Characterization of a single strong tissue-specific enhancer downstream from the three human genes encoding placental lactogen

1994 ◽  
Vol 14 (1) ◽  
pp. 93-103
Author(s):  
P Jacquemin ◽  
C Oury ◽  
B Peers ◽  
A Morin ◽  
A Belayew ◽  
...  
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Chromosome 17 ◽  
Placental Lactogen ◽  
Cell Nuclei ◽  
Enhancer Activity ◽  
Protein Binding Sites ◽  
Human Genes ◽  
Genes Encoding

The human genes coding for growth hormone (hGH) and placental lactogen (choriosomatomammotropic hormone [hCS]) are clustered on chromosome 17 in the following order: 5' hGH-N hCS-L hCS-A hGH-V hCS-B 3'. So far, a single placenta-specific enhancer has been identified in the locus, 2 kb downstream from the hCS-B gene, and shown to comprise one in vitro binding site for a nuclear protein. We here provide evidence that the hCS-B enhancer is more complex: (i) protection against DNase I digestion in the 3' flanking region of the hCS-B gene reveals four binding sites (DF-1, DF-2, DF-3, and DF-4) for nuclear proteins from either placental or HeLa cells, and (ii) placenta-specific enhancer activity can be fully exerted in transient expression experiments by a 126-bp fragment comprising the DF-3 and DF-4 protein-binding sites. By dissecting this region, we show that enhancer activity is mediated by a synergy between DF-3 and DF-4. Competitions with various oligonucleotides in footprinting and gel retardation experiments indicate that the same protein or set of proteins, different in HeLa and placenta cell nuclei, interacts with sites DF-2, DF-3, and DF-4. We also studied the regions of the hCS-L and hCS-A genes which are highly similar to the hCS-B enhancer. Although they each present the same four protein-binding sites, they exhibit only minor enhancer activity.

scholarly journals Characterization of a single strong tissue-specific enhancer downstream from the three human genes encoding placental lactogen.

1994 ◽  
Vol 14 (1) ◽  
pp. 93-103 ◽  
Author(s):  
P Jacquemin ◽  
C Oury ◽  
B Peers ◽  
A Morin ◽  
A Belayew ◽  
...  
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Chromosome 17 ◽  
Placental Lactogen ◽  
Cell Nuclei ◽  
Enhancer Activity ◽  
Protein Binding Sites ◽  
Human Genes ◽  
Genes Encoding

The human genes coding for growth hormone (hGH) and placental lactogen (choriosomatomammotropic hormone [hCS]) are clustered on chromosome 17 in the following order: 5' hGH-N hCS-L hCS-A hGH-V hCS-B 3'. So far, a single placenta-specific enhancer has been identified in the locus, 2 kb downstream from the hCS-B gene, and shown to comprise one in vitro binding site for a nuclear protein. We here provide evidence that the hCS-B enhancer is more complex: (i) protection against DNase I digestion in the 3' flanking region of the hCS-B gene reveals four binding sites (DF-1, DF-2, DF-3, and DF-4) for nuclear proteins from either placental or HeLa cells, and (ii) placenta-specific enhancer activity can be fully exerted in transient expression experiments by a 126-bp fragment comprising the DF-3 and DF-4 protein-binding sites. By dissecting this region, we show that enhancer activity is mediated by a synergy between DF-3 and DF-4. Competitions with various oligonucleotides in footprinting and gel retardation experiments indicate that the same protein or set of proteins, different in HeLa and placenta cell nuclei, interacts with sites DF-2, DF-3, and DF-4. We also studied the regions of the hCS-L and hCS-A genes which are highly similar to the hCS-B enhancer. Although they each present the same four protein-binding sites, they exhibit only minor enhancer activity.

EVALUATION OF TISSUE STEROID BINDING IN VITRO

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S223-S246 ◽  
Author(s):  
C. R. Wira ◽  
H. Rochefort ◽  
E. E. Baulieu
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Experimental System ◽  
Specific Protein ◽  
Coupling Mechanism ◽  
Target Tissue ◽  
Protein Binding Sites ◽  
Definition Of ◽  
Hormone Specificity

ABSTRACT The definition of a RECEPTOR* in terms of a receptive site, an executive site and a coupling mechanism, is followed by a general consideration of four binding criteria, which include hormone specificity, tissue specificity, high affinity and saturation, essential for distinguishing between specific and nonspecific binding. Experimental approaches are proposed for choosing an experimental system (either organized or soluble) and detecting the presence of protein binding sites. Techniques are then presented for evaluating the specific protein binding sites (receptors) in terms of the four criteria. This is followed by a brief consideration of how receptors may be located in cells and characterized when extracted. Finally various examples of oestrogen, androgen, progestagen, glucocorticoid and mineralocorticoid binding to their respective target tissues are presented, to illustrate how researchers have identified specific corticoid and mineralocorticoid binding in their respective target tissue receptors.

scholarly journals Transcription signals and protein binding sites for sericin gene transcription in vitro

1989 ◽  
Vol 264 (31) ◽  
pp. 18707-18713 ◽  
Author(s):  
K Matsuno ◽  
C C Hui ◽  
S Takiya ◽  
T Suzuki ◽  
K Ueno ◽  
...  
Keyword(s):  
Protein Binding ◽  
Gene Transcription ◽  
Binding Sites ◽  
Protein Binding Sites ◽  
Transcription In Vitro ◽  
Transcription Signals

scholarly journals Regulation of the human T-cell receptor alpha gene enhancer: multiple ubiquitous and T-cell-specific nuclear proteins interact with four hypomethylated enhancer elements.

1990 ◽  
Vol 10 (9) ◽  
pp. 4720-4727 ◽  
Author(s):  
I C Ho ◽  
J M Leiden
Keyword(s):  
T Cell ◽  
Protein Binding ◽  
Binding Sites ◽  
Nuclear Protein ◽  
Cell Receptor ◽  
Enhancer Activity ◽  
Protein Binding Sites ◽  
Human T Cell ◽  
Alpha Gene ◽  
Enhancer Function

Transcription of human T-cell receptor (TCR) alpha genes is regulated by a T-cell-specific transcriptional enhancer that is located 4.5 kilobases 3' of the C alpha gene segment. Previous studies have demonstrated that this enhancer contains at least five nuclear protein-binding sites called T alpha 1 to T alpha 5. In the studies described in this report, we have determined the molecular requirements for human TCR alpha enhancer function. In vitro mutagenesis and deletion analyses demonstrated that full enhancer activity is retained in a 116-base-pair fragment containing the T alpha 1 and T alpha 2 nuclear protein-binding sites and that both of these sites are required for full enhancer function. Functional enhancer activity requires that the T alpha 1 and T alpha 2 binding sites be separated by more than 15 and fewer than 85 base pairs. However, the sequence of this spacer region and the relative phase of the two binding sites on the DNA helix do not affect enhancer function. Deletion and mutation analyses demonstrated that the T alpha 3 and T alpha 4 nuclear protein-binding sites are not necessary or sufficient for TCR alpha enhancer activity. However, a fragment containing these two sites was able to compensate for T alpha 1 and T alpha 2 mutations that otherwise abolished enhancer activity. Electrophoretic mobility shift analyses of the TCR alpha enhancer binding proteins revealed that the T alpha 1, T alpha 3, and T alpha 4 binding proteins are expressed in a variety of T-cell and non-T-cell tumor cell lines. In contrast, one of the two T alpha 2 binding activities was detected only in T-cell nuclear extracts. The activity of the TCR alpha enhancer does not appear to be regulated solely at the level of DNA methylation on that the enhancer sequences were found to be identically hypomethylated in B and T cells as compared with fibroblasts. Taken together, these results suggest that TCR alpha enhancer activity is regulated by the interaction of multiple T-cell-specific and ubiquitous nuclear proteins with partially redundant cis-acting enhancer elements that are hypomethylated in cells of the lymphoid lineage.

An Improved Method for Identifying Specific DNA-Protein-Binding Sites In Vitro

2017 ◽  
Vol 59 (2-3) ◽  
pp. 59-65
Author(s):  
Liangyan Wang ◽  
Huizhi Lu ◽  
Yunguang Wang ◽  
Su Yang ◽  
Hong Xu ◽  
...  
Keyword(s):  
Protein Binding ◽  
Binding Sites ◽  
Improved Method ◽  
Protein Binding Sites

Identification and characterization of a T-cell-specific enhancer adjacent to the murine CD4 gene

1991 ◽  
Vol 11 (11) ◽  
pp. 5506-5515
Author(s):  
S Sawada ◽  
D R Littman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Minor Effect ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Protein Binding Sites ◽  
Factor Binding

Expression of the CD4 and CD8 glycoproteins is a tightly regulated process tied to the maturation of functionally distinct classes of thymocytes. Therefore, understanding of the mechanism of expression of the genes encoding CD4 and CD8 is likely to yield important insight into regulation of the differentiated functions of T cells. Here, we report the identification of a T-cell-specific enhancer in a DNase I-hypersensitive region about 13 kb 5' of the transcription initiation site of the murine CD4 gene. Within the minimal enhancer element, at least three nuclear protein binding sites were identified by DNase I footprint analysis. One site contains the consensus motif for TCF-1 alpha/LEF-1, a recently identified HMG box transcription factor primarily expressed in pre-B and T cells. By Southwestern (DNA-protein) blotting and binding competition analyses, the protein binding to this site was found to be indistinguishable from TCF-1 alpha/LEF-1. Mutagenesis of this site resulted in loss of factor binding but had a relatively minor effect on enhancer activity. In contrast, mutations in another site, containing two consensus binding motifs for basic helix-loop-helix proteins, abolished factor binding and dramatically reduced enhancer activity. None of the protein binding sites had activity on its own, suggesting that the CD4 enhancer requires the interaction of multiple regulatory sites.

scholarly journals Identification and characterization of a T-cell-specific enhancer adjacent to the murine CD4 gene.

1991 ◽  
Vol 11 (11) ◽  
pp. 5506-5515 ◽  
Author(s):  
S Sawada ◽  
D R Littman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Protein Binding ◽  
Binding Sites ◽  
Dnase I ◽  
Minor Effect ◽  
Enhancer Activity ◽  
Enhancer Element ◽  
Protein Binding Sites ◽  
Factor Binding

Expression of the CD4 and CD8 glycoproteins is a tightly regulated process tied to the maturation of functionally distinct classes of thymocytes. Therefore, understanding of the mechanism of expression of the genes encoding CD4 and CD8 is likely to yield important insight into regulation of the differentiated functions of T cells. Here, we report the identification of a T-cell-specific enhancer in a DNase I-hypersensitive region about 13 kb 5' of the transcription initiation site of the murine CD4 gene. Within the minimal enhancer element, at least three nuclear protein binding sites were identified by DNase I footprint analysis. One site contains the consensus motif for TCF-1 alpha/LEF-1, a recently identified HMG box transcription factor primarily expressed in pre-B and T cells. By Southwestern (DNA-protein) blotting and binding competition analyses, the protein binding to this site was found to be indistinguishable from TCF-1 alpha/LEF-1. Mutagenesis of this site resulted in loss of factor binding but had a relatively minor effect on enhancer activity. In contrast, mutations in another site, containing two consensus binding motifs for basic helix-loop-helix proteins, abolished factor binding and dramatically reduced enhancer activity. None of the protein binding sites had activity on its own, suggesting that the CD4 enhancer requires the interaction of multiple regulatory sites.

scholarly journals Conserved Structure Modules within the lncRNA SChLAP1 Mediate Protein Recognition Implicated in Aggressive Prostate Cancer

2021 ◽  
Author(s):  
Emily J McFadden ◽  
James P Falese ◽  
Amanda E Hargrove
Keyword(s):  
Prostate Cancer ◽  
Secondary Structure ◽  
Protein Binding ◽  
Binding Sites ◽  
Cancer Metastasis ◽  
Magnesium Concentration ◽  
Purification Method ◽  
Aggressive Prostate Cancer ◽  
Protein Binding Sites

The lncRNA Second Chromosome Locus Associated with Prostate 1 (SChLAP1) was previously identified as a predictive biomarker and driver of aggressive prostate cancer. Recent work suggests that SChLAP1 may bind the SWI/SNF chromatin remodeling complex to promote prostate cancer metastasis, though the exact role of SWI/SNF recognition is debated. To date, there are no detailed biochemical studies of apo SChLAP1 or the SChLAP1:SWI/SNF complex. Herein, we report the first secondary structure model of SChLAP1 utilizing SHAPE-MaP both in vitro and in cellulo. Comparison of the in vitro and in cellulo data via ΔSHAPE identified putative protein binding sites within SChLAP1, specifically to evolutionarily conserved exons of the transcript. We also demonstrate that global SChLAP1 secondary structure is sensitive to both purification method and magnesium concentration. Further, we identified a 3'-fragment of SChLAP1 (SChLAP1Frag) that harbors multiple potential protein binding sites and presents a robustly folded secondary structure, supporting a functional role for this region. This work lays the foundation for future efforts in selective targeting and disruption of the SChLAP1:protein interface and the development of new therapeutic avenues in prostate cancer treatment.

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