Different sequence requirements for expression in erythroid and megakaryocytic cells within a regulatory element upstream of the GATA-1 gene

Development ◽  
1999 ◽  
Vol 126 (12) ◽  
pp. 2799-2811 ◽  
Author(s):  
P. Vyas ◽  
M.A. McDevitt ◽  
A.B. Cantor ◽  
S.G. Katz ◽  
Y. Fujiwara ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Base Pair ◽  
Regulatory Element ◽  
Point Mutations ◽  
Comparative Sequence Analysis ◽  
Gata Factor ◽  
Base Pairs ◽  
Cis Acting ◽  
E Box ◽  
High Level

The lineage-restricted transcription factor GATA-1 is required for differentiation of erythroid and megakaryocytic cells. We have localized a 317 base pair cis-acting regulatory element, HS I, associated with a hematopoietic-specific DNase I hypersensitive site, which lies approx. 3.7 kilobases upstream of the murine hematopoietic-specific GATA-1 IE promoter. HS I directs high-level expression of reporter GATA-1/lacZ genes to primitive and definitive erythroid cells and megakaryocytes in transgenic mice. Comparative sequence analysis of HS I between human and mouse shows approx. 63% nucleotide identity with a more conserved core of 169 base pairs (86% identity). This core contains a GATA site separated by 10 base pairs from an E-box motif. The composite motif binds a multi-protein hematopoietic-specific transcription factor complex which includes GATA-1, SCL/tal-1, E2A, Lmo2 and Ldb-1. Point mutations of the GATA site abolishes HS I function, whereas mutation of the E-box motif still allows reporter gene expression in both lineages. Strict dependence of HS I activity on a GATA site implies that assembly of a protein complex containing a GATA-factor, presumably GATA-1 or GATA-2, is critical to activating or maintaining its function. Further dissection of the 317 base pair region demonstrates that, whereas all 317 base pairs are required for expression in megakaryocytes, only the 5′ 62 base pairs are needed for erythroid-specific reporter expression. These findings demonstrate differential lineage requirements for expression within the HS I element.

scholarly journals Isolation and characterization of the Beadex locus of Drosophila melanogaster: a putative cis-acting negative regulatory element for the heldup-a gene.

1984 ◽  
Vol 4 (7) ◽  
pp. 1343-1353 ◽  
Author(s):  
W W Mattox ◽  
N Davidson
Keyword(s):  
Drosophila Melanogaster ◽  
Base Pair ◽  
Regulatory Element ◽  
P Element ◽  
Functional Domain ◽  
Lambda Phage ◽  
Base Pairs ◽  
Negative Regulatory Element ◽  
Cis Acting ◽  
Isolation And Characterization

We isolated recombinant lambda phage clones spanning 49 kilobases of DNA which contain the Beadex and heldup-a loci of Drosophila melanogaster. These cloned DNAs were used to analyze the structure of eight dominant mutant alleles of the Beadex locus which show increased gene activity. A region, only 700 base pairs in length, is altered in each of these mutants. Six of the mutations have DNA insertions within this segment. Most of these insertions resemble retrovirus-like transposable elements. In one case (Beadex2) the inserted sequences are homologous to the gypsy transposon family. The other two Beadex alleles were induced by hybrid dysgenesis and suffered deletions which included at least part of the 700-base-pair segment. These deletions appear to have resulted from imprecise excision or deletion of a nearby P element found in the wild-type parental strain. Analysis of one heldup-a allele (heldup-aD30r) indicates that a similar P element-mediated event is responsible for this lesion. In this mutant, deletion of sequences no more than 1,600 base pairs from the Beadex locus accompanies the loss of heldup-a function. The deleted sequences in heldup-aD30r include the entire 700-base-pair segment within which at least part of the Beadex locus resides, yet these flies have no Beadex phenotype. This indicates that a functional heldup-a gene is necessary for expression of the Beadex phenotype. Together, these results suggest that the Beadex functional domain is contained within a short segment of DNA near the heldup-a gene and support the hypothesis that the Beadex locus functions as a cis-acting negative regulatory element for the heldup-a gene.

scholarly journals Isolation and characterization of the Beadex locus of Drosophila melanogaster: a putative cis-acting negative regulatory element for the heldup-a gene

1984 ◽  
Vol 4 (7) ◽  
pp. 1343-1353
Author(s):  
W W Mattox ◽  
N Davidson
Keyword(s):  
Drosophila Melanogaster ◽  
Base Pair ◽  
Regulatory Element ◽  
P Element ◽  
Functional Domain ◽  
Lambda Phage ◽  
Base Pairs ◽  
Negative Regulatory Element ◽  
Cis Acting ◽  
Isolation And Characterization

We isolated recombinant lambda phage clones spanning 49 kilobases of DNA which contain the Beadex and heldup-a loci of Drosophila melanogaster. These cloned DNAs were used to analyze the structure of eight dominant mutant alleles of the Beadex locus which show increased gene activity. A region, only 700 base pairs in length, is altered in each of these mutants. Six of the mutations have DNA insertions within this segment. Most of these insertions resemble retrovirus-like transposable elements. In one case (Beadex2) the inserted sequences are homologous to the gypsy transposon family. The other two Beadex alleles were induced by hybrid dysgenesis and suffered deletions which included at least part of the 700-base-pair segment. These deletions appear to have resulted from imprecise excision or deletion of a nearby P element found in the wild-type parental strain. Analysis of one heldup-a allele (heldup-aD30r) indicates that a similar P element-mediated event is responsible for this lesion. In this mutant, deletion of sequences no more than 1,600 base pairs from the Beadex locus accompanies the loss of heldup-a function. The deleted sequences in heldup-aD30r include the entire 700-base-pair segment within which at least part of the Beadex locus resides, yet these flies have no Beadex phenotype. This indicates that a functional heldup-a gene is necessary for expression of the Beadex phenotype. Together, these results suggest that the Beadex functional domain is contained within a short segment of DNA near the heldup-a gene and support the hypothesis that the Beadex locus functions as a cis-acting negative regulatory element for the heldup-a gene.

Tc, an unusual promoter element required for constitutive transcription of the yeast HIS3 gene

1990 ◽  
Vol 10 (9) ◽  
pp. 4447-4455
Author(s):  
S Mahadevan ◽  
K Struhl
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Base Pair ◽  
Point Mutations ◽  
Proximal Promoter ◽  
Promoter Element ◽  
Tata Element ◽  
Typical Sequence ◽  
His3 Gene ◽  
Transcriptional Stimulation

Tc is the proximal promoter element required for constitutive his3 transcription that occurs in the absence of the canonical TATA element (TR) and is initiated from the +1 site. The TC element, unlike TR, does not respond to transcriptional stimulation by the GCN4 or GAL4 activator protein. Analysis of deletion, substitution, and point mutations indicates that Tc mapped between nucleotides -54 and -83 and is a sequence-dependent element because it could not be functionally replaced by other DNA sequences. However, in contrast to the behavior of typical promoter elements, it was surprisingly difficult to eliminate Tc function by base pair substitutions. Of 15 derivatives averaging four substitutions in the Tc region and representing 40% of all possible single changes, only 1 inactivated the Tc element. Moreover, the phenotypes of mutant and hybrid elements indicated that inactivation of Tc required multiple changes. The spacing between Tc and the initiation region could be varied over a 30-base-pair range without significantly affecting the level of transcription from the +1 site. From these results, we consider it possible that Tc may not interact with TFIID or some other typical sequence-specific transcription factor, but instead might influence transcription, either directly or indirectly, by its DNA structure.

Identification of a signal transduction response sequence element necessary for induction of a Dictyostelium discoideum gene by extracellular cyclic AMP

1989 ◽  
Vol 9 (11) ◽  
pp. 4660-4669
Author(s):  
J Pavlovic ◽  
B Haribabu ◽  
R P Dottin
Keyword(s):  
Signal Transduction ◽  
Cyclic Amp ◽  
Dictyostelium Discoideum ◽  
Regulatory Element ◽  
Response Sequence ◽  
Base Pairs ◽  
Camp Response Element ◽  
Sequence Element ◽  
Cis Acting ◽  
Gene Coding

The signal transduction pathways that lead to gene induction are being intensively investigated in Dictyostelium discoideum. We have identified by deletion and transformation analysis a sequence element necessary for induction of a gene coding for uridine diphosphoglucose pyrophosphorylase (UDPGP1) of D. discoideum in response to extracellular cyclic AMP (cAMP). This regulatory element is located 380 base pairs upstream of the transcription start site and contains a G+C-rich partially palindromic sequence. It is not required for transcription per se but is required for induction of the gene in response to the stimulus of extracellular cAMP. The cAMP response sequence is also required for induction of the gene during normal development. A second A+T-rich cis-acting region located immediately downstream of the cAMP response sequence appears to be essential for the basal level of expression of the UDPGP1 gene. The position of the cAMP response element coincides with a DNase I-hypersensitive site that is observed when the UDPGP1 gene is actively transcribed.

scholarly journals Spo0A-Dependent Activation of an Extended −10 Region Promoter in Bacillus subtilis

2006 ◽  
Vol 188 (4) ◽  
pp. 1411-1418 ◽  
Author(s):  
Guangnan Chen ◽  
Amrita Kumar ◽  
Travis H. Wyman ◽  
Charles P. Moran
Keyword(s):  
Bacillus Subtilis ◽  
Base Pair ◽  
Promoter Activity ◽  
Mutational Analysis ◽  
Dna Binding Protein ◽  
Base Pairs ◽  
Dnase I Footprinting ◽  
Level Of Activity ◽  
High Level ◽  
Activity Dependent

ABSTRACT At the onset of endospore formation in Bacillus subtilis the DNA-binding protein Spo0A directly activates transcription from promoters of about 40 genes. One of these promoters, Pskf, controls expression of an operon encoding a killing factor that acts on sibling cells. AbrB-mediated repression of Pskf provides one level of security ensuring that this promoter is not activated prematurely. However, Spo0A also appears to activate the promoter directly, since Spo0A is required for Pskf activity in a ΔabrB strain. Here we investigate the mechanism of Pskf activation. DNase I footprinting was used to determine the locations at which Spo0A bound to the promoter, and mutations in these sites were found to significantly reduce promoter activity. The sequence near the −10 region of the promoter was found to be similar to those of extended −10 region promoters, which contain a TRTGn motif. Mutational analysis showed that this extended −10 region, as well as other base pairs in the −10 region, is required for Spo0A-dependent activation of the promoter. We found that a substitution of the consensus base pair for the nonconsensus base pair at position −9 of Pskf produced a promoter that was active constitutively in both ΔabrB and Δspo0A ΔabrB strains. Therefore, the base pair at position −9 of Pskf makes its activity dependent on Spo0A binding, and the extended −10 region motif of the promoter contributes to its high level of activity.

scholarly journals How affinity of the ELT-2 GATA factor binding to cis-acting regulatory sites controls Caenorhabditis elegans intestinal gene transcription

Development ◽  
2020 ◽  
Vol 147 (14) ◽  
pp. dev190330
Author(s):  
Brett R. Lancaster ◽  
James D. McGhee
Keyword(s):  
Transcription Factor ◽  
Caenorhabditis Elegans ◽  
Binding Affinity ◽  
Experimental System ◽  
Quantitative Relationship ◽  
Gata Factor ◽  
Unknown Parameters ◽  
Cis Acting ◽  
Complicated Model

ABSTRACTWe define a quantitative relationship between the affinity with which the intestine-specific GATA factor ELT-2 binds to cis-acting regulatory motifs and the resulting transcription of asp-1, a target gene representative of genes involved in Caenorhabditis elegans intestine differentiation. By establishing an experimental system that allows unknown parameters (e.g. the influence of chromatin) to effectively cancel out, we show that levels of asp-1 transcripts increase monotonically with increasing binding affinity of ELT-2 to variant promoter TGATAA sites. The shape of the response curve reveals that the product of the unbound ELT-2 concentration in vivo [i.e. (ELT-2free) or ELT-2 ‘activity’] and the largest ELT-XXTGATAAXX association constant (Kmax) lies between five and ten. We suggest that this (unitless) product [Kmax×(ELT-2free) or the equivalent product for any other transcription factor] provides an important quantitative descriptor of transcription-factor/regulatory-motif interaction in development, evolution and genetic disease. A more complicated model than simple binding affinity is necessary to explain the fact that ELT-2 appears to discriminate in vivo against equal-affinity binding sites that contain AGATAA instead of TGATAA.

Identification of an evolutionarily conserved 110 base-pair cis-acting regulatory sequence that governs Wnt-1 expression in the murine neural plate

Development ◽  
1998 ◽  
Vol 125 (14) ◽  
pp. 2735-2746 ◽  
Author(s):  
D.H. Rowitch ◽  
Y. Echelard ◽  
P.S. Danielian ◽  
K. Gellner ◽  
S. Brenner ◽  
...  
Keyword(s):  
Base Pair ◽  
Regulatory Element ◽  
Regulatory Region ◽  
Neural Plate ◽  
Homologous Region ◽  
Regulatory Sequence ◽  
Embryonic Mouse ◽  
Cis Acting ◽  
Evolutionarily Conserved

The generation of anterior-posterior polarity in the vertebrate brain requires the establishment of regional domains of gene expression at early somite stages. Wnt-1 encodes a signal that is expressed in the developing midbrain and is essential for midbrain and anterior hindbrain development. Previous work identified a 5.5 kilobase region located downstream of the Wnt-1 coding sequence which is necessary and sufficient for Wnt-1 expression in vivo. Using a transgenic mouse reporter assay, we have now identified a 110 base pair regulatory sequence within the 5.5 kilobase enhancer, which is sufficient for expression of a lacZ reporter in the approximate Wnt-1 pattern at neural plate stages. Multimers of this element driving Wnt-1 expression can partially rescue the midbrain-hindbrain phenotype of Wnt-1(−/−) embryos. The possibility that this region represents an evolutionarily conserved regulatory module is suggested by the identification of a highly homologous region located downstream of the wnt-1 gene in the pufferfish (Fugu rubripes). These sequences are capable of appropriate temporal and spatial activation of a reporter gene in the embryonic mouse midbrain; although, later aspects of the Wnt-1 expression pattern are absent. Genetic evidence has implicated Pax transcription factors in the regulation of Wnt-1. Although Pax-2 binds to the 110 base pair murine regulatory element in vitro, the location of the binding sites could not be precisely established and mutation of two putative low affinity sites did not abolish activation of a Wnt-1 reporter transgene in vivo. Thus, it is unlikely that Pax proteins regulate Wnt-1 by direct interactions with this cis-acting regulatory region. Our analysis of the 110 base pair minimal regulatory element suggests that Wnt-1 regulation is complex, involving different regulatory interactions for activation and the later maintenance of transgene expression in the dorsal midbrain and ventral diencephalon, and at the midbrain-hindbrain junction.

Regulation of the chicken embryonic myosin light-chain (L23) gene: existence of a common regulatory element shared by myosin alkali light-chain genes

1990 ◽  
Vol 10 (6) ◽  
pp. 2562-2569
Author(s):  
T Uetsuki ◽  
Y Nabeshima ◽  
A Fujisawa-Sehara ◽  
Y Nabeshima
Keyword(s):  
Light Chain ◽  
Base Pair ◽  
Regulatory Element ◽  
Common Factor ◽  
Protein S ◽  
Nuclear Extract ◽  
Initiation Site ◽  
Promoter Regions ◽  
Cis Acting ◽  
Carg Box

The transcriptional regulation of the chicken myosin alkali light-chain (MLC) L23 gene was analyzed. Two different types of cis-regulatory regions were identified: one was a silencerlike region located between 3.7 and 2.7 kilobases upstream of the mRNA initiation site, and the other was essential for the expression of L23 in skeletal muscle cells and was located between 106 and 91 base pairs upstream of the cap site. This 16-base-pair cis-acting element was designated as the MLC box since it is well conserved in various muscle-specific MLC promoter regions. The activity of the MLC box showed tissue specificity. To analyze the relationship between the nucleotide sequence and the activity of the MLC box precisely, mutation analysis was performed. The 16-base-pair sequence was indispensable for the active transcription of L23 gene, and the MLC box could function in either orientation. The inverted sequence of the MLC box was similar to the sequence of the alpha-actin CArG box. By using a gel mobility retardation assay, the nuclear protein(s) that binds to both MLC box and CArG box was detected with nuclear extract prepared from chicken embryonic breast muscle. These observations imply that a common factor regulates the coordinate expression of these contractile proteins in muscle differentiation.

Regulation of vitellogenin gene expression in transgenic Caenorhabditis elegans: short sequences required for activation of the vit-2 promoter

1992 ◽  
Vol 12 (4) ◽  
pp. 1652-1662
Author(s):  
M MacMorris ◽  
S Broverman ◽  
S Greenspoon ◽  
K Lea ◽  
C Madej ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Comparative Sequence Analysis ◽  
Recognition Sequence ◽  
Directed Mutation ◽  
Regulated Expression ◽  
Adult Hermaphrodite ◽  
Specific Regulation ◽  
High Level

The Caenorhabditis elegans vitellogenin genes are subject to sex-, stage-, and tissue-specific regulation: they are expressed solely in the adult hermaphrodite intestine. Comparative sequence analysis of the DNA immediately upstream of these genes revealed the presence of two repeated heptameric elements, vit promoter element 1 (VPE1) and VPE2. VPE1 has the consensus sequence TGTCAAT, while VPE2, CTGATAA, shares the recognition sequence of the GATA family of transcription factors. We report here a functional analysis of the VPEs within the 5'-flanking region of the vit-2 gene using stable transgenic lines. The 247 upstream bp containing the VPEs was sufficient for high-level, regulated expression. Furthermore, none of the four deletion mutations or eight point mutations tested resulted in expression of the reporter gene in larvae, males, or inappropriate hermaphrodite tissues. Mutation of the VPE1 closest to the TATA box inactivated the promoter, in spite of the fact that four additional close matches to the VPE1 consensus sequence are present within the 5'-flanking 200 bp. Each of these upstream VPE1-like sequences could be mutated without loss of high-level transgene expression, suggesting that if these VPE1 sequences play a role in regulating vit-2, their effects are more subtle. A site-directed mutation in the overlapping VPE1 and VPE2 at -98 was sufficient to inactivate the promoter, indicating that one or both of these VPEs must be present for activation of vit-2 transcription. Similarly, a small perturbation of the VPE2 at -150 resulted in reduction of fp155 expression, while a more extensive mutation in this element eliminated expression. On the other hand, deletion of this VPE2 and all upstream DNA still permitted correctly regulated expression, although at a very low level, suggesting that this VPE2 performs an important role in activation of vit-2 expression but may not be absolutely required. The results, taken together, demonstrate that both VPE1 and VPE2 are sites for activation of the vit-2 promoter.

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