Abstract P257: Isoform-Specific Effects of the Transcription Factor Sister-of-Mammalian Grainyhead on Endothelial Cells and in Vivo

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Arne Mlynek ◽  
Margarete Lukosz ◽  
Martin Graf ◽  
Christoph Winkler ◽  
Judith Haendeler ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Protein Isoforms ◽  
Expression Vectors ◽  
Luciferase Expression ◽  
Reporter System ◽  
Active Transcription

Apoptosis and reduced migratory capacity of human endothelial cells (EC) are hallmarks for the development of atherosclerosis. TNFalpha has been described as one apoptotic stimulus, which is increased during cardiovascular disease. However, recent findings support the hypothesis that TNFalpha can induce survival genes before committing cells to apoptosis. In a screen for anti-apoptotic genes regulated by TNFalpha we have identified the transcription factor Sister-of-Mammalian Grainyhead/Grainyhead-like 3 (SOM/GRHL3). In humans two RNAs are transcribed from the gene, one of which is alternatively spliced, yielding the protein isoforms SOM1 and SOM3, the latter being an N-terminally truncated version. We have found that both isoforms are expressed in EC. Since nothing is known about the function of these proteins in EC, we investigated their functional properties and role in migration and apoptosis. To analyze their transcription factor activity we established a SOM-dependent reporter system by inserting tandem SOM binding sites and corresponding mutants upstream of a minimal promoter driving luciferase expression. To assess transcriptional activation by SOM1 and SOM3 we cotransfected these reporters with expression vectors for both proteins. In contrast to previously published work, in which isolated SOM domains fused to a Gal4 DNA binding domain were used, we found that both full length proteins are active transcription factors. We next investigated the influence of SOM1 and SOM3 on EC functions. Surprisingly, overexpression of isoform 1 induced migration and inhibited apoptosis, whereas isoform 3 had opposite effects. Along the same lines, SOM1, but not SOM3 activated endothelial nitric oxide synthase and Akt. To investigate whether these isoforms have different functions also in vivo, we overexpressed them in zebrafish embryos. SOM3 but not SOM1 overexpression led to increased lethality, a strong reduction in normal phenotype and a 10 fold higher frequency in heavy deformations. The effects observed on EC migration and apoptosis as well as on zebrafish development suggest that these isoforms activate different sets of target genes, which we are currently identifying by microarray analysis.

Abstract 267: Opposing Functions of Two Splice Variants of the Human Transcription Factor Grainyhead-like 3 in Endothelial Cells And in vivo

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Joachim Altschmied ◽  
Nicole Büchner ◽  
Sascha Jakob ◽  
Sabrina Farrokh ◽  
Christine Goy ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Target Genes ◽  
Splice Variants ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Active Transcription ◽  
Human Transcription Factor

Grainyhead-like 3 (GRHL3) is a member of the evolutionary conserved Grainyhead family of transcription factors. In humans, three isoforms are derived from differential first exon usage and alternative splicing, which differ only in their N-terminus. Isoform 2, the only variant also present in mouse, is required for endothelial cell (EC) migration and protects against apoptosis. The functions of the human specific isoforms 1 and 3, which are derived from an alternatively spliced pre-mRNA, have not yet been investigated, although all three isoforms are expressed in EC. Therefore, we have assessed their effects on EC migration and apoptosis. Overexpression of the two proteins had opposite effects on EC migration, with isoform 1 acting pro-migratory. This protein also protected EC against apoptosis in an eNOS-dependent manner, whereas isoform 3 had no effect. These opposing outcomes with respect to apoptosis EC were corroborated by isoform-specific knockdowns. With reporter assays using a GRHL3-specific luciferase reporter we demonstrated that both are active transcription factors. Microarray analyses revealed that they induce divergent target gene sets in EC. Two validated targets, Akt2 and Mxi1, which are upregulated by isoform1, are regulators of Akt1-, and thus eNOS-phosphorylation and apoptosis, which could explain the effects of this protein on these processes. In vivo, overexpression of isoform 3 in zebrafish embryos resulted in increased lethality and severe deformations, while isoform 1 had no deleterious effect. In conclusion, our data demonstrate that the splice variant derived isoforms 1 and 3 of the human transcription factor GRHL3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.

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.

Abstract 234: Defining Domains In The Transcription Factor Grainyhead-like 3 Required For Protective Functions In The Endothelium

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Sabrina Farrokh ◽  
Niloofar Ale-Agha ◽  
Judith Haendeler ◽  
Joachim Altschmied
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Large Scale ◽  
Target Genes ◽  
Intracellular Localization ◽  
Expression Vectors ◽  
Luciferase Reporter ◽  
Deletion Mutants ◽  
Nuclear Localization Signals ◽  
Apoptosis Level

Important aspects during aging of human endothelial cells (EC) are an increased apoptosis level and a reduced migratory capacity. In a screening for anti-apoptotic genes we have identified the transcription factor Grainyhead-like 3 (GRHL3) and demonstrated its anti-apoptotic and pro-migratory effects after overexpression and knockdown in EC. To define the domains in GRHL3 responsible for these - potentially also extranuclear - functions we have cloned large scale deletion mutants and mutants with deletions of putative nuclear localization signals (NLS) and analyzed them for their intracellular localization and functional properties. Immunostaining of transfected EC were used to examine the subcellular distribution. Two mutants with deletions in a bioinformatically predicted bipartite NLS were localized predominantly in the cytoplasm. To corroborate these data, lysates of the cells were fractionated biochemically. Western blotting confirmed the immunostaining data, indicating that we have identified the major NLS in GRHL3. To analyze the transcriptional properties of these mutants, we constructed a GRHL3-specific luciferase reporter containing a tandem GRHL3 binding sites in front of a minimal promoter and cotransfected it with expression vectors for the GRHL3 deletion mutants. Besides the previously described activation domain we identified another region required for transcriptional upregulation of target genes in the N-terminal half of the protein. We will now use these mutants to further dissect the regions in GRHL3 necessary for its pro-migratory and anti-apoptotic activities in endothelial cells. This will also allow us to investigate if all of these functions are mediated solely by the activation of target genes or by other mechanisms coupled to a non-nuclear function of GRHL3.

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

scholarly journals Transcriptional Activation in Yeast Cells Lacking Transcription Factor IIA

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 1573-1581 ◽  
Author(s):  
Susanna Chou ◽  
Sukalyan Chatterjee ◽  
Mark Lee ◽  
Kevin Struhl
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Large Subunit ◽  
Yeast Cells ◽  
Specific Cell ◽  
Wild Type ◽  
Activation Domains ◽  
Cycle Arrest ◽  
General Transcription Factor

Abstract The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID. Biochemical experiments suggest that TFIIA is important in the response to transcriptional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex. Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to <1% of the wild-type level. Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16. However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary. These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA.

scholarly journals Regulatory Network Analysis in Estradiol-Treated Human Endothelial Cells

2021 ◽  
Vol 22 (15) ◽  
pp. 8193
Author(s):  
Daniel Pérez-Cremades ◽  
Ana B. Paes ◽  
Xavier Vidal-Gómez ◽  
Ana Mompeón ◽  
Carlos Hermenegildo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Regulatory Network ◽  
Mirna Target ◽  
Target Genes ◽  
Functional Characterization ◽  
Human Endothelial Cells ◽  
Mrna Targets ◽  
Canonical Pathways

Background/Aims: Estrogen has been reported to have beneficial effects on vascular biology through direct actions on endothelium. Together with transcription factors, miRNAs are the major drivers of gene expression and signaling networks. The objective of this study was to identify a comprehensive regulatory network (miRNA-transcription factor-downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol. Methods: miRNA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17β-estradiol (E2) (1 nmol/L, 24 h). miRNA–mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miRNA-regulated genes. Transcription factor downstream target genes were predicted by consensus transcription factor binding sites in the promoter region of E2-regulated genes by using JASPAR and TRANSFAC tools in Enrichr software. Results: miRNA–target pairings were filtered by using differentially expressed miRNAs and mRNAs characterized by a regulatory relationship according to miRNA target prediction databases. The analysis identified 588 miRNA–target interactions between 102 miRNAs and 588 targets. Specifically, 63 upregulated miRNAs interacted with 295 downregulated targets, while 39 downregulated miRNAs were paired with 293 upregulated mRNA targets. Functional characterization of miRNA/mRNA association analysis highlighted hypoxia signaling, integrin, ephrin receptor signaling and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Transcription factors and downstream genes analysis revealed eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhesion molecule binding pathways. Conclusion: This study identifies regulatory networks obtained by integrative microarray analysis and provides additional insights into the way estradiol could regulate endothelial function in human endothelial cells.

scholarly journals Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

2015 ◽  
Vol 36 (6) ◽  
pp. 913-922 ◽  
Author(s):  
Nallani Vijay Kumar ◽  
Jianbo Yang ◽  
Jitesh K. Pillai ◽  
Swati Rawat ◽  
Carlos Solano ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Transcriptional Response ◽  
Transcriptional Regulator ◽  
Yeast Protein ◽  
Content Type ◽  
Arsenic Tolerance ◽  
Arsenic Sensing

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.

Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells

Blood ◽  
2010 ◽  
Vol 116 (6) ◽  
pp. 909-914 ◽  
Author(s):  
Enid Yi Ni Lam ◽  
Christopher J. Hall ◽  
Philip S. Crosier ◽  
Kathryn E. Crosier ◽  
Maria Vega Flores
Keyword(s):  
Endothelial Cells ◽  
Transcription Factor ◽  
Fluorescent Protein ◽  
Living Organism ◽  
Time Lapse ◽  
Dorsal Aorta ◽  
Transgenic Zebrafish ◽  
Hematopoietic Stem ◽  
Green Fluorescent

Abstract Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

scholarly journals CHOP Enhancement of Gene Transcription by Interactions with Jun/Fos AP-1 Complex Proteins

1999 ◽  
Vol 19 (11) ◽  
pp. 7589-7599 ◽  
Author(s):  
Mariano Ubeda ◽  
Mario Vallejo ◽  
Joel F. Habener
Keyword(s):  
Transcription Factor ◽  
Gene Transcription ◽  
Stress Responses ◽  
Leucine Zipper ◽  
Target Genes ◽  
Cellular Stress ◽  
Dominant Negative ◽  
Dimerization Domain ◽  
Mobility Shift

ABSTRACT The transcription factor CHOP (C/EBP homologous protein 10) is a bZIP protein induced by a variety of stimuli that evoke cellular stress responses and has been shown to arrest cell growth and to promote programmed cell death. CHOP cannot form homodimers but forms stable heterodimers with the C/EBP family of activating transcription factors. Although initially characterized as a dominant negative inhibitor of C/EBPs in the activation of gene transcription, CHOP-C/EBP can activate certain target genes. Here we show that CHOP interacts with members of the immediate-early response, growth-promoting AP-1 transcription factor family, JunD, c-Jun, and c-Fos, to activate promoter elements in the somatostatin, JunD, and collagenase genes. The leucine zipper dimerization domain is required for interactions with AP-1 proteins and transactivation of transcription. Analyses by electrophoretic mobility shift assays and by an in vivo mammalian two-hybrid system for protein-protein interactions indicate that CHOP interacts with AP-1 proteins inside cells and suggest that it is recruited to the AP-1 complex by a tethering mechanism rather than by direct binding of DNA. Thus, CHOP not only is a negative or a positive regulator of C/EBP target genes but also, when tethered to AP-1 factors, can activate AP-1 target genes. These findings establish the existence of a new mechanism by which CHOP regulates gene expression when cells are exposed to cellular stress.

scholarly journals Identification of target genes and a unique cis element regulated by IRF-8 in developing macrophages

Blood ◽  
2005 ◽  
Vol 106 (6) ◽  
pp. 1938-1947 ◽  
Author(s):  
Tomohiko Tamura ◽  
Pratima Thotakura ◽  
Tetsuya S. Tanaka ◽  
Minoru S. H. Ko ◽  
Keiko Ozato
Keyword(s):  
Transcription Factor ◽  
Cystatin C ◽  
Target Genes ◽  
De Novo ◽  
Consensus Sequence ◽  
Binding Motif ◽  
Microarray Gene Expression ◽  
Cathepsin C ◽  
Cis Element

Abstract Interferon regulatory factor-8 (IRF-8)/interferon consensus sequence–binding protein (ICSBP) is a transcription factor that controls myeloid-cell development. Microarray gene expression analysis of Irf-8-/- myeloid progenitor cells expressing an IRF-8/estrogen receptor chimera (which differentiate into macrophages after addition of estradiol) was used to identify 69 genes altered by IRF-8 during early differentiation (62 up-regulated and 7 down-regulated). Among them, 4 lysosomal/endosomal enzyme-related genes (cystatin C, cathepsin C, lysozyme, and prosaposin) did not require de novo protein synthesis for induction, suggesting that they were direct targets of IRF-8. We developed a reporter assay system employing a self-inactivating retrovirus and analyzed the cystatin C and cathepsin C promoters. We found that a unique cis element mediates IRF-8–induced activation of both promoters. Similar elements were also found in other IRF-8 target genes with a consensus sequence (GAAANN[N]GGAA) comprising a core IRF-binding motif and an Ets-binding motif; this sequence is similar but distinct from the previously reported Ets/IRF composite element. Chromatin immunoprecipitation assays demonstrated that IRF-8 and the PU.1 Ets transcription factor bind to this element in vivo. Collectively, these data indicate that IRF-8 stimulates transcription of target genes through a novel cis element to specify macrophage differentiation.

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