Biomedical subjects

D Wotton

Publications and source records attributed to D Wotton.

27 records · Page 2Linked to original sources

PubMed1994

Cooperative binding of Ets-1 and core binding factor to DNA.

Two phorbol ester-inducible elements (beta E2 and beta E3) within the human T-cell receptor beta gene enhancer each contain consensus binding sites for the Ets and core binding factor (CBF) transcription factor families. Recombinant Ets-1 and purified CBF bound individually to beta E2 and beta E3, in which the Ets and core sites are directly adjacent. In this report, we show that CBF and Ets-1 bind together to beta E2 and beta E3 and that Ets-1-CBF-DNA complexes are favored over the binding of either protein alone to beta E2. Formation of Ets-1-CBF-DNA complexes increased the affinity of Ets-1-DNA interactions and decreased the rate of dissociation of CBF from DNA. Ets-1-CBF-DNA complexes were not observed when either the Ets or core site was mutated. The spatial requirements for the cooperative interaction of Ets-1 and CBF were analyzed by oligonucleotide mutagenesis and binding site selection experiments. Core and Ets sites were coselected, and there appeared to be little constraint on the relative orientation and spacing of the two sites. These results demonstrate that CBF and Ets-1 form a high-affinity DNA-binding complex when both of their cognate sites are present and that the relative spacing and orientation of the two sites are unimportant. Ets and core sites are found in several T-cell-specific enhancers, suggesting that this interaction is of general importance in T-cell-specific transcription.

Animals↗

PubMed1993

Activity of both Raf and Ras is necessary for activation of transcription of the human T cell receptor beta gene by protein kinase C, Ras plays multiple roles.

Transcription of the T cell receptor beta gene is up-regulated during T cell activation. We have previously defined the elements within the TcR beta gene enhancer responsible for increased transcription in response to phorbol esters, which mimic part of the pathway for T cell activation. Using a reporter construct (beta E2 x 4) containing four copies of this inducible element, activation was achieved by the addition of phytohemagglutinin or phorbol esters. Activation was observed after 2 h of treatment, was maximal by 8 h, and could be blocked by the protein synthesis inhibitor cycloheximide. Coexpression of v-Ha-Ras-, v-Raf-, and v-Src-activated beta E2 x 4, and constitutively active mutants of protein kinase C also increased transcription from this construct. Calcium ionophore generated signals which synergized with both Ras and protein kinase C. Expression of a truncated Raf protein which has been shown to act in a dominant negative manner, was able to inhibit activation of beta E2 x 4, demonstrating that Raf plays an important role in T cell signaling. A dominant negative mutant of v-Ha-Ras inhibited all methods of activation tested, including transfection with v-Raf. Thus, Ras activity appears to be necessary at more than one point in the transduction of signals from T cell receptor to nucleus.

Actins↗

PubMed1993

Regulation of human T cell receptor beta gene expression by Ets-1.

Expression of the human TcR beta gene is controlled by an enhancer located 6kb 3' to the C beta 2 gene segment. The activity of this enhancer has been shown to be inducible with phorbol esters. Within the enhancer the beta E2 element is responsible for the major part of the inducibility, multimerised beta E2 alone is also highly phorbol ester inducible. The beta E2 element contains a consensus ets-binding site as well as a core motif, and we have shown that the beta E2 ets site binds both Ets-1 and Ets-2 in vitro and that purified core binding factor (CBF) can bind the core site present in beta E2. Mutations which specifically disrupt Ets-1 and Ets-2 binding abolish inducibility as well as reducing activity, whereas mutants which cannot bind CBF have only reduced basal activity. In Jurkat, which has a high level of endogenous Ets-1, multimerized beta E2 was inactive unless treated with PMA. However when transfected into cells with no detectable Ets-1 the beta E2 multimer was highly active in the absence of PMA. Co-transfection of an Ets-1 expression construct with the full enhancer into Jurkat cells led to a repression of enhancer activity, suggesting a repressive role for Ets-1. Co-transfection of Ets-1 was also able to repress strongly the activity of the beta E2 multimer. Repression of activity from both the full enhancer construct and the beta E2 multimer was most dramatic in the presence of PMA, suggesting that Ets-1 could block TcR beta activation. The Ets-1 expression construct used transactivated the HTLV-1 LTR which has also been shown to bind Ets-1. The repression of beta E2 activity by Ets-1 appears therefore to be specific. In conclusion, the combination of ets and core sites in beta E2 constitutes a novel inducible element, which is specifically transrepressed by Ets-1.

Base Sequence↗

PubMed1992

A phorbol ester response element within the human T-cell receptor beta-chain enhancer.

The activity of the T-cell receptor beta-chain gene enhancer is increased by activators of the protein kinase C pathway during T-cell activation. Analysis of mutant enhancer constructs identified two elements, beta E2 and beta E3, conferring phorbol ester inducibility. Multimerized beta E2 acted in isolation as a phorbol ester-responsive element. Both beta E2 and beta E3, which contain a consensus Ets-binding site, were shown to bind directly to the product of the c-ets-1 protooncogene. Both regions also bound a second factor, core-binding factor. Mutation of the beta E2 Ets site abolished the inducibility of the beta E2 multimer. beta E2 and beta E3 Ets site mutations also profoundly affected activity and inducibility of the enhancer. In contrast, enhancer activity but not its inducibility was affected by mutation of the beta E2 core-binding factor site. Cotransfection studies showed that Ets-1 specifically repressed activity of the multimerized beta E2 element and the complete T-cell receptor beta-chain enhancer. These data show that the T-cell receptor beta-chain enhancer responds to protein kinase C-mediated activation signals via a functional domain, composed of two elements, which contains binding sites for Ets transcription factors and which is negatively regulated by Ets-1.

Base Sequence↗

PubMed1991

Identification and functional analysis of the transcriptional enhancer of the human T cell receptor beta gene.

The productive rearrangement and transcription of T cell receptor (TcR) beta genes is confined to T lymphocytes and is subject to both tissue-specific and developmental regulation. In addition to their function in transcriptional control, cis-acting elements are likely to play a role in the regulation of the rearrangement process. In this report we describe the location of a strong and inducible transcriptional enhancer 3' to the human TcR C beta 2 gene segment. The core enhancer, defined by deletion analysis using a transient transfection assay, resided within 362 bp of DNA. This enhancer core was able to activate transcription from a heterologous promoter and functioned well in T and B lymphocytes, but only minimally in HeLa cells. In contrast, a longer fragment containing the enhancer core showed marked T cell specificity. The enhancer was highly inducible by phorbol esters, the molecular basis for the inducibility residing within a 118-bp region of the enhancer core. This inducibility may be important in modulation of TcR beta gene expression during T cell differentiation and/or activation.

Base Sequence↗

PubMed1990

A 3' transcriptional enhancer regulates tissue-specific expression of the human CD2 gene.

A strong lymphocyte-specific transcriptional enhancer was identified within a DNase I hypersensitive site at the 3' end of the human CD2 gene. Full activity, in a transient expression assay, was contained within a region of 550 bp (minimal enhancer). T cells which express CD2 could use the enhancer to activate transcription from the reporter gene chloramphenicol acetyltransferase in the context of a heterologous promoter. Lower levels of transcription were detected in non-CD2-expressing T cells and in B cells. In contrast, the enhancer did not function in the epithelial cell line HeLa or in Colo 320 HSR, a cell line of neuroendocrine origin. Low levels of enhancement were detectable from two core regions, which acted synergistically with other cis-acting sequences to generate the complete enhancer. DNase I footprinting studies identified six cis-acting sequences to which proteins bound. Five of these sequence motifs were novel; the sixth was a canonical cAMP response element. Topoisomerase II, and scaffold attachment region consensus sequences were also found within an A/T-rich area downstream of the minimal enhancer. Neither region was bound to the nuclear matrix. The CD2 enhancer is modular in structure, it is constructed of novel cis-acting sequences and it is a major component of the regulatory system that controls expression of the CD2 gene.

Base Sequence↗

PubMed1990

DNase hypersensitivity and methylation of the human CD3G and D genes during T-cell development.

The mouse and human CD3G and D genes are organized in opposite transcriptional orientation, their 5' ends being separated by about 1.6 kilobases (kb) of DNA. The molecular basis of the tissue-specific regulation of expression of the human CD3G and D genes were examined using DNase I hypersensitivity and CpG methylation analysis. Two T cell-specific DNase I hypersensitivity sites were defined within the intergenic region. A third hypersensitive site (DHS3) was detected 0.4 kb 3' to the CD3D gene. This latter site was present in all T cells, but was absent in all other committed cell types examined. DHS3 was also detected in the lympho-myeloid progenitor cell KG1, but was absent when this line was induced to differentiate to the macrophage lineage. The intergenic region was undermethylated in T cells expressing CD3, but was in general more extensively methylated in other cell types. Importantly, however, in KG1 sublines which express the CD3 genes the intergenic region remains extensively methylated. These results define areas 3' to the D gene and within the intergenic region which contain regulatory elements that influence both CD3D and G expression. They further show that transcription from the CD3D and G genes may occur initially from a methylated promoter. Significantly, the 3' regulatory region was shown to adopt an open chromatin structure prior to lineage commitment and before CD3 transcription.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1989

Chromatin configuration of the human CD2 gene locus during T-cell development.

To investigate the molecular basis for the tissue-specific expression of the human CD2 gene, its chromatin configuration was assessed by determining DNase I hypersensitivity and the degree of methylation during T-cell lineage commitment and development. Tissue-specific DNase I-hypersensitive sites were found within the 5' promoter region and a region 3' of the gene essential for gene expression. DNase I hypersensitivity of the 5' region correlated strictly with transcriptional activity, whereas hypersensitivity of the 3' region correlated with T-cell progenitor activity or lineage commitment but not necessarily with transcription. Hha I and Hpa II sites around the 5' and 3' regions were undermethylated in CD2-expressing T cells but were more extensively methylated in other cell types. These results define likely regulatory elements both upstream and downstream of the CD2 gene that control its tissue-specific expression. Further, they show that the 3' regulatory region adopts an open chromatin configuration prior to lineage commitment and during early stages of T-cell development before the CD2 gene is transcribed.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1988

The structure of the human CD2 gene and its expression in transgenic mice.

We report the genomic organization of the human CD2 gene and its expression in transgenic mice. A 28.5 kb segment of DNA consisting of 4.5 kb 5' flanking sequences, 15 kb containing the gene's five exons and 9 kb of 3' flanking sequences can direct the expression of the CD2 gene only on thymocytes, circulating T cells and megakaryocytes of the transgenic mice. The expression of each copy of the human CD2 transgene appears to be as high as the endogenous mouse CD2 gene and as high as the expression on the surface of human T lymphocytes, independent of the site of integration and dependent on the copy number of genes that have integrated.

Amino Acid Sequence↗