A G-string positive cis-regulatory element in the LpS1 promoter binds two distinct nuclear factors distributed non-uniformly in Lytechinus pictus embryos

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1345-1355 ◽  
Author(s):  
M. Xiang ◽  
S.Y. Lu ◽  
M. Musso ◽  
G. Karsenty ◽  
W.H. Klein
Keyword(s):  
Binding Site ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Mobility Shift ◽  
Specific Expression ◽  
Lytechinus Pictus ◽  
Nuclear Extracts ◽  
Nuclear Factors ◽  
Electrophoretic Mobility Shift Assays

The LpS1 alpha and beta genes of Lytechinus pictus are activated at the late cleavage stage of embryogenesis, with LpS1 mRNAs accumulating only in lineages contributing to aboral ectoderm. We had shown previously that 762 bp of 5' flanking DNA from the LpS1 beta gene was sufficient for proper temporal and aboral ectoderm specific expression. In the present study, we identified a strong positive cis-regulatory element at −70 bp to −75 bp in the LpS1 beta promoter with the sequence (G)6 and a similar, more distal cis-element at −721 bp to −726 bp. The proximal ‘G-string’ element interacted with two nuclear factors, one specific to ectoderm and one to endoderm/mesoderm nuclear extracts, whereas the distal G-string element interacted only with the ectoderm factor. The ectoderm and endoderm/mesoderm G-string factors were distinct based on their migratory behavior in electrophoretic mobility shift assays, binding site specificities, salt optima and EDTA sensitivity. The proximal G-string element shared homology with a binding site for the mammalian transcription factor IF1, a protein that binds to negative cis-regulatory elements in the mouse alpha 1(I) and alpha 2(I) collagen gene promoters. Competition experiments using wild-type and mutant oligonucleotides indicated that the ectoderm G-string factor and IF1 have similar recognition sites. Partially purified IF1 specifically bound to an oligonucleotide containing the proximal G-string of LpS1 beta. From our results, we suggest that the ectoderm G-string factor, a member of the G-rich DNA-binding protein family, activates the LpS1 gene in aboral ectoderm cells by binding to the LpS1 promoter at the proximal G-string site.

scholarly journals Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor.

1990 ◽  
Vol 10 (6) ◽  
pp. 2653-2659 ◽  
Author(s):  
D Kardassis ◽  
M Hadzopoulou-Cladaras ◽  
D P Ramji ◽  
R Cortese ◽  
V I Zannis ◽  
...  
Keyword(s):  
Binding Site ◽  
Regulatory Elements ◽  
Apob Gene ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Nuclear Extracts ◽  
Dna Binding Site ◽  
Gene Definition ◽  
Definition Of ◽  
Electrophoretic Mobility Shift Assays

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

Homeodomain proteins MEIS1 and PBXs regulate the lineage-specific transcription of the platelet factor 4 gene

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4748-4756 ◽  
Author(s):  
Yoshiaki Okada ◽  
Ryohei Nagai ◽  
Takahiro Sato ◽  
Eri Matsuura ◽  
Takashi Minami ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Platelet Factor 4 ◽  
Homeodomain Proteins ◽  
Mobility Shift ◽  
Specific Expression ◽  
Human Cord Blood ◽  
Platelet Factor ◽  
Nuclear Extracts

AbstractPlatelet factor 4 (PF4) is expressed during megakaryocytic differentiation. We previously reported that GATA-1 and ETS-1 regulate the rat PF4 promoter and transactivate the PF4 gene. For the present study, we investigated the regulatory elements and their transcription factors responsible for the lineage-specific expression of the PF4 gene. The promoter activities of deletion constructs were evaluated, and a novel regulatory element termed TME (tandem repeat of MEIS1 binding element) (–219 to –182) was defined. Binding proteins to TME were strongly detected in HEL nuclear extracts by electrophoresis mobility shift assay (EMSA), and they were purified by DNA affinity chromatography. By performing Western blottings and supershift assays, the binding proteins were identified as homeodomain proteins, MEIS1, PBX1B, and PBX2. These factors are expressed in megakaryocytes differentiated from CD34+ cells in human cord blood. MEIS1 and PBXs bind to the TME as MEIS1/PBX complexes and activate the PF4 promoter. In nonmegakaryocytic HepG2 cells, GATA-1 and ETS-1 activate the PF4 promoter approximately 10-fold. Surprisingly, we found that additional expression of both MEIS1 and PBX2 multiplied this major activation another 2-fold. This activation was not observed when MEIS1 binding sites in the TME were disrupted. Furthermore, inhibition of the binding of endogenous MEIS1/PBX complexes to the TME decreased the promoter activity by almost one half, in megakaryocytic HEL cells. Thus, these studies demonstrate that the homeodomain proteins, MEIS1, PBX1B, and PBX2, play an important role in megakaryocytic gene expression.

Characterization of the promoter elements required for hepatic and intestinal transcription of the human apoB gene: definition of the DNA-binding site of a tissue-specific transcriptional factor

1990 ◽  
Vol 10 (6) ◽  
pp. 2653-2659
Author(s):  
D Kardassis ◽  
M Hadzopoulou-Cladaras ◽  
D P Ramji ◽  
R Cortese ◽  
V I Zannis ◽  
...  
Keyword(s):  
Binding Site ◽  
Regulatory Elements ◽  
Apob Gene ◽  
Mobility Shift ◽  
Promoter Elements ◽  
Nuclear Extracts ◽  
Dna Binding Site ◽  
Gene Definition ◽  
Definition Of ◽  
Electrophoretic Mobility Shift Assays

The promoter elements important for intestinal and hepatic transcription of the human apoB gene have been localized downstream of nucleotide -150. Footprinting analysis using hepatic nuclear extracts identified four protected regions, -124 to -100, -97 to -93, -86 to -33, and +33 to +52. Gel electrophoretic mobility shift assays showed that multiple factors interact with the apoB sequence -86 to -33, while the region -88 to -61 binds a single nuclear factor. Methylation interference analysis and nucleotide substitution mutagenesis identified the binding site of the factor between residues -78 and -68. Binding competition experiments indicate that this factor recognizes the regulatory elements of other liver-specific genes.

Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins

1993 ◽  
Vol 13 (11) ◽  
pp. 6690-6701
Author(s):  
H Koizumi ◽  
M F Horta ◽  
B S Youn ◽  
K C Fu ◽  
B S Kwon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Site ◽  
Protein Interactions ◽  
Regulatory Element ◽  
Killer Cell ◽  
Mobility Shift ◽  
Specific Expression ◽  
Native Molecular Mass ◽  
Mobility Shift Assay

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

scholarly journals A Pair of Highly Conserved Two-Component Systems Participates in the Regulation of the Hypervariable FIR Proteins in Different Legionella Species

2007 ◽  
Vol 189 (9) ◽  
pp. 3382-3391 ◽  
Author(s):  
Michal Feldman ◽  
Gil Segal
Keyword(s):  
Binding Site ◽  
Legionella Pneumophila ◽  
Response Regulator ◽  
Regulatory Element ◽  
Expression System ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Mobility Shift ◽  
Type Iv ◽  
Essential Components

ABSTRACT Legionella pneumophila and other pathogenic Legionella species multiply inside protozoa and human macrophages by using the Icm/Dot type IV secretion system. The IcmQ protein, which possesses pore-forming activity, and IcmR, which functions as its chaperone, are two essential components of this system. It was previously shown that in 29 Legionella species, a large hypervariable-gene family (fir genes) is located upstream from a conserved icmQ gene, but although nonhomologous, the FIR proteins were found to function similarly together with their corresponding IcmQ proteins. Alignment of the regulatory regions of 29 fir genes revealed that they can be divided into three regulatory groups; the first group contains a binding site for the CpxR response regulator, which was previously shown to regulate the L. pneumophila fir gene (icmR); the second group, which includes most of the fir genes, contains the CpxR binding site and an additional regulatory element that was identified here as a PmrA binding site; and the third group contains only the PmrA binding site. Analysis of the regulatory region of two fir genes, which included substitutions in the CpxR and PmrA consensus sequences, a controlled expression system, as well as examination of direct binding with mobility shift assays, revealed that both CpxR and PmrA positively regulate the expression of the fir genes that contain both regulatory elements. The change in the regulation of the fir genes that occurred during the course of evolution might be required for the adaptation of the different Legionella species to their specific environmental hosts.

scholarly journals Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins.

1993 ◽  
Vol 13 (11) ◽  
pp. 6690-6701 ◽  
Author(s):  
H Koizumi ◽  
M F Horta ◽  
B S Youn ◽  
K C Fu ◽  
B S Kwon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Site ◽  
Protein Interactions ◽  
Regulatory Element ◽  
Killer Cell ◽  
Mobility Shift ◽  
Specific Expression ◽  
Native Molecular Mass ◽  
Mobility Shift Assay

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

scholarly journals The GC-box is critical for high level expression of the testis-specific Hsp70.2/Hst70 gene.

2007 ◽  
Vol 54 (1) ◽  
pp. 107-112 ◽  
Author(s):  
Wiesława Widłak ◽  
Natalia Vydra ◽  
Volha Dudaladava ◽  
Dorota Scieglińska ◽  
Bolesław Winiarski ◽  
...  
Keyword(s):  
Regulatory Element ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Specific Expression ◽  
Transcription Start Sites ◽  
Nuclear Extracts ◽  
Efficient Expression ◽  
Gc Box ◽  
Specific Expression Pattern ◽  
High Level

The Hsp70.2/Hst70 gene, which belongs to the 70 kDa heat-shock protein (HSP) family, is expressed specifically in primary spermatocytes and spermatids. The regulatory elements required for a high level of testis-specific expression of the gene are placed between the two major transcription start sites T1 and T2 (approximately 350 and 115 bp upstream of the starting ATG codon). Here we have shown that sequences proximal to the exon1/intron splicing site in the 5' untranslated region of the Hsp70.2/Hst70 gene, which include a highly conserved element called box B, are required for efficient expression of the chloramphenicol acetyltransferase reporter gene in testes of transgenic mice. However, in spite of the drastically reduced overall activity, the stage-specific expression pattern of the transgene was preserved after removal of these sequences. We have also shown that GC-box located downstream of the box B (approximately 210 bp upstream of the starting ATG codon) is indispensable for efficient expression of the Hsp70.2/Hst70 gene promoter in spermatogenic cells. The GC-box specifically binds proteins present in nuclear extracts from testes (putatively Sp1-like factors). A change in the pattern of such GC-box-interacting factors corresponds to activation of the Hsp70.2/Hst70 gene, confirming the importance of this regulatory element.

Identification and characterization of an Alu-containing, T-cell-specific enhancer located in the last intron of the human CD8 alpha gene

1993 ◽  
Vol 13 (11) ◽  
pp. 7056-7070
Author(s):  
J E Hambor ◽  
J Mennone ◽  
M E Coon ◽  
J H Hanke ◽  
P Kavathas
Keyword(s):  
T Cell ◽  
Binding Sites ◽  
Close Association ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Transcriptional Enhancer ◽  
Mobility Shift ◽  
Specific Expression ◽  
Hypersensitive Sites ◽  
Alpha Gene

Expression of the human CD8 alpha gene is restricted to cells of the lymphoid lineage and developmentally regulated during thymopoiesis. As an initial step towards understanding the molecular basis for tissue-specific expression of this gene, we surveyed the surrounding chromatin structure for potential cis-acting regulatory regions by DNase I hypersensitivity mapping and found four hypersensitive sites, three of which were T cell restricted. By using a reporter-based expression approach, a T-cell-specific enhancer was identified by its close association with a prominent T-cell-restricted hypersensitive sites in the last intron of the CD8 alpha gene. Deletion studies demonstrated that the minimal enhancer is adjacent to a negative regulatory element. DNA sequence analysis of the minimal enhancer revealed a striking cluster of consensus binding sites for Ets-1, TCF-1, CRE, GATA-3, LyF-1, and bHLH proteins which were verified by electrophoretic mobility shift assays. In addition, the 5' end of the enhancer was composed of an Alu repeat which contained the GATA-3, bHLH, and LyF-1 binding sites. Site-directed mutation of the Ets-1 and GATA-3 sites dramatically reduced enhancer activity. The functional importance of the other binding sites only became apparent when combinations of mutations were analyzed. Taken together, these results suggest that the human CD8 alpha gene is regulated by the interaction of multiple T-cell nuclear proteins with a transcriptional enhancer located in the last intron of the gene. Comparison of the CD8 alpha enhancer with other recently identified T-cell-specific regulatory elements suggests that a common set of transcription factors regulates several T-cell genes.

scholarly journals Differential regulation of the human nidogen gene promoter region by a novel cell-type-specific silencer element

1999 ◽  
Vol 338 (2) ◽  
pp. 343-350 ◽  
Author(s):  
Marlies ZEDLACHER ◽  
Marion SCHMOLL ◽  
Katrin ZIMMERMANN ◽  
Oliver HORSTKORTE ◽  
Roswitha NISCHT
Keyword(s):  
Gene Expression ◽  
Promoter Region ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Epidermal Keratinocytes ◽  
Mobility Shift ◽  
Sv40 Promoter ◽  
Specific Expression ◽  
Regulatory Domains ◽  
Silencer Element

Transfection analyses of the human nidogen promoter region in nidogen-producing fibroblasts from adult skin revealed multiple positive and negative cis-acting elements controlling nidogen gene expression. Characterization of the positive regulatory domains by gel mobility-shift assays and co-transfection studies in Drosophila SL2 cells unequivocally demonstrated that Sp1-like transcription factors are essential for a high expression of the human nidogen gene. Analysis of the negative regulatory domains identified a novel silencer element between nt -1333 and -1322, which is bound by a distinct nuclear factor, by using extracts from adult but not from embryonal fibroblasts. In embryonal fibroblasts, which express significantly higher amounts of nidogen mRNA as compared with adult fibroblasts, this inhibitory nidogen promoter region did not affect nidogen and SV40 promoter activities. The silencer element seems to be active only in nidogen-producing cells. Therefore this regulatory element might function in vivo to limit nidogen gene expression in response to external stimuli. However, none of the identified regulatory elements, including the silencer, contribute significantly to cell-specific expression of the human nidogen gene. Instead we provide evidence that gene expression in epidermal keratinocytes that are not producing nidogen is repressed by methylation-specific and chromatin-dependent mechanisms.

scholarly journals Identification and characterization of an Alu-containing, T-cell-specific enhancer located in the last intron of the human CD8 alpha gene.

1993 ◽  
Vol 13 (11) ◽  
pp. 7056-7070 ◽  
Author(s):  
J E Hambor ◽  
J Mennone ◽  
M E Coon ◽  
J H Hanke ◽  
P Kavathas
Keyword(s):  
T Cell ◽  
Binding Sites ◽  
Close Association ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Transcriptional Enhancer ◽  
Mobility Shift ◽  
Specific Expression ◽  
Hypersensitive Sites ◽  
Alpha Gene

Expression of the human CD8 alpha gene is restricted to cells of the lymphoid lineage and developmentally regulated during thymopoiesis. As an initial step towards understanding the molecular basis for tissue-specific expression of this gene, we surveyed the surrounding chromatin structure for potential cis-acting regulatory regions by DNase I hypersensitivity mapping and found four hypersensitive sites, three of which were T cell restricted. By using a reporter-based expression approach, a T-cell-specific enhancer was identified by its close association with a prominent T-cell-restricted hypersensitive sites in the last intron of the CD8 alpha gene. Deletion studies demonstrated that the minimal enhancer is adjacent to a negative regulatory element. DNA sequence analysis of the minimal enhancer revealed a striking cluster of consensus binding sites for Ets-1, TCF-1, CRE, GATA-3, LyF-1, and bHLH proteins which were verified by electrophoretic mobility shift assays. In addition, the 5' end of the enhancer was composed of an Alu repeat which contained the GATA-3, bHLH, and LyF-1 binding sites. Site-directed mutation of the Ets-1 and GATA-3 sites dramatically reduced enhancer activity. The functional importance of the other binding sites only became apparent when combinations of mutations were analyzed. Taken together, these results suggest that the human CD8 alpha gene is regulated by the interaction of multiple T-cell nuclear proteins with a transcriptional enhancer located in the last intron of the gene. Comparison of the CD8 alpha enhancer with other recently identified T-cell-specific regulatory elements suggests that a common set of transcription factors regulates several T-cell genes.

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