scholarly journals Identification of a Functional Androgen-Response Element in the Exon 1-Coding Sequence of the Cystatin-Related Protein Gene crp2

1997 ◽  
Vol 11 (8) ◽  
pp. 1033-1043 ◽  
Author(s):  
A. Devos ◽  
F. Claessens ◽  
P. Alen ◽  
J. Winderickx ◽  
W. Heyns ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Binding Sites ◽  
Chloramphenicol Acetyltransferase ◽  
Sequence Motif ◽  
Transcription Start Point ◽  
Transcription Start ◽  
Response Element ◽  
Androgen Response Element ◽  
Steroid Dependent ◽  
Intron 1

Abstract Two hormone-responsive segments, one in the region of the promoter and one in intron 1, are identified in two homologous androgen-regulated and differentially expressed rat genes encoding the cystatin-related proteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA-binding domain on the promoter-containing fragments reveals an AR-binding site downstream of the transcription start point in the crp2 gene (ARBSd/crp2,+ 40/+63). It displays an androgen response element-like sequence motif 5′-AGAAGAaaaTGTACA-3′ and overlaps with the ATG translation start codon. A double-stranded oligonucleotide containing this sequence forms a DNA-protein complex with the full-length AR synthesized by vaccinia, as seen in band shift assays. Additional AR-binding sites, ARBSu/crp1 and ARBSu/crp2, occur 5′ upstream of the transcription start point and are located at an identical position (−142/−120) in crp1 and crp2. The AR affinity for these two slightly different sequence motifs is relatively weak. The biological function of all three AR-binding sites as transcription control elements has been studied. The ARBSd/crp2 element clearly shows androgen-response element characteristics. The contribution of the common upstream element to the androgen-dependent control of reporter gene transcription is less clear. The transcription of a reporter gene construct containing the crp2 footprint fragment crp2F (−273/+88) is hormonally regulated as determined by transfection into the human breast cancer cell line T-47D. Androgens, but also glucocorticoids, efficiently stimulate steroid-dependent transcription of the chloramphenicol acetyltransferase gene. Mutation of the 5′-TGTACA-3′ sequence in ARBSd/crp2 destroys the AR binding and abolishes the androgen-dependent synthesis of chloramphenicol acetyltransferase. A large fragment derived from intron 1 of the crp1 and crp2 gene can also provide the androgen-dependent transcription of chimeric constructs in T-47D cells. However, the induction measured is less than the one observed with crp2F (−273/+88), and this activity seems to reside in several subfragments that each display a low but consistent androgen responsiveness.

Identification of an Androgen-Dependent Enhancer within the Prostate Stem Cell Antigen Gene

2002 ◽  
Vol 16 (10) ◽  
pp. 2323-2337 ◽  
Author(s):  
Anjali Jain ◽  
Amanda Lam ◽  
Igor Vivanco ◽  
Michael F. Carey ◽  
Robert E. Reiter
Keyword(s):  
Stem Cell ◽  
Binding Sites ◽  
Mutational Analysis ◽  
Diagnostic Marker ◽  
Prostate Stem Cell Antigen ◽  
Transcription Start ◽  
Cell Antigen ◽  
Androgen Response Element ◽  
Deoxyribonuclease I ◽  
High Affinity Binding Site

Abstract Prostate stem cell antigen (PSCA) is emerging as an important diagnostic marker and therapeutic target in prostate cancer. Previous studies indicated that PSCA was directly regulated by androgens, but the mechanism has not been elucidated. Here we describe the identification of a compact cell-specific and androgen-responsive enhancer between 2.7 and 3 kb upstream of the transcription start site. The enhancer functions autonomously when positioned immediately adjacent to a minimal promoter. Deoxyribonuclease I footprinting analysis with recombinant androgen receptor (AR) reveals that the enhancer contains two AR binding sites at one end. Mutational analysis of the AR binding sites revealed the importance of the higher affinity one. The dissociation constant of the high affinity binding site (androgen response element I) was determined to be approximately 87 nm. The remainder of the enhancer contains elements that function synergistically with the AR. We discuss the structural organization of the PSCA enhancer and compare it with that found in other AR-regulated genes.

A sequence motif distinct from Hox binding sites controls the specificity of a Hox response element

Development ◽  
1999 ◽  
Vol 126 (24) ◽  
pp. 5581-5589 ◽  
Author(s):  
X. Li ◽  
A. Veraksa ◽  
W. McGinnis
Keyword(s):  
Binding Sites ◽  
Site Response ◽  
Sequence Motif ◽  
Hox Proteins ◽  
Sequence Element ◽  
Response Element ◽  
Cofactor Binding ◽  
Dna Binding Sites ◽  
Activation Response ◽  
Hox Protein

Hox transcription factors, in combination with cofactors such as PBC proteins, provide diverse developmental fates to cells on the anteroposterior body axis of animal embryos. However, the mechanisms by which the different Hox proteins and their cofactors generate those diverse fates remain unclear. Recent findings have provided support for a model where the DNA binding sites that directly interact with Hox-PBC heterodimers determine which member of the Hox protein family occupies and thereby regulates a given target element. In the experiments reported here, we test the function of chimeric Hox response elements and, surprisingly, find evidence that runs counter to this view. A 21 bp cofactor binding sequence from an embryonic Deformed Hox response element, containing no Hox or Hox-PBC binding sites, was combined with single or multimeric sites that bind heterodimers of Labial-type Hox and PBC proteins. Normally, multimerized Labial-PBC binding sites are sufficient to trigger a Labial-specific activation response in either Drosophila or mouse embryos. Here we find that the 21 bp sequence element plays an important role in Deformed specificity, as it is capable of switching a Labial-PBC binding site/response element to a Deformed response element. Thus, cofactor binding sites that are separate and distinct from homeodomain binding sites can dictate the regulatory specificity of a Hox response element.

scholarly journals Identification of a Class of Chromatin Boundary Elements

1998 ◽  
Vol 18 (12) ◽  
pp. 7478-7486 ◽  
Author(s):  
Olivier Cuvier ◽  
Craig M. Hart ◽  
Ulrich K. Laemmli
Keyword(s):  
Reporter Gene ◽  
Binding Sites ◽  
Point Mutations ◽  
Boundary Elements ◽  
Sequence Motif ◽  
Sequence Motifs ◽  
Position Effects ◽  
Sequence Homologies ◽  
Genetic Activity ◽  
Chromatin Boundary

ABSTRACT Boundary elements are thought to define the ends of functionally independent domains of genetic activity. An assay for boundary activity based on this concept measures the ability to insulate a bracketed, chromosomally integrated reporter gene from position effects. Despite their presumed importance, the few examples identified to date apparently do not share sequence motifs or DNA binding proteins. TheDrosophila protein BEAF binds the scs′ boundary element of the 87A7 hsp70 locus and roughly half of polytene chromosome interband loci. To see if these sites represent a class of boundary elements that have BEAF in common, we have isolated and studied several genomic BEAF binding sites as candidate boundary elements (cBEs). BEAF binds with high affinity to clustered, variably arranged CGATA motifs present in these cBEs. No other sequence homologies were found. Two cBEs were tested and found to confer position-independent expression on a mini-whitereporter gene in transgenic flies. Furthermore, point mutations in CGATA motifs that eliminate binding by BEAF also eliminate the ability to confer position-independent expression. Taken together, these findings suggest that clustered CGATA motifs are a hallmark of a BEAF-utilizing class of boundary elements found at many loci. This is the first example of a class of boundary elements that share a sequence motif and a binding protein.

sae is essential for expression of the staphylococcal adhesins Eap and Emp

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 1789-1800 ◽  
Author(s):  
Niamh Harraghy ◽  
Jan Kormanec ◽  
Christiane Wolz ◽  
Dagmar Homerova ◽  
Christiane Goerke ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Extracellular Matrix ◽  
Virulence Factor ◽  
Eukaryotic Cells ◽  
Transcription Start Point ◽  
Chronic Infections ◽  
Transcription Start ◽  
Regulatory Mutants ◽  
Global Regulators ◽  
Glucose Analysis

Eap and Emp are two Staphylococcus aureus adhesins initially described as extracellular matrix binding proteins. Eap has since emerged as being important in adherence to and invasion of eukaryotic cells, as well as being described as an immunomodulator and virulence factor in chronic infections. This paper describes the mapping of the transcription start point of the eap and emp promoters. Moreover, using reporter-gene assays and real-time PCR in defined regulatory mutants, environmental conditions and global regulators affecting expression of eap and emp were investigated. Marked differences were found in expression of eap and emp between strain Newman and the 8325 derivatives SH1000 and 8325-4. Moreover, both genes were repressed in the presence of glucose. Analysis of expression of both genes in various regulatory mutants revealed that sarA and agr were involved in their regulation, but the data suggested that there were additional regulators of both genes. In a sae mutant, expression of both genes was severely repressed. sae expression was also reduced in the presence of glucose, suggesting that repression of eap and emp in glucose-containing medium may, in part, be a consequence of a decrease in expression of sae.

scholarly journals Genome-Wide Identification of Transcription Start Sites, Promoters and Transcription Factor Binding Sites in E. coli

PLoS ONE ◽  
2009 ◽  
Vol 4 (10) ◽  
pp. e7526 ◽  
Author(s):  
Alfredo Mendoza-Vargas ◽  
Leticia Olvera ◽  
Maricela Olvera ◽  
Ricardo Grande ◽  
Leticia Vega-Alvarado ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Transcription Factor Binding Sites ◽  
Transcription Factor Binding ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
E Coli ◽  
Factor Binding ◽  
Genome Wide

The correlations of the function and positional distribution of the cis-elements CArG around the TSS in the genes of Mus musculus

Genome ◽  
2009 ◽  
Vol 52 (3) ◽  
pp. 217-221 ◽  
Author(s):  
Xia Shen ◽  
Bruce Walsh ◽  
Jing J. Li ◽  
Hong X. Pang ◽  
Wen J. Wang ◽  
...  
Keyword(s):  
Binding Sites ◽  
Copy Number ◽  
Serum Response Factor ◽  
Sequence Data ◽  
Positional Distribution ◽  
Cis Elements ◽  
Transcription Start ◽  
Data Set ◽  
Control Set ◽  
Serum Response

While many studies of cis-elements CArG bound by serum response factor (SRF) are in progress, little is known about the positional distribution of the functional CArG elements around the transcription start site (TSS) of genes that they influence. We use a validated CArG data set to calculate the distance distribution of functional CArG elements around the TSS. Distances between adjacent CArGs were also analyzed. We compare these distributions with those derived using a control set of randomly selected CArGs (that were not experimentally validated for function). Our results show that most functional CArG elements (108 of 152, 71%) exist upstream of the annotated TSS, with copy number increasing as one moves closer to the TSS. Moreover, the average number of the CArG elements in the CArG-containing genes is significantly more than that in the control genes. Our study extends earlier bioinformatic analyses of functional CArG elements and provides an application of comparative sequence data to the identification of transcription factor binding sites.

A 182 bp fragment of the mouse pro alpha 1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice

1995 ◽  
Vol 108 (12) ◽  
pp. 3677-3684 ◽  
Author(s):  
G. Zhou ◽  
S. Garofalo ◽  
K. Mukhopadhyay ◽  
V. Lefebvre ◽  
C.N. Smith ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Reporter Gene ◽  
Type Ii Collagen ◽  
Mouse Embryos ◽  
Collagen Gene ◽  
Specific Expression ◽  
Intact Mouse ◽  
Endogenous Gene ◽  
Intron 1 ◽  
Beta Galactosidase

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro alpha 1(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a beta-galactosidase reporter gene. A construction containing a 3,000 bp promoter and a 3,020 bp intron 1 fragment directed high levels of beta-galactosidase expression specifically to chondrocytes. Expression of the transgene coincided with the temporal expression of the endogenous gene at all stages of embryonic development. Successive deletions of intron 1 delineated a 182 bp fragment which targeted beta-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. Transgenic mice harboring a 309 bp Col2a1 promoter lacking intron 1 tester sequences showed no beta-galactosidase expression in chondrocytes. Reduction of the 182 bp fragment to a 73 bp subfragment surrounding a decamer sequence previously reported to be involved in chondrocyte specificity, resulted in loss of transgene expression in chondrocytes. When the Col2a1 promoter was replaced with a minimal beta-globin promoter, the 182 bp intron 1 sequence was still able to target expression of the transgene to chondrocytes. We conclude that a 182 bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression on a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression.

Estrogen and progesterone receptor-binding sites on the chicken vitellogenin II gene: synergism of steroid hormone action

1988 ◽  
Vol 8 (12) ◽  
pp. 5323-5330
Author(s):  
A C Cato ◽  
E Heitlinger ◽  
H Ponta ◽  
L Klein-Hitpass ◽  
G U Ryffel ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Receptor Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Chloramphenicol Acetyltransferase ◽  
Synergistic Action ◽  
Gene Fragment ◽  
T47d Cells ◽  
Vitellogenin Gene

The chicken vitellogenin II gene is transcriptionally activated by estrogens. In transient transfection experiments in human T47D cells that contain receptors for various steroids, we showed estradiol, progestin, and androgen responses of a chimeric chicken vitellogenin II construct. This construct consists of DNA sequences from -626 to -590 upstream of the start of transcription of the chicken vitellogenin gene linked to the herpes simplex virus thymidine kinase promoter driving the transcription of the bacterial chloramphenicol acetyltransferase gene. Treatment of the transfected T47D cells with a combination of estradiol and the progestin R5020 led to a superinduction of chloramphenicol acetyltransferase activity, showing a synergistic action of these two steroids. This synergism was not observed upon treatment of the transfected cells with estradiol and the androgen dihydrotestosterone. Using point mutations in the vitellogenin gene fragment, we showed in functional and in in vitro DNase I footprinting assays with a purified progesterone receptor that, for the synergistic action of estradiol and R5020 to occur, the progesterone receptor must be bound to the vitellogenin gene fragment. The progesterone receptor-binding site was localized at -610 to -590, close to the consensus sequence (-626 to -613) for estrogen receptor binding and function. We therefore demonstrate here that two different steroid hormones can be functionally synergistic through the interaction of their corresponding receptors with two different binding sites adjacent to one another.

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