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 Postrecruitment Function of Yeast Med6 Protein during the Transcriptional Activation by Mediator Complex

2018 ◽  
Vol 2018 ◽  
pp. 1-9
Author(s):  
Gwang Sik Kim ◽  
Young Chul Lee
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Reporter Gene Expression ◽  
Temperature Sensitive ◽  
Internal Deletion ◽  
Cell Extracts ◽  
Evolutionarily Conserved

Med6 protein (Med6p) is a hallmark component of evolutionarily conserved Mediator complexes, and the genuine role of Med6p in Mediator functions remains elusive. For the functional analysis ofSaccharomyces cerevisiaeMed6p (scMed6p), we generated a series of scMed6p mutants harboring a small internal deletion. Genetic analysis of these mutants revealed that three regions (amino acids 33–42 (Δ2), 125–134 (Δ5), and 157–166 (Δ6)) of scMed6p are required for cell viability and are located at highly conserved regions of Med6 homologs. Notably, the Med6p-Δ2 mutant was barely detectable in whole-cell extracts and purified Mediator, suggesting a loss of Mediator association and concurrent rapid degradation. Consistent with this, the recombinant forms of Med6p having these mutations partially (Δ2) restore or fail (Δ5 and Δ6) to restore in vitro transcriptional defects caused by temperature-sensitivemed6mutation. In an artificial recruitment assay, Mediator containing a LexA-fused wild-type Med6p or Med6p-Δ5 was recruited to thelexAoperator region with TBP and activated reporter gene expression. However, the recruitment of Mediator containing LexA-Med6p-Δ6 tolexAoperator region resulted in neither TBP recruitment nor reporter gene expression. This result demonstrates a pivotal role of Med6p in the postrecruitment function of Mediator, which is essential for transcriptional activation by Mediator.

scholarly journals Purα and Purβ Collaborate with Sp3 To Negatively Regulate β-Myosin Heavy Chain Gene Expression duringSkeletal Muscle Inactivity

2006 ◽  
Vol 27 (4) ◽  
pp. 1531-1543 ◽  
Author(s):  
Juan Ji ◽  
Gretchen L. Tsika ◽  
Hansjörg Rindt ◽  
Kathy L. Schreiber ◽  
John J. McCarthy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Myosin Heavy Chain ◽  
Reporter Gene ◽  
Heavy Chain ◽  
Weight Bearing ◽  
Ectopic Expression ◽  
Reporter Gene Expression ◽  
Proximal Promoter ◽  
C2c12 Myotubes ◽  
Adult Skeletal Muscle

ABSTRACT Adult skeletal muscle retains the capability of transcriptional reprogramming. This attribute is readily observable in the non-weight-bearing (NWB) soleus muscle, which undergoes a slow-to-fast fiber type transition concurrent with decreased β-myosin heavy chain (βMyHC) gene expression. Our previous work showed that Sp3 contributes to decreased βMyHC gene expression under NWB conditions. In this study, we demonstrate that physical and functional interactions between Sp3, Purα, and Purβ proteins mediate repression of βMyHC expression under NWB conditions. Binding of Purα or Purβ to the single-stranded βMyHC distal negative regulatory element-sense strand (dβNRE-S) element is markedly increased under NWB conditions. Ectopic expression of Purα and Purβ decreasedβ MyHC reporter gene expression, while mutation of the dβNRE-S element increased expression in C2C12 myotubes. The dβNRE-S element conferred Pur-dependent decreased expression on a minimal thymidine kinase promoter. Short interfering RNA sequences specific for Sp3 or for Purα and Purβ decreased endogenous Sp3 and Pur protein levels and increased βMyHC reporter gene expression in C2C12 myotubes. Immunoprecipitation assays revealed an association between endogenous Purα, Purβ, and Sp3, while chromatin immunoprecipitation assays demonstrated Purα, Purβ, and Sp3 binding to the βMyHC proximal promoter region harboring the dβNRE-S and C-rich elements in vivo. These data demonstrate that Pur proteins collaborate with Sp3 to regulate a transcriptional program that enables muscle cells to remodel their phenotype.

Transforming growth factor-beta stimulates laminin α3A gene expression through cooperation between AP-1 binding sites

1998 ◽  
Vol 16 ◽  
pp. S22
Author(s):  
Thierry Virolle ◽  
Marie-Noëlle Monthouel ◽  
Jean-Paul Ortonne ◽  
Guerrino Meneguzzi ◽  
Daniel Aberdam
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Binding Sites ◽  
Transforming Growth Factor ◽  
Growth Factor Beta

scholarly journals Growth Hormone Regulation of Insulin-Like Growth Factor-I Gene Expression May Be Mediated by Multiple Distal Signal Transducer and Activator of Transcription 5 Binding Sites

Endocrinology ◽  
2008 ◽  
Vol 149 (5) ◽  
pp. 2230-2240 ◽  
Author(s):  
Satyanaryana Eleswarapu ◽  
Zhiliang Gu ◽  
Honglin Jiang
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Binding Sites ◽  
Mouse Liver ◽  
Consensus Sequence ◽  
Reporter Gene Expression ◽  
Hormone Regulation ◽  
Signal Transducer ◽  
Igf I ◽  
I Gene

The transcription factor signal transducer and activator of transcription (STAT)-5 mediates GH stimulation of IGF-I gene expression in the liver. Previous studies suggested that STAT5 might exert this effect by binding to an IGF-I intron 2 region and a distal 5′-flanking region each containing two STAT5 binding sites. Here we report the identification of three additional chromosomal regions containing a total of five putative STAT5 binding sites that may mediate GH-induced STAT5 activation of IGF-I gene expression in the mouse liver. By comparing an 170-kb mouse genomic DNA containing the IGF-I gene with the corresponding human sequence, we identified 19 putative STAT5 binding sites that bear the consensus sequence of STAT5 binding site and are conserved across the two species. Chromatin immunoprecipitation assays indicated that five chromosomal regions containing a total of nine of the 19 putative STAT5 binding sites were bound by STAT5 in the mouse liver in response to GH administration and that these bindings preceded or coincided with GH-increased IGF-I gene transcription. Two of the five chromosomal regions correspond to those previously identified in other species, and the three new chromosomal regions that contain a total of five putative STAT5 binding sites are IGF-I intron 3 regions located at least 26 kb from the transcription start site. Gel-shift assays confirmed the binding of the five new putative STAT5 binding sites as well as three of the four previously identified STAT5 binding sites to GH-activated STAT5 from the mouse liver. Cotransfection analyses indicated that, although each of the five chromosomal regions was able to mediate STAT5 activation of reporter gene expression, together they mediated greater STAT5 activation of reporter gene expression in response to GH. Overall, these results suggest that GH-induced STAT5 activation of IGF-I gene expression in the mouse liver might be collectively mediated by at least eight STAT5 binding sites located in distal intronic and 5′-flanking regions of the IGF-I gene.

A Drosophila growth factor homolog, decapentaplegic, regulates homeotic gene expression within and across germ layers during midgut morphogenesis

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1041-1050 ◽  
Author(s):  
G.E. Panganiban ◽  
R. Reuter ◽  
M.P. Scott ◽  
F.M. Hoffmann
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Homeotic Gene ◽  
Homeotic Genes ◽  
Germ Layers ◽  
Visceral Mesoderm ◽  
Endodermal Cells ◽  
Homeotic Gene Expression

The decapentaplegic (dpp) gene product, a member of the transforming growth factor-beta family, is required in Drosophila embryos for normal gastrulation and the establishment of dorsal-ventral polarity in the embryo. dpp is also expressed at specific positions in the visceral mesoderm along the developing midgut. We find that mutations that eliminate the visceral mesoderm expression of dpp lead to defects in midgut morphogenesis and alter the spatially localized expression of the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), and Antennapedia (Antp) in the visceral mesoderm. The extracellular dpp protein migrates from the visceral mesoderm across the apposing endodermal cell layer in a region of the endoderm that expresses the homeotic gene labial (lab). Mesodermal expression of dpp is required for the expression of lab in these endodermal cells indicating that dpp mediates an inductive interaction between the two germ layers. We propose that extracellular dpp protein regulates gut morphogenesis, in part, by regulating homeotic gene expression in the visceral mesoderm and endoderm of the developing midgut.

scholarly journals Cell Signaling Switches HOX-PBX Complexes from Repressors to Activators of Transcription Mediated by Histone Deacetylases and Histone Acetyltransferases

2000 ◽  
Vol 20 (22) ◽  
pp. 8623-8633 ◽  
Author(s):  
Maya Saleh ◽  
Isabel Rambaldi ◽  
Xiang-Jiao Yang ◽  
Mark S. Featherstone
Keyword(s):  
Gene Expression ◽  
Cell Signaling ◽  
Reporter Gene ◽  
Binding Sites ◽  
Histone Deacetylases ◽  
Trichostatin A ◽  
Cell Aggregation ◽  
Reporter Gene Expression ◽  
Hek293 Cells ◽  
Creb Binding Protein

ABSTRACT The Hoxb1 autoregulatory element comprises three HOX-PBX binding sites. Despite the presence of HOXB1 and PBX1, this enhancer fails to activate reporter gene expression in retinoic acid-treated P19 cell monolayers. Activation requires cell aggregation in addition to RA. This suggests that HOX-PBX complexes may repress transcription under some conditions. Consistent with this, multimerized HOX-PBX binding sites repress reporter gene expression in HEK293 cells. We provide a mechanistic basis for repressor function by demonstrating that a corepressor complex, including histone deacetylases (HDACs) 1 and 3, mSIN3B, and N-CoR/SMRT, interacts with PBX1A. We map a site of interaction with HDAC1 to the PBX1 N terminus and show that the PBX partner is required for repression by the HOX-PBX complex. Treatment with the deacetylase inhibitor trichostatin A not only relieves repression but also converts the HOX-PBX complex to a net activator of transcription. We show that this activation function is mediated by the recruitment of the coactivator CREB-binding protein by the HOX partner. Interestingly, HOX-PBX complexes are switched from transcriptional repressors to activators in response to protein kinase A signaling or cell aggregation. Together, our results suggest a model whereby the HOX-PBX complex can act as a repressor or activator of transcription via association with corepressors and coactivators. The model implies that cell signaling is a direct determinant of HOX-PBX function in the patterning of the animal embryo.

Direct regulation of the muscle-identity gene apterous by a Hox protein in the somatic mesoderm

Development ◽  
2001 ◽  
Vol 128 (8) ◽  
pp. 1221-1230 ◽  
Author(s):  
M. Capovilla ◽  
Z. Kambris ◽  
J. Botas
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Binding Sites ◽  
Muscle Development ◽  
Hox Genes ◽  
Enhancer Element ◽  
Hox Gene ◽  
Control Segment ◽  
Hox Protein ◽  
Direct Regulation

Hox genes control segment identity in the mesoderm as well as in other tissues. Most evidence indicates that Hox genes act cell-autonomously in muscle development, although this remains a controversial issue. We show that apterous expression in the somatic mesoderm is under direct Hox control. We have identified a small enhancer element of apterous (apME680) that regulates reporter gene expression in the LT1-4 muscle progenitors. We show that the product of the Hox gene Antennapedia is present in the somatic mesoderm of the second and third thoracic segments. Through complementary alterations in the Antennapedia protein and in its binding sites on apME680, we show that Antennapedia positively regulates apterous in a direct manner, demonstrating unambiguously its cell-autonomous role in muscle development. Finally, we determine that LT1-4 muscles contain more nuclei in the thorax than in the abdomen and we propose that one of the segmental differences under Hox control is the number of myoblasts allocated to the formation of specific muscles in different segments.

scholarly journals Development of Rice Stripe Tenuivirus Minireplicon Reverse Genetics Systems Suitable for Analyses of Viral Replication and Intercellular Movement

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiaoyan Zhang ◽  
Kai Sun ◽  
Yan Liang ◽  
Shuo Wang ◽  
Kaili Wu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Reverse Genetics ◽  
Ectopic Expression ◽  
Cell Movement ◽  
Rice Stripe Virus ◽  
Reporter Gene Expression ◽  
N Protein ◽  
Intercellular Movement ◽  
Negative Sense

Rice stripe virus (RSV), a tenuivirus with four negative-sense/ambisense genome segments, is one of the most devastating viral pathogens affecting rice production in many Asian countries. Despite extensive research, our understanding of RSV infection cycles and pathogenesis has been severely impaired by the lack of reverse genetics tools. In this study, we have engineered RSV minireplicon (MR)/minigenome cassettes with reporter genes substituted for the viral open reading frames in the negative-sense RNA1 or the ambisense RNA2-4 segments. After delivery to Nicotiana benthamiana leaves via agroinfiltration, MR reporter gene expression was detected only when the codon-optimized large viral RNA polymerase protein (L) was coexpressed with the nucleocapsid (N) protein. MR activity was also critically dependent on the coexpressed viral suppressors of RNA silencing, but ectopic expression of the RSV-encoded NS3 silencing suppressor drastically decreased reporter gene expression. We also developed intercellular movement-competent MR systems with the movement protein expressed either in cis from an RNA4-based MR or in trans from a binary plasmid. Finally, we generated multicomponent replicon systems by expressing the N and L proteins directly from complementary-sense RNA1 and RNA3 derivatives, which enhanced reporter gene expression, permitted autonomous replication and intercellular movement, and reduced the number of plasmids required for delivery. In summary, this work enables reverse genetics analyses of RSV replication, transcription, and cell-to-cell movement and provides a platform for engineering more complex recombinant systems.

scholarly journals Strategic Attack on Host Cell Gene Expression during Adenovirus Infection

2003 ◽  
Vol 77 (20) ◽  
pp. 11006-11015 ◽  
Author(s):  
Hongxing Zhao ◽  
Fredrik Granberg ◽  
Ludmila Elfineh ◽  
Ulf Pettersson ◽  
Catharina Svensson
Keyword(s):  
Gene Expression ◽  
Transforming Growth Factor Beta ◽  
Transcriptional Activation ◽  
Transforming Growth Factor ◽  
Cell Cycle Control ◽  
Adenovirus Type ◽  
Major Pathway ◽  
Amino Terminal ◽  
Cellular Genes ◽  
Genes Encoding

ABSTRACT To understand the interaction between the virus and its host, we used three sources of cDNA microarrays to examine the expression of 12,309 unique genes at 6 h postinfection of HeLa cells with high multiplicities of adenovirus type 2. Seventy-six genes with significantly changed expression ratios were identified, suggesting that adenovirus only modulates expression of a limited set of cellular genes. Quantitative real-time PCR analyses on selected genes were performed to confirm the microarray results. Significantly, a pronounced transcriptional activation by the promiscuous E1A-289R transcriptional activator was not apparent. Instead, promoter sequences in 45% of the upregulated genes harbored a potential E2F binding site, suggesting that the ability of the amino-terminal domain of E1A to regulate E2F-dependent transcription may be a major pathway for regulation of cellular gene expression. CDC25A was the only upregulated gene directly involved in cell cycle control. In contrast, several genes implicated in cell growth arrest were repressed. The transforming growth factor beta superfamily was specifically affected in the expression of both the upstream ligand and an intracellular regulator. In agreement with previous reports, adenovirus also targeted the innate immune response by downregulating several cytokines, including CLL2, CXCL1, and interleukin-6. Finally, stress response genes encoding GADD45B, ATF3, and TP53AP1 were upregulated. Importantly, we also found a novel countermeasure—activation of the apoptosis inhibitor survivin.

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