Characterization of motifs which are critical for activity of the cyclic AMP-responsive transcription factor CREB

1991 ◽  
Vol 11 (3) ◽  
pp. 1306-1312
Author(s):  
G A Gonzalez ◽  
P Menzel ◽  
J Leonard ◽  
W H Fischer ◽  
M R Montminy
Keyword(s):  
Transcription Factor ◽  
Cyclic Amp ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Transactivation Domain ◽  
Hormonal Stimulation ◽  
Eukaryotic Genes ◽  
Teratocarcinoma Cells ◽  
A Site ◽  
F9 Teratocarcinoma

Cyclic AMP mediates the hormonal stimulation of a number of eukaryotic genes by directing the protein kinase A (PK-A)-dependent phosphorylation of transcription factor CREB. We have previously determined that although phosphorylation at Ser-133 is critical for induction, this site does not appear to participate directly in transactivation. To test the hypothesis that CREB ultimately activates transcription through domains that are distinct from the PK-A site, we constructed a series of CREB mutants and evaluated them by transient assays in F9 teratocarcinoma cells. Remarkably, a glutamine-rich region near the N terminus appeared to be important for PK-A-mediated induction of CREB since removal of this domain caused a marked reduction in CREB activity. A second region consisting of a short acidic motif (DLSSD) C terminal to the PK-A site also appeared to synergize with the phosphorylation motif to permit transcriptional activation. Biochemical experiments with purified recombinant CREB protein further demonstrate that the transactivation domain is more sensitive to trypsin digestion than are the DNA-binding and dimerization domains, suggesting that the activator region may be structured to permit interactions with other proteins in the RNA polymerase II complex.

scholarly journals Characterization of motifs which are critical for activity of the cyclic AMP-responsive transcription factor CREB.

1991 ◽  
Vol 11 (3) ◽  
pp. 1306-1312 ◽  
Author(s):  
G A Gonzalez ◽  
P Menzel ◽  
J Leonard ◽  
W H Fischer ◽  
M R Montminy
Keyword(s):  
Transcription Factor ◽  
Cyclic Amp ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Transactivation Domain ◽  
Hormonal Stimulation ◽  
Eukaryotic Genes ◽  
Teratocarcinoma Cells ◽  
A Site ◽  
F9 Teratocarcinoma

Cyclic AMP mediates the hormonal stimulation of a number of eukaryotic genes by directing the protein kinase A (PK-A)-dependent phosphorylation of transcription factor CREB. We have previously determined that although phosphorylation at Ser-133 is critical for induction, this site does not appear to participate directly in transactivation. To test the hypothesis that CREB ultimately activates transcription through domains that are distinct from the PK-A site, we constructed a series of CREB mutants and evaluated them by transient assays in F9 teratocarcinoma cells. Remarkably, a glutamine-rich region near the N terminus appeared to be important for PK-A-mediated induction of CREB since removal of this domain caused a marked reduction in CREB activity. A second region consisting of a short acidic motif (DLSSD) C terminal to the PK-A site also appeared to synergize with the phosphorylation motif to permit transcriptional activation. Biochemical experiments with purified recombinant CREB protein further demonstrate that the transactivation domain is more sensitive to trypsin digestion than are the DNA-binding and dimerization domains, suggesting that the activator region may be structured to permit interactions with other proteins in the RNA polymerase II complex.

Crystal structure of the human transcription elongation factor DSIF hSpt4 subunit in complex with the hSpt5 dimerization interface

2009 ◽  
Vol 425 (2) ◽  
pp. 373-380 ◽  
Author(s):  
Sabine Wenzel ◽  
Berta M. Martins ◽  
Paul Rösch ◽  
Birgitta M. Wöhrl
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Structural Similarity ◽  
Transcription Elongation Factor

The eukaryotic transcription elongation factor DSIF [DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) sensitivity-inducing factor] is composed of two subunits, hSpt4 and hSpt5, which are homologous to the yeast factors Spt4 and Spt5. DSIF is involved in regulating the processivity of RNA polymerase II and plays an essential role in transcriptional activation of eukaryotes. At several eukaryotic promoters, DSIF, together with NELF (negative elongation factor), leads to promoter-proximal pausing of RNA polymerase II. In the present paper we describe the crystal structure of hSpt4 in complex with the dimerization region of hSpt5 (amino acids 176–273) at a resolution of 1.55 Å (1 Å=0.1 nm). The heterodimer shows high structural similarity to its homologue from Saccharomyces cerevisiae. Furthermore, hSpt5-NGN is structurally similar to the NTD (N-terminal domain) of the bacterial transcription factor NusG. A homologue for hSpt4 has not yet been found in bacteria. However, the archaeal transcription factor RpoE” appears to be distantly related. Although a comparison of the NusG-NTD of Escherichia coli with hSpt5 revealed a similarity of the three-dimensional structures, interaction of E. coli NusG-NTD with hSpt4 could not be observed by NMR titration experiments. A conserved glutamate residue, which was shown to be crucial for dimerization in yeast, is also involved in the human heterodimer, but is substituted for a glutamine residue in Escherichia coli NusG. However, exchanging the glutamine for glutamate proved not to be sufficient to induce hSpt4 binding.

Manipulating Higher Order Chromatin Structure of the β-Globin Locus by Targeted Tethering of a “looping” Factor

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 647-647
Author(s):  
Wulan Deng ◽  
Philip D Gregory ◽  
Andreas Reik ◽  
Gerd Blobel
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Conflicts Of Interest ◽  
Chromatin Loop ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Loop Formation ◽  
Pol Ii ◽  
Globin Promoter

Abstract Abstract 647 The mammalian β-globin locus is under the coordinated control of multiple transcription factors to ensure the correct expression of the globin genes during development. The distal β-globin locus control region (LCR) physically interacts with β-like globin promoters to form developmentally dynamic chromatin loops. The hematopoietic transcription factor GATA-1 and its associated cofactor Ldb1 bind to the LCR and the β-globin promoter and are essential for loop formation and β-globin expression. However, the molecular basis of chromatin looping and its cause-effect relationship with transcriptional activation are unclear. Here, we examined whether Ldb1 is an effector of GATA-1 during loop formation. Specifically, we tested whether artificial tethering of Ldb1 to the endogenous β-globin promoter and LCR can substitute for GATA-1 function. Ldb1 was fused to artificial zinc finger proteins (ZFP) designed to bind to the LCR and β-globin promoter. Ldb1-ZFPs were introduced pairwise into murine GATA-1 null erythroid cells in which the β-globin locus is relaxed and transcriptionally silent. In vivo binding of the Ldb1-ZFPs to their targets was verified by ChIP assay. Strikingly, expression of Ldb1-ZFPs but not Ldb1 or ZFPs alone led to substantial activation of β-globin transcription in the absence of GATA-1. Moreover, chromosome conformation capture experiments showed that Ldb1-ZFPs triggered physical association between the LCR and the β-globin promoter. Recruitment of RNA polymerase II (Pol II) and its phosphorylation at serine 5 are critical LCR-dependent regulatory steps in β-globin transcription. We found that Ldb1-ZFP expression facilitated Pol II recruitment at the β-globin promoter and serine 5 phosphorylation to the same level as GATA-1-expressing erythroid cells. This is consistent with an Ldb1-ZFP-induced LCR-β-globin promoter chromatin loop. In concert, these results indicate that Ldb1 is a critical effector for GATA-1 by mediating enhancer-promoter loops. In broader terms, our results suggest that chromatin loop formation can be sufficient for gene activation in the absence of an essential transcription factor. We are currently in the process of examining whether targeting of the LCR to embryonic and fetal globin genes can be used to activate them in adult cells. Targeted chromatin loop formation may provide a method to activate fetal or adult hemoglobin expression in individuals with β-thalassemia or sickle cell anemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Paired-Domain Transcription Factor Pax8 Binds to the Upstream Enhancer of the Rat Sodium/Iodide Symporter Gene and Participates in Both Thyroid-Specific and Cyclic-AMP-Dependent Transcription

1999 ◽  
Vol 19 (3) ◽  
pp. 2051-2060 ◽  
Author(s):  
Makoto Ohno ◽  
Mariastella Zannini ◽  
Orlie Levy ◽  
Nancy Carrasco ◽  
Roberto di Lauro
Keyword(s):  
Transcription Factor ◽  
Cyclic Amp ◽  
Transcriptional Activation ◽  
Mutational Analysis ◽  
Thyroid Stimulating Hormone ◽  
Dependent Manner ◽  
Sodium Iodide Symporter ◽  
Paired Domain ◽  
Gene Encoding ◽  
Responsive Element

ABSTRACT The gene encoding the Na/I symporter (NIS) is expressed at high levels only in thyroid follicular cells, where its expression is regulated by the thyroid-stimulating hormone via the second messenger, cyclic AMP (cAMP). In this study, we demonstrate the presence of an enhancer that is located between nucleotides −2264 and −2495 in the 5′-flanking region of the NIS gene and that recapitulates the most relevant aspects of NIS regulation. When fused to either its own or a heterologous promoter, the NIS upstream enhancer, which we call NUE, stimulates transcription in a thyroid-specific and cAMP-dependent manner. The activity of NUE depends on the four most relevant sites, identified by mutational analysis. The thyroid-specific transcription factor Pax8 binds at two of these sites. Mutations that interfere with Pax8 binding also decrease transcriptional activity of the NUE. Furthermore, expression of Pax8 in nonthyroid cells results in transcriptional activation of NUE, strongly suggesting that the paired-domain protein Pax8 plays an important role in NUE activity. The NUE responds to cAMP in both protein kinase A-dependent and -independent manners, indicating that this enhancer could represent a novel type of cAMP responsive element. Such a cAMP response requires Pax8 but also depends on the integrity of a cAMP responsive element (CRE)-like sequence, thus suggesting a functional interaction between Pax8 and factors binding at the CRE-like site.

scholarly journals In vivo binding of NF-κB to the IκBβ promoter is insufficient for transcriptional activation

2006 ◽  
Vol 400 (1) ◽  
pp. 115-125 ◽  
Author(s):  
Bryan D. Griffin ◽  
Paul N. Moynagh
Keyword(s):  
Gene Regulation ◽  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Nuclear Factor Κb ◽  
Transcriptional Initiation ◽  
Il Interleukin

Despite certain structural and biochemical similarities, differences exist in the function of the NF-κB (nuclear factor κB) inhibitory proteins IκBα (inhibitory κBα) and IκBβ. The functional disparity arises in part from variance at the level of gene regulation, and in particular from the substantial induction of IκBα, but not IκBβ, gene expression post-NF-κB activation. In the present study, we probe the differential effects of IL (interleukin)-1β on induction of IκBα and perform the first characterization of the human IκBβ promoter. A consensus NF-κB-binding site, capable of binding NF-κB both in vitro and in vivo, is found in the IκBβ gene 5′ flanking region. However, the IκBβ promoter was not substantially activated by pro-inflammatory cytokines, such as IL-1β and tumour necrosis factor α, that are known to cause strong activation of NF-κB. Furthermore, in contrast with IκBα, NF-κB activation did not increase expression of endogenous IκBβ as assessed by analysis of mRNA and protein levels. Unlike κB-responsive promoters, IκBβ promoter-bound p65 inefficiently recruits RNA polymerase II, which stalls at the promoter. We present evidence that this stalling is likely due to the absence of transcription factor IIH engagement, a prerequisite for RNA polymerase II phosphorylation and transcriptional initiation. Differences in the conformation of promoter-bound NF-κB may underlie the variation in the ability to engage the basal transcriptional apparatus at the IκBβ and κB-responsive promoters. This accounts for the differential expression of IκB family members in response to NF-κB activation and furthers our understanding of the mechanisms involved in transcription factor activity and IκBβ gene regulation.

scholarly journals Heat Shock Element Architecture Is an Important Determinant in the Temperature and Transactivation Domain Requirements for Heat Shock Transcription Factor

1998 ◽  
Vol 18 (11) ◽  
pp. 6340-6352 ◽  
Author(s):  
Nicholas Santoro ◽  
Nina Johansson ◽  
Dennis J. Thiele
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Transcriptional Activation ◽  
Single Gene ◽  
Limited Proteolysis ◽  
Normal Growth ◽  
Heat Shock Transcription Factor ◽  
Transactivation Domain ◽  
Heat Shock Element

ABSTRACT The baker’s yeast Saccharomyces cerevisiae possesses a single gene encoding heat shock transcription factor (HSF), which is required for the activation of genes that participate in stress protection as well as normal growth and viability. Yeast HSF (yHSF) contains two distinct transcriptional activation regions located at the amino and carboxyl termini. Activation of the yeast metallothionein gene, CUP1, depends on a nonconsensus heat shock element (HSE), occurs at higher temperatures than other heat shock-responsive genes, and is highly dependent on the carboxyl-terminal transactivation domain (CTA) of yHSF. The results described here show that the noncanonical (or gapped) spacing of GAA units in the CUP1HSE (HSE1) functions to limit the magnitude of CUP1transcriptional activation in response to heat and oxidative stress. The spacing in HSE1 modulates the dependence for transcriptional activation by both stresses on the yHSF CTA. Furthermore, a previously uncharacterized HSE in the CUP1 promoter, HSE2, modulates the magnitude of the transcriptional activation of CUP1, via HSE1, in response to stress. In vitro DNase I footprinting experiments suggest that the occupation of HSE2 by yHSF strongly influences the manner in which yHSF occupies HSE1. Limited proteolysis assays show that HSF adopts a distinct protease-sensitive conformation when bound to the CUP1HSE1, providing evidence that the HSE influences DNA-bound HSF conformation. Together, these results suggest that CUP1regulation is distinct from that of other classic heat shock genes through the interaction of yHSF with two nonconsensus HSEs. Consistent with this view, we have identified other gene targets of yHSF containing HSEs with sequence and spacing features similar to those ofCUP1 HSE1 and show a correlation between the spacing of the GAA units and the relative dependence on the yHSF CTA.

Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor

1988 ◽  
Vol 8 (4) ◽  
pp. 1765-1774
Author(s):  
K Leong ◽  
L Brunet ◽  
A J Berk
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Tata Box ◽  
Late Promoter ◽  
Cell Extracts ◽  
Infected Cells ◽  
Major Late Promoter ◽  
E1a Protein

Extracts of adenovirus-infected HeLa cells have 5- to 10-fold-higher activity for transcription from the major late promoter in vitro than do extracts of mock-infected or E1A mutant-infected cells (K. Leong and A. J. Berk, Proc. Natl. Acad. Sci. USA 83:5844-5848, 1986). In this study, we analyzed extracts from mock-infected cells and from cells infected with an E1A mutant, pm975, which expresses principally the large E1A protein responsible for the stimulation of transcription. These extracts were fractionated by phosphocellulose chromatography, a procedure which separates factors required for transcription from this promoter (J. D. Dignam, B. S. Shastry, and R. G. Roeder, Methods Enzymol. 101:582-589, 1983), allowing the quantitative assay of individual factors (M. Samuels, A. Fire, and P. A. Sharp, J. Biol. Chem. 257:14419-14427, 1982). Fractions eluted with 0.04, 0.35, and 0.6 M KCl, which contained RNA polymerase II, the upstream factor MLTF, and three general polymerase II transcription factors, had similar activities when prepared from virus-infected or from mock-infected cells. The sequence-specific DNA-binding activity of MLTF was also similar in the virus-infected- and mock-infected-cell extracts. In contrast, the 1.0 M KCl fraction prepared from virus-infected cells consistently exhibited activity severalfold higher than that of the equivalent fraction prepared in parallel from mock-infected cells. E1A protein eluted principally (greater than 80%) in the 0.35 M KCl fraction. Results of others (M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985) have shown that the 1.0 M KCl fraction, containing 2 to 5% of the unfractionated protein extract, contains a factor which binds specifically to the major late promoter TATA box. These results, together with a recent genetic analysis of the E1B promoter which demonstrated that the TATA box was required for its efficient transcriptional activation (transactivation) by E1A (L. Wu, D. S. E. Rosser, M. Schmidt, and A. J. Berk, Nature (London) 326:512-515, 1987), are consistent with the model that E1A protein indirectly activates the TATA box transcription factor. Consistent with this model was the finding that mutants of the major late promoter containing only the TATA box and cap site region were transcribed at higher rates with extracts from virus-infected cells than with extracts from mock-infected cells. Other models consistent with the results are also discussed.

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