A 5′ Regulatory Sequence Containing Two Ets Motifs Controls the Expression of the Wiskott-Aldrich Syndrome Protein (WASP) Gene in Human Hematopoietic Cells

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4554-4560 ◽  
Author(s):  
A. Petrella ◽  
I. Doti ◽  
V. Agosti ◽  
P. Carandente Giarrusso ◽  
D. Vitale ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Jurkat Cells ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Specific Expression ◽  
Wiskott Aldrich Syndrome ◽  
Nuclear Extracts ◽  
Ets Family ◽  
Wasp Gene ◽  
Syndrome Protein

The recently-identified Wiskott-Aldrich syndrome protein gene (WASP) is responsible for the Wiskott-Aldrich X-linked immunodeficiency as well as for isolated X-linked thrombocytopenia (XLT). To characterize the regulatory sequences of the WASP gene, we have isolated, sequenced and functionally analyzed a 1.6-Kb DNA fragment upstream of the WASP coding sequence. Transfection experiments showed that this fragment is capable of directing efficient expression of the reporter chloramphenicol acetyltransferase (CAT) gene in all human hematopoietic cell lines tested. Progressive 5′ deletions showed that the minimal sequence required for hematopoietic-specific expression consists of 137 bp upstream of the transcription start site. This contains potential binding sites for several hematopoietic transcription factors and, in particular, two Ets-1 consensus that proved able to specifically bind to proteins present in nuclear extracts of Jurkat cells. Overexpression of Ets-1 in HeLa resulted in transactivation of the CAT reporter gene under the control of WASP regulatory sequences. Disruption of the Ets-binding sequences by side-directed mutagenesis abolished CAT expression in Jurkat cells, indicating that transcription factors of the Ets family play a key role in the control of WASP transcription.

A 5′ Regulatory Sequence Containing Two Ets Motifs Controls the Expression of the Wiskott-Aldrich Syndrome Protein (WASP) Gene in Human Hematopoietic Cells

Blood ◽  
1998 ◽  
Vol 91 (12) ◽  
pp. 4554-4560 ◽  
Author(s):  
A. Petrella ◽  
I. Doti ◽  
V. Agosti ◽  
P. Carandente Giarrusso ◽  
D. Vitale ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Jurkat Cells ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Specific Expression ◽  
Wiskott Aldrich Syndrome ◽  
Nuclear Extracts ◽  
Ets Family ◽  
Wasp Gene ◽  
Syndrome Protein

Abstract The recently-identified Wiskott-Aldrich syndrome protein gene (WASP) is responsible for the Wiskott-Aldrich X-linked immunodeficiency as well as for isolated X-linked thrombocytopenia (XLT). To characterize the regulatory sequences of the WASP gene, we have isolated, sequenced and functionally analyzed a 1.6-Kb DNA fragment upstream of the WASP coding sequence. Transfection experiments showed that this fragment is capable of directing efficient expression of the reporter chloramphenicol acetyltransferase (CAT) gene in all human hematopoietic cell lines tested. Progressive 5′ deletions showed that the minimal sequence required for hematopoietic-specific expression consists of 137 bp upstream of the transcription start site. This contains potential binding sites for several hematopoietic transcription factors and, in particular, two Ets-1 consensus that proved able to specifically bind to proteins present in nuclear extracts of Jurkat cells. Overexpression of Ets-1 in HeLa resulted in transactivation of the CAT reporter gene under the control of WASP regulatory sequences. Disruption of the Ets-binding sequences by side-directed mutagenesis abolished CAT expression in Jurkat cells, indicating that transcription factors of the Ets family play a key role in the control of WASP transcription.

scholarly journals A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor.

1994 ◽  
Vol 14 (2) ◽  
pp. 1383-1394 ◽  
Author(s):  
Y H Lee ◽  
M Yano ◽  
S Y Liu ◽  
E Matsunaga ◽  
P F Johnson ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Ternary Complex ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Mrna Levels ◽  
Binding Studies ◽  
Mobility Shift ◽  
Specific Expression ◽  
Nuclear Extracts

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

The Mouse Homolog of the Wiskott–Aldrich Syndrome Protein (WASP) Gene Is Highly Conserved and Maps near the Scurfy (sf) Mutation on the X Chromosome

Genomics ◽  
1995 ◽  
Vol 29 (2) ◽  
pp. 471-477 ◽  
Author(s):  
JONATHAN M.J. DERRY ◽  
PHILIPP WIEDEMANN ◽  
PATRICK BLAIR ◽  
YUKER WANG ◽  
JULIE A. KERNS ◽  
...  
Keyword(s):  
X Chromosome ◽  
Mouse Homolog ◽  
Wiskott Aldrich Syndrome ◽  
Wasp Gene ◽  
Syndrome Protein

scholarly journals Successful renal transplantation in a patient with a Wiskott-Aldrich syndrome protein (WASP) gene mutation

2015 ◽  
Vol 28 (8) ◽  
pp. 1005-1009 ◽  
Author(s):  
Zita Chovancova ◽  
Milan Kuman ◽  
Marcela Vlkova ◽  
Jiri Litzman
Keyword(s):  
Renal Transplantation ◽  
Gene Mutation ◽  
Wiskott Aldrich Syndrome ◽  
Wasp Gene ◽  
Syndrome Protein

scholarly journals Identification of a DNA sequence involved in osteoblast-specific gene expression via interaction with helix-loop-helix (HLH)-type transcription factors.

1994 ◽  
Vol 126 (3) ◽  
pp. 773-782 ◽  
Author(s):  
M Tamura ◽  
M Noda
Keyword(s):  
Transcription Factors ◽  
Promoter Region ◽  
Gene Promoter ◽  
Binding Activity ◽  
Specific Gene ◽  
Specific Expression ◽  
Helix Loop Helix ◽  
Nuclear Extracts ◽  
Osteocalcin Gene ◽  
E Box

To elucidate regulatory mechanism(s) underlying differentiation of osteoblasts, we examined involvement of helix-loop-helix (HLH)-type transcription factors in osteoblast-specific expression of a phenotypic marker gene which encodes osteocalcin, a major noncollagenous bone matrix protein, exclusively expressed in osteoblasts. Overexpression of a dominant negative HLH protein, Id-1, decreased the activity of the 1.1-kb osteocalcin gene promoter cotransfected into rat osteoblastic osteosarcoma ROS17/2.8 cells. Analysis of deletion mutants revealed that a 264-bp fragment of osteocalcin promoter (-198 to +66) was sufficient for the Id-1-dependent suppression. Furthermore, the activity of the same promoter fragment (-198 to +66) was enhanced when antisense Id-1 expression vector was cotransfected. This osteocalcin gene promoter region contains two sites of an E-box motif, a consensus binding site for HLH proteins, which we refer to as OCE1 (CACATG, at -102) and OCE2 (CAGCTG, at -149), respectively. Mutagenesis in OCE1 but not OCE2 led to greater than 50% reduction in transcriptional activity of the osteocalcin gene promoter. Electrophoresis mobility shift assay indicated that factors in nuclear extracts prepared from ROS17/2.8 cells bound to the 30-bp oligonucleotide probe containing the E-box motif of OCE1. This binding was competed out by OCE1 oligonucleotide but neither by OCmE1 oligonucleotide in which E-box motif was mutated nor by OCE2. The OCE1-binding activity in the nuclear extracts of ROS17/2.8 cells was reduced by 70% when bacterially expressed Id-1 protein was added to the reaction mixture, suggesting the involvement of HLH proteins in the DNA/protein complex formation. In contrast to the osteoblast-like cells, OCE1-binding activity in the nuclear extracts of C3H10T1/2 fibroblasts was very low. However, when these fibroblasts were treated with recombinant human bone morphogenetic protein-2 which induced expression of osteocalcin as well as other phenotypic markers of osteoblasts, OCE1-binding activity was increased approximately 40-fold, indicating that OCE1 would be involved in the tissue-specific expression of the osteocalcin gene. These findings indicated for the first time that osteoblast-specific gene transcription is regulated via the interaction between certain E-box binding transcription factor(s) in osteoblasts and the OCE1 sequence in the promoter region of the osteocalcin gene.

Mutations of the Wiskott-Aldrich Syndrome Protein (WASP): hotspots, effect on transcription, and translation and phenotype/genotype correlation

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 4010-4019 ◽  
Author(s):  
Yinzhu Jin ◽  
Cinzia Mazza ◽  
Jacinda R. Christie ◽  
Silvia Giliani ◽  
Maurilia Fiorini ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Strong Association ◽  
Wiskott Aldrich Syndrome ◽  
Cytoskeleton Organization ◽  
Number Of Patients ◽  
Increased Risk ◽  
Mutational Hotspots ◽  
Actin Cytoskeleton Organization ◽  
Wasp Gene ◽  
Syndrome Protein

Abstract The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immune deficiency disorder characterized by thrombocytopenia, small platelet size, eczema, recurrent infections, and increased risk of autoimmune disorders and malignancies. X-linked thrombocytopenia (XLT) is an allelic variant of WAS which presents with a milder phenotype, generally limited to thrombocytopenia. WAS and XLT are caused by mutations of the Wiskott-Aldrich syndrome protein (WASP) gene which encodes a 502-amino acid protein, named WASP. WASP is thought to play a role in actin cytoskeleton organization and cell signaling. Here, we report the identification of 141 unique mutations, 71 not previously reported, from 227 WAS/XLT families with a total of 262 affected members. When possible we studied the effects of these mutations on transcription, RNA splicing, and protein expression. By analyzing a large number of patients with WAS/XLT at the molecular level we identified 5 mutational hotspots in the WASP gene and have been able to establish a strong association between genotype and phenotype. (Blood. 2004;104:4010-4019)

scholarly journals S100A8/S100A9 cytokine acts as a transcriptional coactivator during breast cellular transformation

2021 ◽  
Vol 7 (1) ◽  
pp. eabe5357
Author(s):  
Ruisheng Song ◽  
Kevin Struhl
Keyword(s):  
Transcription Factors ◽  
Transcriptional Activation ◽  
Cellular Transformation ◽  
Extracellular Proteins ◽  
Sequence Motifs ◽  
Transcriptional Coactivator ◽  
Specific Expression ◽  
Calcium Dependent ◽  
Nuclear Extracts ◽  
Dna Sequence Motifs

Cytokines are extracellular proteins that convey messages between cells by interacting with cognate receptors at the cell surface and triggering signaling pathways that alter gene expression and other phenotypes in an autocrine or paracrine manner. Here, we show that the calcium-dependent cytokines S100A8 and S100A9 are recruited to numerous promoters and enhancers in a model of breast cellular transformation. This recruitment is associated with multiple DNA sequence motifs recognized by DNA binding transcription factors that are linked to transcriptional activation and are important for transformation. The cytokines interact with these transcription factors in nuclear extracts, and they activate transcription when artificially recruited to a target promoter. Nuclear-specific expression of S100A8/A9 promotes oncogenic transcription and leads to enhanced breast transformation phenotype. These results suggest that, in addition to its classical cytokine function, S100A8/A9 can act as a transcriptional coactivator.

A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor

1994 ◽  
Vol 14 (2) ◽  
pp. 1383-1394
Author(s):  
Y H Lee ◽  
M Yano ◽  
S Y Liu ◽  
E Matsunaga ◽  
P F Johnson ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Ternary Complex ◽  
Sequence Similarity ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Mrna Levels ◽  
Binding Studies ◽  
Mobility Shift ◽  
Specific Expression ◽  
Nuclear Extracts

The rat CYP2D5 gene encodes a cytochrome P450 and is expressed in liver cells. Its expression commences a few days after birth, and maximal mRNA levels are achieved when animals reach puberty. Transfection and DNA binding studies were performed to investigate the mechanism controlling developmentally programmed, liver-specific expression of CYP2D5. Transfection studies using a series of CYP2D5 upstream DNA chloramphenicol acetyltransferase gene fusion constructs identified a segment of DNA between nucleotides -55 and -156 that conferred transcriptional activity in HepG2 cells. Activity was markedly increased by cotransfection with a vector expressing C/EBP beta but was unaffected by vectors producing other liver-enriched transcription factors (C/EBP alpha, HNF-1 alpha, and DBP). DNase I footprinting revealed a region protected by both HepG2 and liver cell nuclear extracts between nucleotides -83 and -112. This region displayed some sequence similarity to the Sp1 consensus sequence and was able to bind the Sp1 protein, as assessed by a gel mobility shift assay. The role of Sp1 in CYP2D5 transcription was confirmed by trans activation of the 2D5-CAT construct in Drosophila melanogaster cells by using an Sp1 expression vector. C/EBP beta alone was unable to directly bind the -83 to -112 region of the promoter but was able to produce a ternary complex when combined with HepG2 nuclear extracts or recombinant human Sp1. C/EBP alpha was unable to substitute for C/EBP beta in forming this ternary complex. A poor C/EBP binding site is present adjacent to the Sp1 site, and mutagenesis of this site abolished formation of the ternary complex with the CYP2D5 regulatory region. These result establish that two transcription factors can work in conjunction, possibly by protein-protein interaction, to activate the CYP2D5 gene.

scholarly journals Global properties of regulatory sequences are predicted by transcription factor recognition mechanisms

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zain M. Patel ◽  
Timothy R. Hughes
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Binding Sites ◽  
Transcription Factor Binding Sites ◽  
Transcription Factor Binding ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Factor Binding ◽  
Global Properties ◽  
Regulatory Sites

Abstract Background Mammalian genomes contain millions of putative regulatory sequences, which are delineated by binding of multiple transcription factors. The degree to which spacing and orientation constraints among transcription factor binding sites contribute to the recognition and identity of regulatory sequence is an unresolved but important question that impacts our understanding of genome function and evolution. Global mechanisms that underlie phenomena including the size of regulatory sequences, their uniqueness, and their evolutionary turnover remain poorly described. Results Here, we ask whether models incorporating different degrees of spacing and orientation constraints among transcription factor binding sites are broadly consistent with several global properties of regulatory sequence. These properties include length, sequence diversity, turnover rate, and dominance of specific TFs in regulatory site identity and cell type specification. Models with and without spacing and orientation constraints are generally consistent with all observed properties of regulatory sequence, and with regulatory sequences being fundamentally small (~ 1 nucleosome). Uniqueness of regulatory regions and their rapid evolutionary turnover are expected under all models examined. An intriguing issue we identify is that the complexity of eukaryotic regulatory sites must scale with the number of active transcription factors, in order to accomplish observed specificity. Conclusions Models of transcription factor binding with or without spacing and orientation constraints predict that regulatory sequences should be fundamentally short, unique, and turn over rapidly. We posit that the existence of master regulators may be, in part, a consequence of evolutionary pressure to limit the complexity and increase evolvability of regulatory sites.

scholarly journals Mutational screening of WASP gene in patients with Wiskott-Aldrich syndrome

2021 ◽  
Vol 19 (1) ◽  
pp. 61-68
Author(s):  
Luong Thi Lan Anh ◽  
Nguyen Thanh Hoa ◽  
Nguyen Hai Ha ◽  
Dang Ton Nguyen
Keyword(s):  
Viral Infections ◽  
Clinical Symptoms ◽  
Data Set ◽  
Wiskott Aldrich Syndrome ◽  
Immunodeficiency Disorder ◽  
Increased Risk ◽  
Wasp Gene ◽  
Families With Children ◽  
Syndrome Protein ◽  
Flanking Regions

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive immunodeficiency disorder characterized by thrombocytopenia and small-sized platelets, eczema, recurrent bacterial and viral infections, higher incidence of autoimmunity and an increased risk of malignancies. WAS occurs due to the mutation or loss of Wiskott-Aldrich Syndrome Protein (WASP) gene located on Xp11.22 – p11.23 of the short arm of the X chromosome. The absence of functional WASP leads to severe clinical symptoms that results in the deaths of patients if they are not diagnosed and treated early. The objective of the study was to identify mutations in the WASP gene of families with children diagnosed with WAS.The whole coding sequence and the intron-exon flanking regions of the WASP were sequenced by Sanger method. Two cases of children who has WAS were found tocarrymutations in the WASP gene. A c.702insAC mutation leadeda frameshift at position of codon 236 and terminated the protein at the position of codon 262 was identified in patient WA007 and a c.91G>A mutation that transformed glutamic acid to lysineat codon 31 was determined in patient WA010.This study provides a data set and screening of mutations in theWASP gene inVietnamese patientsto further identify the genetic causes and contribute to the clinical management and genetic counseling for the affected families.

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