scholarly journals Temporal Activation of the Sea Urchin Late H1 Gene Requires Stage-Specific Phosphorylation of the Embryonic Transcription Factor SSAP

1999 ◽  
Vol 19 (5) ◽  
pp. 3684-3695 ◽  
Author(s):  
Zhe Li ◽  
Geoffrey Childs
Keyword(s):  
Protein Kinase ◽  
Dna Binding ◽  
Sea Urchin ◽  
Transcriptional Activation ◽  
Gene Activation ◽  
Protein Kinase Activity ◽  
Transactivation Domain ◽  
Blastula Stage ◽  
Enhancer Element

ABSTRACT Stage-specific activator protein (SSAP) is a 41-kDa polypeptide that binds to embryonic enhancer elements of the sea urchin late H1 gene. These enhancer elements mediate the transcriptional activation of the late H1 gene in a temporally specific manner at the mid-blastula stage of embryogenesis. Although SSAP can transactivate the late H1 gene only at late stages of the development, it resides in the sea urchin nucleus and maintains DNA binding activity throughout early embryogenesis. In addition, it has been shown that SSAP undergoes a conversion from a 41-kDa monomer to a ∼80- to 100-kDa dimer when the late H1 gene is activated. We have demonstrated that SSAP is differentially phosphorylated during embryogenesis. Serine 87, a cyclic AMP-dependent protein kinase consensus site located in the N-terminal DNA binding domain, is constitutively phosphorylated. At the mid-blastula stage of embryogenesis, temporally correlated with SSAP dimer formation and late H1 gene activation, a threonine residue in the C-terminal transactivation domain is phosphorylated. This phosphorylation can be catalyzed by a break-ended double-stranded DNA-activated protein kinase activity from the sea urchin nucleus in vitro. Microinjection of synthetic SSAP mRNAs encoding either serine or threonine phosphorylation mutants results in the failure to transactivate reporter genes that contain the enhancer element, suggesting that both serine and threonine phosphorylation of SSAP are required for the activation of the late H1 gene. Furthermore, SSAP can undergo blastula-stage-specific homodimerization through its GQ-rich transactivation domain. The late-specific threonine phosphorylation in this domain is essential for the dimer assembly. These observations indicate that temporally regulated SSAP activation is promoted by threonine phosphorylation on its transactivation domain, which triggers the formation of a transcriptionally active SSAP homodimer.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

scholarly journals Purification and characterization of the stage-specific embryonic enhancer-binding protein SSAP-1.

1993 ◽  
Vol 13 (3) ◽  
pp. 1746-1758 ◽  
Author(s):  
D J DeAngelo ◽  
J DeFalco ◽  
G Childs
Keyword(s):  
Gene Expression ◽  
Molecular Weight ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Gene Activation ◽  
Upstream Sequence ◽  
Blastula Stage ◽  
Sequence Element ◽  
Enhancer Element ◽  
Beta Gene

We have demonstrated that a highly conserved segment of DNA between positions -288 and -317 (upstream sequence element IV [USE IV]) is largely responsible for the transcriptional activation of the sea urchin H1-beta histone gene during the blastula stage of embryogenesis. This sequence is capable of acting as an embryonic enhancer element, activating target genes in a stage-specific manner. Nuclear extracts prepared from developmentally-staged organisms before and after the gene is activated all contain a factor which specifically binds to the enhancer. We have purified a 43-kDa polypeptide which binds to and footprints the USE IV enhancer element. We refer to this protein as stage-specific activator protein 1 (SSAP-1). Early in development before the enhancer is active, SSAP appears as a 43-kDa monomer, but it undergoes a change in its molecular weight beginning at about 12 h postfertilization (early blastula) which precisely parallels the increase in H1-beta gene expression. Modified SSAP has an apparent molecular mass of approximately 90 to 100 kDa and contains at least one 43-kDa SSAP polypeptide. Thus, it is the disappearance of the 43-kDa species and the appearance of the 90- to 100-kDa species which coincide with the H1-beta gene activation. The correlation between the change in molecular weight of SSAP and the stage-specific activation of H1-beta gene expression strongly suggests that this higher-molecular-weight form of SSAP is directly responsible for the blastula stage-specific transcriptional activation of the late H1 gene.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

scholarly journals The embryonic enhancer-binding protein SSAP contains a novel DNA-binding domain which has homology to several RNA-binding proteins.

1995 ◽  
Vol 15 (3) ◽  
pp. 1254-1264 ◽  
Author(s):  
D J DeAngelo ◽  
J DeFalco ◽  
L Rybacki ◽  
G Childs
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Sea Urchin ◽  
Rna Processing ◽  
Histone H1 ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Blastula Stage ◽  
Enhancer Element ◽  
Specific Manner

Stage-specific activator protein (SSAP) is a 43-kDa polypeptide that binds to an enhancer element of the sea urchin late histone H1 gene. This enhancer element mediates the transcriptional activation of the late histone H1 gene in a temporally specific manner at the mid-blastula stage of embryogenesis. We have cloned cDNAs encoding SSAP by using polyclonal antibodies raised against purified SSAP to screen expression libraries. SSAP is unrelated to previously characterized transcription factors; however, it exhibits striking homology to a large family of proteins involved in RNA processing. The protein is a sequence-specific DNA-binding protein that recognizes both single- and double-stranded DNA. The DNA-binding domain of the protein was localized to the conserved RNA recognition motif (RRM). In addition to tandem copies of this conserved domain, SSAP contains a central domain that is rich in glutamine and glycine and a C-terminal domain that is enriched in serine, threonine, and basic amino acids. Overexpression of SSAP in sea urchin embryos by microinjection of either synthetic mRNA or an SSAP expression vector results in four- to eightfold transactivation of target reporter genes that contain the enhancer sequence. Transactivation occurs beginning only at the mid-blastula stage of development, suggesting that SSAP must be modified in a stage-specific manner in order to activate transcription. In addition, there are a number of other RRM-containing proteins that contain glutamine-rich regions which are postulated to function in the regulation of RNA processing. Instead, we suggest that SSAP is a member of a family of glutamine-rich RRM proteins which constitute a novel class of transcription factors.

scholarly journals The erythroid Krüppel-like factor transactivation domain is a critical component for cell-specific inducibility of a beta-globin promoter.

1995 ◽  
Vol 15 (2) ◽  
pp. 852-860 ◽  
Author(s):  
J J Bieker ◽  
C M Southwood
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Erythroid Cell ◽  
Transactivation Domain ◽  
Beta Globin ◽  
Cell Type Specificity ◽  
Globin Promoter ◽  
Murine Erythroleukemia

Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific DNA-binding protein that activates transcription from the beta-globin CACCC element, a functionally important and evolutionarily conserved component of globin as well as other erythroid cell-specific promoters and enhancers. We have attempted to elucidate the molecular role of EKLF in erythrocyte-specific transcriptional activation. First, in vivo and in vitro analyses have been used to demonstrate that the level of activation by EKLF is dependent on the orientation and number of CACCC elements, that EKLF contains separable activation and DNA-binding domains, and that the EKLF proline-rich region is a potent activator in CV-1 cells when fused to a nonrelated DNA-binding module. Second, we have established a transient assay in murine erythroleukemia cells in which reproducible levels of a reporter can be induced when linked to a locus control region enhancer-beta-globin promoter and in which induction is abolished when the promoter CAC site is mutated to a GAL site. Third, we demonstrate that the EKLF transactivation region, when fused to the GAL DNA-binding domain, can restore inducibility to this mutated construct and that this inducibility exhibits activator-, promoter-, and cell-type specificity. These results demonstrate that EKLF provides a crucial transactivation function for globin expression and further reinforce the idea that EKLF is an important regulator of CACCC element-directed transcription in erythroid cells.

An embryonic enhancer determines the temporal activation of a sea urchin late H1 gene

1989 ◽  
Vol 9 (6) ◽  
pp. 2315-2321
Author(s):  
Z C Lai ◽  
D J DeAngelo ◽  
M DiLiberto ◽  
G Childs
Keyword(s):  
Sea Urchin ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Competition Assay ◽  
Mobility Shift ◽  
Enhancer Element

Normal development requires that individual genes be expressed in their correct temporal patterns, but the mechanisms regulating this process during early embryogenesis are poorly understood. We have studied the early and late sea urchin histone genes during embryogenesis to address the molecular mechanisms controlling temporal gene expression. By measuring the changes in expression of cloned H1-beta DNA constructs after microinjection into fertilized one-cell zygotes, we demonstrated that a highly conserved 30-base-pair segment of DNA between positions -288 and -317 (USE IV) is responsible for the transcriptional activation of this late histone gene at the late blastula stage. In this report, we demonstrate that an oligonucleotide corresponding to USE IV acts as an embryonic enhancer element capable of activating the simian virus 40 early promoter in a stage-specific manner. Using an in vivo competition assay and in vitro DNase I footprinting and mobility shift assays, we also identified a protein(s) that interacts with this enhancer. Results of the competition assay suggested that this factor acts to stimulate transcription of the H1-beta gene. The factor was found to be stored in mature eggs as well as in all embryonic stages examined. The mobility of the factor found in eggs, however, differed from that of the embryonic form, which suggested that posttranslational modification occurs after fertilization.

scholarly journals Contributions to gene activation by multiple functions of Bicoid

1999 ◽  
Vol 338 (2) ◽  
pp. 447-455 ◽  
Author(s):  
Xiuguang MA ◽  
Dong YUAN ◽  
Tom SCARBOROUGH ◽  
Jun MA
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Gene Activation ◽  
Dimerization Domain ◽  
Dna Complexes ◽  
Activation Domain ◽  
Enhancer Element ◽  
Multiple Functions ◽  
Binding Function ◽  
Morphogenetic Protein

Bicoid is a Drosophila morphogenetic protein required for the development of anterior structures in the embryo. To gain a better understanding of how Bicoid works as a transcriptional activator, we systematically analysed various functions of Bicoid required for gene activation. We provide evidence suggesting that Bicoid is an intrinsically weak activator. First, our biochemical experiments demonstrate that the Bicoid–DNA complexes are very unstable, suggesting a weak DNA-binding function of Bicoid. This idea is further supported by our experiments demonstrating that the same number of LexA–Bicoid fusion molecules can activate transcription more effectively from LexA sites than from Bicoid sites. Secondly, we demonstrate that transcriptional activation by the weak activator Bicoid is readily influenced by the local enhancer environment. These influences are decreased when the Bicoid function is enforced by attaching to it either a known dimerization domain or the strong activation domain VP16. VP16 can also compensate for the loss of some Bicoid sites in an enhancer element. Our experiments demonstrate that the outcome of transcriptional activation by Bicoid is determined by multiple weak functions that are interconnected, a finding that can further help us to understand how this morphogenetic protein achieves its molecular functions.

scholarly journals Yeast Mpk1 Mitogen-Activated Protein Kinase Activates Transcription through Swi4/Swi6 by a Noncatalytic Mechanism That Requires Upstream Signal

2008 ◽  
Vol 28 (8) ◽  
pp. 2579-2589 ◽  
Author(s):  
Ki-Young Kim ◽  
Andrew W. Truman ◽  
David E. Levin
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Kinase Activity ◽  
Wall Stress ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase Activity ◽  
Mitogen Activated Protein

ABSTRACT The cell wall integrity mitogen-activated protein kinase (MAPK) cascade of Saccharomyces cerevisiae drives changes in gene expression in response to cell wall stress. We show that the MAPK of this pathway (Mpk1) and its pseudokinase paralog (Mlp1) use a noncatalytic mechanism to activate transcription of the FKS2 gene. Transcriptional activation of FKS2 was dependent on the Swi4/Swi6 (SBF) transcription factor and on an activating signal to Mpk1 but not on protein kinase activity. Activated (phosphorylated) Mpk1 and Mlp1 were detected in a complex with Swi4 and Swi6 at the FKS2 promoter. Mpk1 association with Swi4 in vivo required phosphorylation of Mpk1. Promoter association of Mpk1 and the Swi4 DNA-binding subunit of SBF were codependent but did not require Swi6, indicating that the MAPK confers DNA-binding ability to Swi4. Based on these data, we propose a model in which phosphorylated Mpk1 or Mlp1 forms a dimeric complex with Swi4 that is competent to associate with the FKS2 promoter. This complex then recruits Swi6 to activate transcription. Finally, we show that human ERK5, a functional ortholog of Mpk1, is similarly capable of driving FKS2 expression in the absence of protein kinase activity, suggesting that this mammalian MAPK may also have a noncatalytic function in vivo.

scholarly journals Acetylation-Mediated Transcriptional Activation of the ETS Protein ER81 by p300, P/CAF, and HER2/Neu

2003 ◽  
Vol 23 (17) ◽  
pp. 6243-6254 ◽  
Author(s):  
Apollina Goel ◽  
Ralf Janknecht
Keyword(s):  
Protein Kinase ◽  
Transcriptional Activation ◽  
Binding Activity ◽  
Tumor Formation ◽  
Mitogen Activated Protein Kinase ◽  
Transactivation Domain ◽  
Dna Binding Activity ◽  
Mitogen Activated Protein

ABSTRACT The regulated expression of the ETS transcription factor ER81 is a prerequisite for normal development, and its dysregulation contributes to neoplasia. Here, we demonstrate that ER81 is acetylated by two coactivators/acetyltransferases, p300 and p300- and CBP-associated factor (P/CAF) in vitro and in vivo. Whereas p300 acetylates two lysine residues (K33 and K116) within the ER81 N-terminal transactivation domain, P/CAF targets only K116. Acetylation of ER81 not only enhances its ability to transactivate but also increases its DNA binding activity and in vivo half-life. Furthermore, oncogenic HER2/Neu, which induces phosphorylation and thereby activation of ER81, was less able to activate acetylation-deficient ER81 mutants, indicating that both acetyltransferase and protein kinase-specific regulatory mechanisms control ER81 activity. Importantly, HER2/Neu overexpression stimulates the ability of p300 to acetylate ER81, likely by inducing phosphorylation of p300 through the Ras→Raf→mitogen-activated protein kinase pathway. This represents a novel mechanism by which oncogenic HER2/Neu, Ras, or Raf may promote tumor formation by enhancing acetylation not only of ER81 but also of other downstream effector transcription factors as well as histones.

scholarly journals An embryonic enhancer determines the temporal activation of a sea urchin late H1 gene.

1989 ◽  
Vol 9 (6) ◽  
pp. 2315-2321 ◽  
Author(s):  
Z C Lai ◽  
D J DeAngelo ◽  
M DiLiberto ◽  
G Childs
Keyword(s):  
Sea Urchin ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Competition Assay ◽  
Mobility Shift ◽  
Enhancer Element

Normal development requires that individual genes be expressed in their correct temporal patterns, but the mechanisms regulating this process during early embryogenesis are poorly understood. We have studied the early and late sea urchin histone genes during embryogenesis to address the molecular mechanisms controlling temporal gene expression. By measuring the changes in expression of cloned H1-beta DNA constructs after microinjection into fertilized one-cell zygotes, we demonstrated that a highly conserved 30-base-pair segment of DNA between positions -288 and -317 (USE IV) is responsible for the transcriptional activation of this late histone gene at the late blastula stage. In this report, we demonstrate that an oligonucleotide corresponding to USE IV acts as an embryonic enhancer element capable of activating the simian virus 40 early promoter in a stage-specific manner. Using an in vivo competition assay and in vitro DNase I footprinting and mobility shift assays, we also identified a protein(s) that interacts with this enhancer. Results of the competition assay suggested that this factor acts to stimulate transcription of the H1-beta gene. The factor was found to be stored in mature eggs as well as in all embryonic stages examined. The mobility of the factor found in eggs, however, differed from that of the embryonic form, which suggested that posttranslational modification occurs after fertilization.

Sign in / Sign up

Export Citation Format

Share Document