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.

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 Interdependent recruitment of CYC8/TUP1 and the transcriptional activator XYR1 at target promoters is required for induced cellulase gene expression in Trichoderma reesei

PLoS Genetics ◽  
2021 ◽  
Vol 17 (2) ◽  
pp. e1009351
Author(s):  
Lei Wang ◽  
Weixin Zhang ◽  
Yanli Cao ◽  
Fanglin Zheng ◽  
Guolei Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Trichoderma Reesei ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Gene Activation ◽  
Transcriptional Activator ◽  
Cellulase Production ◽  
Gene Promoters ◽  
Cellulase Gene ◽  
Histone H4

Cellulase production in filamentous fungus Trichoderma reesei is highly responsive to various environmental cues involving multiple positive and negative regulators. XYR1 (Xylanase regulator 1) has been identified as the key transcriptional activator of cellulase gene expression in T. reesei. However, the precise mechanism by which XYR1 achieves transcriptional activation of cellulase genes is still not fully understood. Here, we identified the TrCYC8/TUP1 complex as a novel coactivator for XYR1 in T. reesei. CYC8/TUP1 is the first identified transcriptional corepressor complex mediating repression of diverse genes in Saccharomyces cerevisiae. Knockdown of Trcyc8 or Trtup1 resulted in markedly impaired cellulase gene expression in T. reesei. We found that TrCYC8/TUP1 was recruited to cellulase gene promoters upon cellulose induction and this recruitment is dependent on XYR1. We further observed that repressed Trtup1 or Trcyc8 expression caused a strong defect in XYR1 occupancy and loss of histone H4 at cellulase gene promoters. The defects in XYR1 binding and transcriptional activation of target genes in Trtup1 or Trcyc8 repressed cells could not be overcome by XYR1 overexpression. Our results reveal a novel coactivator function for TrCYC8/TUP1 at the level of activator binding, and suggest a mechanism in which interdependent recruitment of XYR1 and TrCYC8/TUP1 to cellulase gene promoters represents an important regulatory circuit in ensuring the induced cellulase gene expression. These findings thus contribute to unveiling the intricate regulatory mechanism underlying XYR1-mediated cellulase gene activation and also provide an important clue that will help further improve cellulase production by T. reesei.

scholarly journals Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charleen Hunt ◽  
Suzanne A. Hartford ◽  
Derek White ◽  
Evangelos Pefanis ◽  
Timothy Hanna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Gene Activation ◽  
Tissue Specific ◽  
Guide Rna ◽  
Synergistic Activation ◽  
Single Transcript

AbstractCRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.

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 HOS15 is a transcriptional corepressor of NPR1-mediated gene activation of plant immunity

2020 ◽  
Vol 117 (48) ◽  
pp. 30805-30815
Author(s):  
Mingzhe Shen ◽  
Chae Jin Lim ◽  
Junghoon Park ◽  
Jeong Eun Kim ◽  
Dongwon Baek ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ubiquitin Ligase ◽  
Transcriptional Activation ◽  
Plant Immunity ◽  
Gene Activation ◽  
Transcriptional Corepressor ◽  
Defense Gene Expression ◽  
Dynamic Regulation ◽  
Defense Gene ◽  
Transcriptional Corepressors

Transcriptional regulation is a complex and pivotal process in living cells. HOS15 is a transcriptional corepressor. Although transcriptional repressors generally have been associated with inactive genes, increasing evidence indicates that, through poorly understood mechanisms, transcriptional corepressors also associate with actively transcribed genes. Here, we show that HOS15 is the substrate receptor for an SCF/CUL1 E3 ubiquitin ligase complex (SCFHOS15) that negatively regulates plant immunity by destabilizing transcriptional activation complexes containing NPR1 and associated transcriptional activators. In unchallenged conditions, HOS15 continuously eliminates NPR1 to prevent inappropriate defense gene expression. Upon defense activation, HOS15 preferentially associates with phosphorylated NPR1 to stimulate rapid degradation of transcriptionally active NPR1 and thus limit the extent of defense gene expression. Our findings indicate that HOS15-mediated ubiquitination and elimination of NPR1 produce effects contrary to those of CUL3-containing ubiquitin ligase that coactivate defense gene expression. Thus, HOS15 plays a key role in the dynamic regulation of pre- and postactivation host defense.

Identification and functional characterization of the human T-cell receptor beta gene transcriptional enhancer: common nuclear proteins interact with the transcriptional regulatory elements of the T-cell receptor alpha and beta genes

1990 ◽  
Vol 10 (10) ◽  
pp. 5486-5495
Author(s):  
L R Gottschalk ◽  
J M Leiden
Keyword(s):  
Gene Expression ◽  
T Cell ◽  
T Cell Receptor ◽  
Binding Sites ◽  
Cell Receptor ◽  
Nuclear Proteins ◽  
Transcriptional Enhancer ◽  
Enhancer Element ◽  
Beta 2 ◽  
Beta Gene

A transcriptional enhancer has been mapped to a region 5.5 kilobases 3' of the C beta 2 gene in the human T-cell receptor (TCR) beta-chain locus. Transient transfections allowed localization of enhancer activity to a 480-base-pair HincII-XbaI restriction enzyme fragment. The TCR beta enhancer was active on both the minimal simian virus 40 promoter and a TCR beta variable gene promoter in both TCR alpha/beta + and TCR gamma/delta + T cells. It displayed significantly less activity in Epstein-Barr virus-transformed B cells and K562 chronic myelogenous leukemia cells and no activity in HeLa fibroblasts. DNA sequence analysis revealed that the enhancer contains a consensus immunoglobulin kappa E2 motif, as well as an AP-1-binding site and a cyclic AMP response element. DNase I footprint analyses using Jurkat T-cell nuclear extracts allowed the identification of five nuclear protein-binding sites, T beta 1 to T beta 5, within the enhancer element. Deletion and in vitro mutagenesis studies demonstrated that the T beta 2- and T beta 3- and T beta 4-binding sites are each required for full transcriptional enhancer activity. In contrast, deletion of the T beta 1- and T beta 5-binding sites had essentially no effect on enhancer function. Electrophoretic mobility shift assays demonstrated that TCR alpha/beta + and TCR gamma/delta + T cells expressed T beta 2-, T beta 3-, and T beta 4-binding activities. In contrast, non-T-cell lines, in which the enhancer was inactive, each lacked expression of at least one of these binding activities. TCR alpha and beta gene expression may be regulated by a common set of T-cell nuclear proteins in that the T beta 2 element binding a set of cyclic AMP response element-binding proteins that are also bound by the T alpha 1 element of the human TCR alpha enhancer and the decamer element present in a large number of human and murine TCR beta promoters. Similarly, the T beta 5 TCR beta-enhancer element and the T alpha 2 TCR alpha-enhancer element bind at least one common T-cell nuclear protein. Taken together, these results suggest that TCR beta gene expression is regulated by the interaction of multiple T cell nuclear proteins with a transcriptional enhancer element located 3' of the C beta 2 gene and that some of these proteins may be involved in the coordinate regulation of TCR alpha and beta gene expression.

decapentaplegic is a direct target of dTcf repression in the Drosophila visceral mesoderm

Development ◽  
2000 ◽  
Vol 127 (17) ◽  
pp. 3695-3702 ◽  
Author(s):  
X. Yang ◽  
M. van Beest ◽  
H. Clevers ◽  
T. Jones ◽  
D.A. Hursh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Transforming Growth Factor Beta ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transforming Growth Factor ◽  
Ectopic Expression ◽  
Reporter Gene Expression ◽  
Enhancer Element ◽  
Visceral Mesoderm

Drosophila T cell factor (dTcf) mediates transcriptional activation in the presence of Wingless signalling and repression in its absence. Wingless signalling is required for the correct expression of decapentaplegic (dpp), a Transforming Growth Factor (beta) family member, in parasegments 3 and 7 of the Drosophila visceral mesoderm. Here we demonstrate that a dpp enhancer element, which directs expression of a reporter gene in the visceral mesoderm in a pattern indistinguishable from dpp, has two functional dTcf binding sites. Mutations that reduce or eliminate Wingless signalling abolish dpp reporter gene expression in parasegment 3 and reduce it in parasegment 7 while ectopic expression of Wingless signalling components expand reporter gene expression anteriorly in the visceral mesoderm. However, mutation of the dTcf binding sites in the dpp enhancer results in ectopic expression of reporter gene expression throughout the visceral mesoderm, with no diminution of expression in the endogenous sites of expression. These results demonstrate that the primary function of dTcf binding to the dpp enhancer is repression throughout the visceral mesoderm and that activation by Wingless signalling is probably not mediated via these dTcf binding sites to facilitate correct dpp expression in the visceral mesoderm.

scholarly journals Gene activation precedes DNA demethylation in response to infection in human dendritic cells

2019 ◽  
Vol 116 (14) ◽  
pp. 6938-6943 ◽  
Author(s):  
Alain Pacis ◽  
Florence Mailhot-Léonard ◽  
Ludovic Tailleux ◽  
Haley E. Randolph ◽  
Vania Yotova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dendritic Cells ◽  
Transcriptional Activation ◽  
Time Course ◽  
Gene Activation ◽  
Dna Demethylation ◽  
Epigenetic Mark ◽  
Distal Enhancer ◽  
Human Dendritic Cells

DNA methylation is considered to be a relatively stable epigenetic mark. However, a growing body of evidence indicates that DNA methylation levels can change rapidly; for example, in innate immune cells facing an infectious agent. Nevertheless, the causal relationship between changes in DNA methylation and gene expression during infection remains to be elucidated. Here, we generated time-course data on DNA methylation, gene expression, and chromatin accessibility patterns during infection of human dendritic cells withMycobacterium tuberculosis. We found that the immune response to infection is accompanied by active demethylation of thousands of CpG sites overlapping distal enhancer elements. However, virtually all changes in gene expression in response to infection occur before detectable changes in DNA methylation, indicating that the observed losses in methylation are a downstream consequence of transcriptional activation. Footprinting analysis revealed that immune-related transcription factors (TFs), such as NF-κB/Rel, are recruited to enhancer elements before the observed losses in methylation, suggesting that DNA demethylation is mediated by TF binding to cis-acting elements. Collectively, our results show that DNA demethylation plays a limited role to the establishment of the core regulatory program engaged upon infection.

scholarly journals The TEA Transcription Factor Tec1 Confers Promoter-Specific Gene Regulation by Ste12-Dependent and -Independent Mechanisms

2010 ◽  
Vol 9 (4) ◽  
pp. 514-531 ◽  
Author(s):  
Barbara Heise ◽  
Julia van der Felden ◽  
Sandra Kern ◽  
Mario Malcher ◽  
Stefan Brückner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Target Genes ◽  
Specific Gene ◽  
Dependent Manner ◽  
A Genome ◽  
Regulated Gene Expression

ABSTRACT In Saccharomyces cerevisiae, the TEA transcription factor Tec1 is known to regulate target genes together with a second transcription factor, Ste12. Tec1-Ste12 complexes can activate transcription through Tec1 binding sites (TCSs), which can be further combined with Ste12 binding sites (PREs) for cooperative DNA binding. However, previous studies have hinted that Tec1 might regulate transcription also without Ste12. Here, we show that in vivo, physiological amounts of Tec1 are sufficient to stimulate TCS-mediated gene expression and transcription of the FLO11 gene in the absence of Ste12. In vitro, Tec1 is able to bind TCS elements with high affinity and specificity without Ste12. Furthermore, Tec1 contains a C-terminal transcriptional activation domain that confers Ste12-independent activation of TCS-regulated gene expression. On a genome-wide scale, we identified 302 Tec1 target genes that constitute two distinct classes. A first class of 254 genes is regulated by Tec1 in a Ste12-dependent manner and is enriched for genes that are bound by Tec1 and Ste12 in vivo. In contrast, a second class of 48 genes can be regulated by Tec1 independently of Ste12 and is enriched for genes that are bound by the stress transcription factors Yap6, Nrg1, Cin5, Skn7, Hsf1, and Msn4. Finally, we find that combinatorial control by Tec1-Ste12 complexes stabilizes Tec1 against degradation. Our study suggests that Tec1 is able to regulate TCS-mediated gene expression by Ste12-dependent and Ste12-independent mechanisms that enable promoter-specific transcriptional control.

The Physiology of Prothrombin Gene Expression Integrates RNA Polyadenylation and Splicing in a Novel Regulatable 3′ RNP-Complex.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1601-1601
Author(s):  
Sven Danckwardt ◽  
Marc Gentzel ◽  
Niels H. Gehring ◽  
Isabelle Kaufmann ◽  
Gabriele Neu-Yilik ◽  
...  
Keyword(s):  
Gene Expression ◽  
Splicing Factors ◽  
Upstream Sequence ◽  
Dependent Manner ◽  
Flanking Sequence ◽  
Gain Of Function ◽  
Sequence Element ◽  
Functional Link ◽  
Rnp Complex ◽  
Prothrombin Gene

Abstract The functional analysis of the common prothrombin (F2) 20210*A allele has recently revealed gain-of-function of 3′end processing as a novel genetic mechanism predisposing to human disease. The general susceptibility of the F2 mRNA for gain-of-function is further exemplified by F2 20209*T and F2 20221*T, and can be explained by an unusual architecture of non-canonical 3′end formation sequence elements: Specifically, the F2 3′ untranslated region (3′UTR) contains a stimulatory upstream sequence element (USE) that compensates for the weak functional activities of the cleavage site and the downstream U-rich element in the F2 3′ flanking sequence. We now show that the F2 USE promotes 3′end formation in a position- and sequence-dependent manner, stimulating the step of mRNA polyadenylation rather than cleavage, and identify specific proteins that interact with the USE. Unexpectedly, the USE RNP includes splicing factors, components of the 3′end processing machinery and AU-rich sequence element-binding proteins (ARE-BP). We demonstrate that the splicing factors U2AF35 and U2AF65, hnRNPI/PTB, PSF/SFPQ and p54nrb/NonO promote 3′end formation via the USE contained in the 3′UTR uncovering a novel and more general functional link between these splicing factors and mRNA 3′ end formation. We propose a model of USE-directed 3′ end processing that involves a novel mRNP that integrates different nuclear pre-mRNA processing steps. Furthermore, the involvement of ARE-BP in this mRNP reveals an intriguing potential for a post-transcriptional regulation of prothrombin gene expression through external stimuli. Our data thus implicate USE-dependent RNP-complex formation in the regulated physiology of prothrombin gene expression specifically and in hemostasis (and other thrombin-dependent processes) more generally.

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