and this correlated with enhanced transactivating capacity. Furthermore, a dominant-negative mutant of ubiquitin preventing polyubiquitination also stimulated androgen receptor-mediated transcription, which in this case could not be enhanced by TSG101. We propose that TSG101 activates androgen receptor-induced transcription by transient stabilization of the monoubiquitinated state, thus revealing a novel regulatory mechanism for nuclear receptors.

Amino Acid Motifs↗
PubMed2006

Human Mediator enhances basal transcription by facilitating recruitment of transcription factor IIB during preinitiation complex assembly.

The multisubunit Mediator is a well established transcription coactivator for gene-specific activators. However, recent studies have shown that, although not essential for basal transcription by purified RNA polymerase II (pol II) and general initiation factors, Mediator is essential for basal transcription in nuclear extracts that contain a more physiological complement of factors (Mittler, G., Kremmer, E., Timmers, H. T., and Meisterernst, M. (2001) EMBO Rep. 2, 808-813; Baek, H. J., Malik, S., Qin, J., and Roeder, R. G. (2002) Mol. Cell. Biol. 22, 2842-2852). Here, mechanistic studies with immobilized DNA templates, purified factors, and factor-depleted HeLa extracts have shown (i) that Mediator enhancement of basal transcription correlates with Mediator-dependent recruitment of pol II and general initiation factors (transcription factor (TF) IIB and TFIIE) to the promoter; (ii) that Mediator and TFIIB, which both interact with pol II, are jointly required for pol II recruitment to the promoter and that TFIIB recruitment is Mediator-dependent, whereas Mediator recruitment is TFIIB-independent; (iii) that a high level of TFIIB can bypass the Mediator requirement for basal transcription and pol II recruitment in nuclear extract, thus indicating a conditional restriction of TFIIB function and a key role of Mediator in overcoming this restriction; and (iv) that an earlier rate-limiting step involves formation of a TFIID-Mediator-promoter complex. These results support a stepwise assembly model, rather than a preformed holoenzyme model, for Mediator-dependent assembly of a basal preinitiation complex and, more important, identify a step involving TFIIB as a key site of action of Mediator.

Binding Sites↗
PubMedDate not supplied

Regulation of globin gene transcription by heme in erythroleukemia cells: analysis of putative heme regulatory motifs in the p45 NF-E2 transcription factor.

The function of the NF-E2 transcription factor, a p45/small Maf heterodimer, was analyzed in the erythroleukemia cell lines MEL and CB3. In contrast to MEL cells, CB3 cells are null for p45 and thus express only extremely low levels of adult globin transcripts upon induction by agents promoting erythroid differentiation. We investigated the response of erythroleukemia cells to hemin treatment. Hemin rapidly induces beta-globin gene transcript levels in MEL cells, but not in CB3 cells. Stable expression of the large p45 NF-E2 subunit in CB3 cells restores hemin mediated beta-globin gene transcription, suggesting that the presence of a functional NF-E2 is required for strong induction of beta-globin mRNA levels by hemin in erythroleukemia cells. We performed mutagenesis of two potential heme-regulatory motifs (HRMs) in p45 NF-E2 and found that the mutated versions are expressed and can still recognize a NF-E2 DNA binding element. In addition, we showed that p45 NF-E2 HRM mutants are able to restore beta-globin gene transcription in CB3 cells upon induction by hemin. Our results suggest that globin gene activation by heme appears to be independent of the putative HRMs in the p45 subunit of the NF-E2 transcription factor.

Animals↗
PubMed1999

The interferon-inducible nucleolar p204 protein binds the ribosomal RNA-specific UBF1 transcription factor and inhibits ribosomal RNA transcription.

p204, a member of the interferon-inducible p200 family of murine proteins, is primarily nucleolar. We generated cell lines in which p204 was inducible by muristerone. This induction resulted in retardation of cell proliferation and inhibition of rRNA transcription in vivo. Interferon treatment, resulting in p204 induction and retardation of proliferation, also caused inhibition of rRNA transcription in vivo. p204 also inhibited rRNA transcription in vitro. This inhibition was overcome by addition of UBF1, the rRNA-specific transcription factor. A direct interaction between p204 and UBF1 was revealed in vitro in pull-down assays, and in vivo by co-immunoprecipitation from cell extracts. UBF1 bound strongly to at least two regions of p204: the N-terminal segment linked to the conserved 200 amino acid a segment, and the conserved 200 amino acid b segment. Cleavage of the a or b segments into two segments (encoded by single exons) resulted in a strong decrease or loss of binding. The inhibition of rRNA transcription by p204 may be due to the inhibition by p204 of the specific DNA binding of UBF1. This was revealed in electrophoretic mobility shift, magnetic bead and footprinting assays. Thus, p204 serves as a mediator of the inhibition of rRNA transcription by interferon.

Animals↗
PubMed1991

Helix-loop-helix transcription factor E47 activates germ-line immunoglobulin heavy-chain gene transcription and rearrangement in a pre-T-cell line.

E47 is a helix-loop-helix transcription factor that binds to sites in the immunoglobulin heavy-chain and kappa light-chain gene enhancers. Other proteins of this type are involved in cell-type determination. A possible role for E47 in B-cell development was tested by overexpressing a cDNA encoding E47 in the pre-T-cell line 2017. We found a dramatic activation of a germ-line heavy-chain gene transcript in these stable transfectants and an equally large induction of immunoglobulin D-to-J rearrangement, the first recognized step in B-cell development. Germ-line kappa light-chain gene transcription and rearrangement were unaffected, but transcription of the recombination-activating genes RAG-1 and RAG-2 and the lymphoid-specific transcription factor Oct-2 was increased. These T cells did not transcribe their rearranged DJ alleles, however, and failed to progress to the next stage of heavy-chain gene assembly, V-to-DJ rearrangement. Because transcription factor E47 can induce pre-T cells to carry out events of B-cell differentiation, it may be a crucial determinant of the earliest stages of B-cell development.

Animals↗
PubMed2000

Role of the TATA binding protein-transcription factor IIB interaction in supporting basal and activated transcription in plant cells.

The TATA binding protein (TBP) and transcription factor IIB (TFIIB) play crucial roles in transcription of class II genes. The requirement for TBP-TFIIB interactions was evaluated in maize cells by introducing mutations into the Arabidopsis TBP (AtTBP2) within the C-terminal stirrup. Protein binding experiments indicated that amino acid residues E-144 and E-146 of AtTBP2 are both essential for TFIIB binding in vitro. Activation domains derived from herpes simplex viral protein VP16, the Drosophila fushi tarazu glutamine-rich domain (ftzQ), and yeast Gal4 were tested in transient assays. TBP-TFIIB interactions were dispensable for basal transcription but were required for activated transcription. In general, activated transcription was more severely inhibited by TBP mutation E-146R than by mutation E-144R. However, these TBP mutations had little effect on activity of the full-length cauliflower mosaic virus 35S and maize ubiquitin promoters, thus demonstrating that strong TBP-TFIIB contacts are not always required for transcription driven by complex promoters.

Animals↗
PubMed1995

Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase II-specific role in basal and activated transcription.

To probe the structure and function of the Saccharomyces cerevisiae general transcription factor TFIIA, we have systematically mutagenized the genes encoding both subunits and analyzed the effects of the mutations both in vivo and in vitro. We found that the central nonconserved region of the large subunit is not essential for function and likely acts as a spacer between the conserved N- and C-terminal regions. Deletion mutagenesis of the large subunit defined a region which is required for TATA binding protein (TBP) interaction. Alanine scanning mutagenesis defined a cluster of four basic residues which are likely required for interaction with DNA in the TBP-DNA complex. Much of the conserved regions of both subunits is required for subunit association, suggesting that these conserved regions fold into compact domains which extensively interact. In vitro transcription performed with extracts from yeast strains with mutations in either the large or the small TFIIA subunit demonstrated that TFIIA stimulates both basal and activated polymerase II (Pol II) transcription. The TFIIA-depleted extracts have normal Pol I and Pol III transcription activity, showing that TFIIA is a Pol II-specific factor. In vivo depletion of TFIIA activity reduced transcription from four different Pol II promoters. Finally, alanine scanning mutagenesis of TFIIA's small subunit has identified at least one mutation which is defective in transcription but which is not defective in subunit association or binding to TBP or TBP-DNA complexes.

Alanine↗
PubMed1999

Paired-homeodomain transcription factor PAX4 acts as a transcriptional repressor in early pancreatic development.

The paired-homeodomain transcription factor PAX4 is expressed in the developing pancreas and along with PAX6 is required for normal development of the endocrine cells. In the absence of PAX4, the numbers of insulin-producing beta cells and somatostatin-producing delta cells are drastically reduced, while the numbers of glucagon-producing alpha cells are increased. To gain insight into PAX4 function, we cloned a full-length Pax4 cDNA from a beta-cell cDNA library and identified a bipartite consensus DNA binding sequence consisting of a homeodomain binding site separated from a paired domain binding site by 15 nucleotides. The paired half of this consensus sequence has similarities to the PAX6 paired domain consensus binding site, and the two proteins bind to common sequences in several islet genes, although with different relative affinities. When expressed in an alpha-cell line, PAX4 represses transcription through the glucagon or insulin promoters or through an isolated PAX4 binding site. This repression is not simply due to competition with the PAX6 transcriptional activator for the same binding site, since PAX4 fused to the unrelated yeast GAL4 DNA binding domain also represses transcription through the GAL4 binding site in the alpha-cell line and to a lesser degree in beta-cell lines and NIH 3T3 cells. Repressor activity maps to more than one domain within the molecule, although the homeodomain and carboxyl terminus give the strongest repression. PAX4 transcriptional regulation apparently plays a role only early in islet development, since Pax4 mRNA as determined by reverse transcriptase PCR peaks at embryonic day 13.5 in the fetal mouse pancreas and is undetectable in adult islets. In summary, PAX4 can function as a transcriptional repressor and is expressed early in pancreatic development, which may allow it to suppress alpha-cell differentiation and permit beta-cell differentiation.

3T3 Cells↗
PubMed1997

PDP1, a novel Drosophila PAR domain bZIP transcription factor expressed in developing mesoderm, endoderm and ectoderm, is a transcriptional regulator of somatic muscle genes.

In vertebrates, transcriptional control of skeletal muscle genes during differentiation is regulated by enhancers that direct the combinatorial binding and/or interaction of MEF2 and the bHLH MyoD family of myogenic factors. We have shown that Drosophila MEF2 plays a role similar to its vertebrate counterpart in the regulation of the Tropomyosin I gene in the development of Drosophila somatic muscles, however, unlike vertebrates, Drosophila MEF2 interacts with a muscle activator region that does not have binding sites for myogenic bHLH-like factors or any other known Drosophila transcription factors. We describe here the isolation and characterization of a component of the muscle activator region that we have named PDP1 (PAR domain protein 1). PDP1 is a novel transcription factor that is highly homologous to the PAR subfamily of mammalian bZIP transcription factors HLF, DBP and VBP/TEF. This is the first member of the PAR subfamily of bZIP transcription factors to be identified in Drosophila. We show that PDP1 is involved in regulating expression of the Tropomyosin I gene in somatic body-wall and pharyngeal muscles by binding to DNA sequences within the muscle activator that are required for activator function. Mutations that eliminate PDP1 binding eliminate muscle activator function and severely reduce expression of a muscle activator plus MEF2 mini-enhancer. These and previous results suggest that PDP1 may function as part of a larger protein/DNA complex that interacts with MEF2 to regulate transcription of Drosophila muscle genes. Furthermore, in addition to being expressed in the mesoderm that gives rise to the somatic muscles, PDP1 is also expressed in the mesodermal fat body, the developing midgut endoderm, the hindgut and Malpighian tubules, and the epidermis and central nervous system, suggesting that PDP1 is also involved in the terminal differentiation of these tissues.

Amino Acid Sequence↗
PubMed1999

3-D organization of ribosomal transcription units after DRB inhibition of RNA polymerase II transcription.

In each bead of the nucleolar necklace, using adenosine analog DRB-treated PtK1 cells, we investigated the three components of rDNA transcription, i.e. the gene, transcription factor UBF and transcripts. In situ hybridization revealed the unraveling and 3-D dispersion of most of the rDNA coding sequences within the nucleus. The signals were small, of similar intensity and tandemly organized in the necklace. This observation is compatible with the fact that they might correspond to single gene units. Active transcription was visualized in these units, demonstrating that they were active functional units. Transcript labeling was not similar for each unit, contrary to UBF labeling. UBF and rRNA transcripts were only partially colocalized, as demonstrated by 3-D image analysis and quantification. As visualized by electron microscopy, the necklace was composed of a small fibrillar center partially surrounded by a dense fibrillar component. The 3-D arrangement of this individual unit in the necklace, investigated both by confocal and electron microscopy in the same cells, showed that the individual beads were linked by a dense fibrillar component. The reversibility of this organization after removal of DRB indicated that the beads in the necklace are certainly the elementary functional domain of the nucleolus. In addition, these results lead us to suggest that the organization of a functional domain, presumably corresponding to a single gene, can be studied by in situ approaches.

Animals↗
PubMed1999

The mitotically phosphorylated form of the transcription termination factor TTF-1 is associated with the repressed rDNA transcription machinery.

The transcription termination factor TTF-1 exerts two functions in ribosomal gene (rDNA) transcription: facilitating initiation and mediating termination of transcription. Using HeLa cells, we show that TTF-1 protein is colocalized with the active transcription machinery in the nucleolus and also with the inactive machinery present in certain mitotic nucleolar organizer regions (NORs) when rDNA transcription is repressed. We also show that TTF-1 is specifically phosphorylated during mitosis in a manner dependent on the cdc2-cyclin B kinase pathway and on an okadaic acid-sensitive phosphatase. Interestingly, the mitotically phosphorylated form of TTF-1 appearing at the G(2)/M transition phase was more easily solubilized than was the interphase form. This indicates that the chromatin-binding affinity of TTF-1 appears to be different in mitotic chromosomes compared to the interphase nucleolus. Correlated with this, the other DNA-binding factor, UBF, which interferes with chromatin conformation in the rDNA promoter, was more strongly bound to rDNA during mitosis than at interphase. The reorganization of the mitotic rDNA promoter might be induced by phosphorylation of certain components of the rDNA transcription machinery and participate in silencing of rDNA during mitosis.

Antibody Specificity↗
PubMed2005

Transcriptional regulation of neuronal genes and its effect on neural functions: transcriptional regulation of neuropeptide Y gene by leptin and its effect on feeding.

Leptin is an adipose tissue-derived secretory hormone that suppresses appetite by inhibition of neuropepeptide Y (NPY) gene expression in arcuate nucleus (ARC) in the hypothalamus. To investigate the transcriptional regulation of NPY gene by leptin, we carried out a luciferase assay using NPY gene promotor plasmid (NPY-luc) in NPY expressing cells such as N18TG2, NG108-15, and PC12 cells. In these cells, the NPY gene was transactivated by leptin through activation of leptin receptor. Leptin-induced transactivation was mediated through the 221-bp region of the NPY gene promotor, which possesses two putative STAT3 binding sites. To investigate the mechanism of in vivo suppression of NPY gene transcription in ARC by leptin, the effect of SOCS members on the leptin-induced transactivation of NPY gene was studied. In vivo SOCS2 and SOCS3 mRNAs were induced in mouse hypothalamus by leptin. Although leptin (125 ng/ml) induced significant increase in NPY gene transcriptional activity in mock-transfected cells, the leptin-induced NPY gene transcriptional activity was completely abolished in SOCS3-transfected cells. SOCS3 also suppressed the basal NPY gene transcription. These finding suggested that leptin inhibits NPY gene transcription in the hypothalamus in vivo and SOCS3 is a negative regulator of the NPY gene.

Animals↗