scholarly journals Identification of in vivo Hox13 binding sites reveals an essential locus controlling zebrafish brachyury expression

Development ◽  
2021 ◽  
Author(s):  
Zhi Ye ◽  
Christopher R. Braden ◽  
Andrea Wills ◽  
David Kimelman
Keyword(s):  
Binding Sites ◽  
Target Genes ◽  
Early Embryogenesis ◽  
Axis Formation ◽  
Direct Connection ◽  
Start Site ◽  
Transcription Start ◽  
Vertebrate Embryo ◽  
Response Site

During early embryogenesis the vertebrate embryo extends from anterior to posterior due to the progressive addition of cells from a posteriorly localized neuromesodermal progenitor (NMp) population. An autoregulatory loop between Wnt and Brachyury/Tbxt is required for the NMps to retain mesodermal potential, and hence normal axis development. We recently showed that the Hox13 genes help to support body axis formation and to maintain the autoregulatory loop, although the direct Hox13 target genes were unknown. Here, using a new method for identifying in vivo transcription factor binding sites, we identified over 500 potential Hox13 targets. Importantly, we found two highly conserved Hox13 binding elements far from the tbxta transcription start site, which also contain a conserved Tcf7/Lef1 (Wnt response) site. We show that the proximal of the two elements is sufficient to confer somitogenesis stage expression to a tbxta promoter that alone only drives NMp expression during gastrulation. Importantly, elimination of this proximal element produces shortened embryos due to aberrant formation of the most posterior somites. Our study provides a potential direct connection between Hox13 and regulation of the Wnt/Brachyury loop.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

scholarly journals Contributions of UP Elements and the Transcription Factor FIS to Expression from the Seven rrn P1 Promoters inEscherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6305-6314 ◽  
Author(s):  
Christine A. Hirvonen ◽  
Wilma Ross ◽  
Christopher E. Wozniak ◽  
Erin Marasco ◽  
Jennifer R. Anthony ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Promoter Activity ◽  
The Other ◽  
Multigene Families ◽  
Inescherichia Coli ◽  
Start Site ◽  
Transcription Start ◽  
Control Of Gene Expression ◽  
Up Elements

ABSTRACT The high activity of the rrnB P1 promoter inEscherichia coli results from acis-acting DNA sequence, the UP element, and atrans-acting transcription factor, FIS. In this study, we examine the effects of FIS and the UP element at the other sixrrn P1 promoters. We find that UP elements are present at all of the rrn P1 promoters, but they make different relative contributions to promoter activity. Similarly, FIS binds upstream of, and activates, all seven rrn P1 promoters but to different extents. The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each rrn P1 promoter. Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most rrn P1 promoters compared to rrnBP1. Our studies indicate that the overall activities of the sevenrrn P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter. These studies have implications for the control of gene expression of unlinked multigene families.

The T-box transcription factor Brachyury regulates expression of eFGF through binding to a non-palindromic response element

Development ◽  
1998 ◽  
Vol 125 (19) ◽  
pp. 3887-3894 ◽  
Author(s):  
E.S. Casey ◽  
M.A. O'Reilly ◽  
F.L. Conlon ◽  
J.C. Smith
Keyword(s):  
Transcription Factor ◽  
Site Selection ◽  
Target Genes ◽  
Biological Function ◽  
Start Site ◽  
Palindromic Sequence ◽  
Vertebrate Development ◽  
Transcription Start ◽  
T Box ◽  
Human And Mouse

Brachyury is a member of the T-box gene family and is required for formation of posterior mesoderm and notochord during vertebrate development. The ability of Brachyury to activate transcription is essential for its biological function, but nothing is known about its target genes. Here we demonstrate that Xenopus Brachyury directly regulates expression of eFGF by binding to an element positioned approximately 1 kb upstream of the eFGF transcription start site. This site comprises half of the palindromic sequence previously identified by binding site selection and is also present in the promoters of the human and mouse homologues of eFGF.

scholarly journals Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Daniel P. Kiesenhofer ◽  
Robert L. Mach ◽  
Astrid R. Mach-Aigner
Keyword(s):  
Trichoderma Reesei ◽  
Transcription Start Site ◽  
Binding Sites ◽  
Inverted Repeat ◽  
Promoter Activity ◽  
Promoter Strength ◽  
Start Site ◽  
Transcription Start ◽  
Content Type ◽  
Sheer Number

ABSTRACTTrichoderma reeseican produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of thecbh1promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in thexyn1promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One suchciselement, an inverted repeat, plays a crucial role in the inducibility of thexyn1promoter. We investigated the impact of the properties of suchciselements by shuffling them by insertion, exchange, deletion, and rearrangement ofciselements in both thecbh1andxyn1promoter. A promoter-reporter assay using theAspergillus nigergoxAgene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase incbh1promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances ofciselements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances ofciselements have large impacts on the strength of thecbh1promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically importantcbh1promoter can be improved byciselement rearrangement.IMPORTANCEIn the present study, we demonstrate that the arrangement ofciselements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances ofciselements to the transcription start site. Furthermore, we found that the configuration ofciselements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement ofciselements is an important factor that should not be overlooked when enhancement of gene expression is desired.

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.

scholarly journals Transcription Regulation by Tandem-Bound FNR at Escherichia coli Promoters

2003 ◽  
Vol 185 (20) ◽  
pp. 5993-6004 ◽  
Author(s):  
Anne M. L. Barnard ◽  
Jeffrey Green ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Transcription Factor ◽  
Binding Site ◽  
Transcription Regulation ◽  
Transcription Start Site ◽  
Promoter Activity ◽  
Start Site ◽  
Transcription Start ◽  
Substitution Mutant

ABSTRACT FNR is an Escherichia coli transcription factor that regulates the transcription of many genes in response to anaerobiosis. We have constructed a series of artificial FNR-dependent promoters, based on the melR promoter, in which a consensus FNR binding site was centered at position −41.5 relative to the transcription start site. A second consensus FNR binding site was introduced at different upstream locations, and promoter activity was assayed in vivo. FNR can activate transcription from these promoters when the upstream FNR binding site is located at many different positions. However, sharp repression is observed when the upstream-bound FNR is located near positions −85 or −95. This repression is relieved by the FNR G74C substitution mutant, previously identified as being defective in transcription repression at the yfiD promoter. A parallel series of artificial FNR-dependent promoters, carrying a consensus FNR binding site at position −61.5 and a second upstream DNA site for FNR, was also constructed. Again, promoter activity was repressed by FNR when the upstream-bound FNR was located at particular positions.

C/ebpα and Pu.1 Are Members of a Critical Regulatory Network Required for Hoxa9-Mediated Immortalization

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 650-650
Author(s):  
Cailin Collins ◽  
Jingya Wang ◽  
Joel Bronstein ◽  
Jay L. Hess
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Target Genes ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Epigenetic Modifications ◽  
Conditional Knockout ◽  
Motif Analysis ◽  
Myeloid Leukemias

Abstract Abstract 650 HOXA9 is a homeodomain-containing transcription factor that plays important roles in both development and hematopoiesis. Deregulation of HOXA9 occurs in a variety of acute lymphoid and myeloid leukemias and plays a key role in their pathogenesis. More than 50% of acute myeloid leukemia (AML) cases show up-regulation of HOXA9, which correlates strongly with poor prognosis. Nearly all cases of AML with mixed lineage leukemia (MLL) translocations have increased HOXA9 expression, as well as cases with mutation of the nucleophosmin gene NPM1, overexpression of CDX2, and fusions of NUP98. Despite the crucial role that HOXA9 plays in development, hematopoiesis and leukemia, its transcriptional targets and mechanisms of action are poorly understood. Previously we identified Hoxa9 and Meis1 binding sites in myeloblastic cells, profiled their epigenetic modifications, and identified the target genes regulated by Hoxa9. Hoxa9 and Meis1 co-bind at hundreds of promoter distal, highly evolutionarily conserved sites showing high levels of histone H3K4 monomethylation and CBP/p300 binding characteristic of enhancers. Hoxa9 association at these sites correlates strongly with increases in histone H3K27 acetylation and activation of downstream target genes, including many proleukemic gene loci. De novo motif analysis of Hoxa9 binding sites shows a marked enrichment of motifs for the transcription factors in the C/EBP and ETS families, and C/ebpα and the ETS transcription factor Pu.1 were found to cobind at Hoxa9-regulated enhancers. Both C/ebpα and Pu.1 are known to play critical roles in the establishment of functional enhancers during normal myeloid development and are mutated or otherwise deregulated in various myeloid leukemias. To determine the importance of co-association of Hoxa9, C/ebpα and Pu.1 at myeloid enhancers, we generated cell lines from C/ebpα and Pu.1 conditional knockout mice (kindly provided by Dr. Daniel Tenen, Harvard University) by immortalization with Hoxa9 and Meis1. In addition we transformed bone marrow with a tamoxifen-regulated form of Hoxa9. Strikingly, loss of C/ebpα or Pu.1, or inactivation of Hoxa9, blocks proliferation and leads to myeloid differentiation. ChIP experiments show that both C/ebpα and Pu.1 remain bound to Hoxa9 binding sites in the absence of Hoxa9. After the loss of Pu.1, both Hoxa9 and C/ebpα dissociate from Hoxa9 binding sites with a corresponding decrease in target gene expression. In contrast, loss of C/ebpα does not lead to an immediate decrease in either Hoxa9 or Pu.1 binding, suggesting that C/ebpα may be playing a regulatory as opposed to a scaffolding role at enhancers. Current work focuses on performing ChIP-seq analysis to assess how C/ebpα and Pu.1 affect Hoxa9 and Meis1 binding and epigenetic modifications genome-wide, and in vivo leukemogenesis assays to confirm the requirement of both Pu.1 and C/ebpα in the establishment and maintenance of leukemias with high levels of Hoxa9. Collectively, our findings implicate C/ebpα and Pu.1 as members of a critical transcription factor network required for Hoxa9-mediated transcriptional regulation in leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Powerful Induction of Divergent tgs1-Rv3131 Genes in Mycobacterium tuberculosis Is Mediated by DevR Interaction with a High-Affinity Site and an Adjacent Cryptic Low-Affinity Site

2009 ◽  
Vol 191 (19) ◽  
pp. 6075-6081 ◽  
Author(s):  
Santosh Chauhan ◽  
Jaya Sivaswami Tyagi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Binding Site ◽  
Binding Sites ◽  
Target Genes ◽  
Promoter Activation ◽  
Transcription Start ◽  
High Affinity ◽  
Synergistic Activation ◽  
Dnase I Footprinting ◽  
High Affinity Site

ABSTRACT DevR activates the transcription of ∼48 genes in response to hypoxia and other stresses and triggers metabolic downshift and dormancy development in Mycobacterium tuberculosis. tgs1 and Rv3131 encode triacylglycerol synthase and a putative nitroreductase, respectively, and both are members of the DevR regulon. This study aimed to understand how a single putative DevR binding site identified previously could sustain powerful induction of divergent tgs1-Rv3131 genes. DNase I footprinting revealed that phosphorylated DevR in fact binds to two sites symmetrically located at −42.5 and −63.5 bp from transcription start points of both genes. DevR first bound to the high-affinity site, P, and cooperatively recruited another DevR molecule to the secondary low-affinity site, S, to activate tgs1-Rv3131 transcription by ∼210- and ∼110-fold, respectively. The presence of a single P site significantly reduced activation of tgs1 expression and abolished Rv3131 activity, reinforcing the requirement of two binding sites for robust expression in both directions. P site inversion abolished tgs1 but not Rv3131 transcription despite DevR occupancy at both sites. The lack of tgs1 expression is most likely due to disruption of its −35 promoter element rather than inversion of the binding site per se. We conclude that (i) an overlap of a DevR binding site and −35 sequence is indispensable for promoter activation, (ii) DevR interaction with two binding sites is obligatory for synergistic activation of tgs1-Rv3131 promoters, and (iii) DevR interaction with binding sites of different affinities offers scope for temporal and differential expression of target genes.

scholarly journals Purines are required at the 5' ends of newly initiated RNAs for optimal RNA polymerase III gene expression.

1996 ◽  
Vol 16 (10) ◽  
pp. 5801-5810 ◽  
Author(s):  
G N Zecherle ◽  
S Whelen ◽  
B D Hall
Keyword(s):  
Rna Polymerase ◽  
Ternary Complex ◽  
Rna Polymerase Iii ◽  
Phenotypic Change ◽  
Start Site ◽  
Transcription Start ◽  
Polymerase Iii ◽  
Nascent Transcripts ◽  
Mutant Genes

We have made specific alterations in the CAACAA element at the transcription start site of a Saccharomyces cerevisiae suppressor tRNA gene. The mutant genes were tested for their ability to suppress the ochre nonsense alleles ade2-1, lys4-1, and met4-1. Many of the mutants showed either no phenotypic change or a weak loss of suppression relative to that of SUP4-o. A 2-bp change, CTCCAA, which alters bases encoding the +1 and +2 nucleotides of pre-tRNA Tyr, had a strong deleterious effect in vivo, as did the more extensive change CTCCTC. In contrast, mutant genes bearing each of the possible single changes at nucleotide +1 retained normal suppression levels. The transcription start point could be shifted in a limited fashion in response to the specific sequences encountered by RNA polymerase III at the start site. ATP was preferentially utilized as the 5' nucleotide in the growing RNA chain, while with start site sequences that precluded utilization of a purine, CTP was greatly preferred to UTP as the +1 nucleotide. Short oligopyrimidine RNAs formed on the CTCCTC allele could be repositioned in the active center of the newly formed ternary complex. Early postinitiation complexes containing short nascent RNAs formed on the CTCCTC mutant were more sensitive to the effects of heparin and produced more abortive transcripts than similar complexes formed on SUP4-o. Our results suggest that the purine-rich sequences at the 5' ends of the nascent transcripts of many genes act to stabilize the early ternary complex.

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