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Query: EC:3.5.4.4 (adenosine deaminase)
 5,136 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have explored the template and factor requirements for in vitro transcription of the GC-rich promoter of the murine adenosine deaminase gene. The core promoter consists of an A-rich sequence (TAAAAAA) 27 base pairs upstream of the initiation site which binds transcription factor IID (TFIID) and a high affinity Sp1 binding site located 27 base pairs further upstream. Multiple upstream elements increased core promoter activity 20-fold and correspond to protected regions in DNase I footprinting assays with purified Sp1 protein. Internal deletion of the TA6 element alone eliminated transcription in spite of the presence of all other promoter elements including four Sp1 binding sites. Recombinant human TFIID supported weak basal transcription in heat-treated nuclear extracts whereas a partially purified TFIID fraction from HeLa cells reconstituted a maximal level of transcription. Inclusion of 12 base pairs immediately adjacent to the proximal Sp1 site resulted in a 5-fold boost in transcriptional activity and corresponds to a second Sp1 binding site. These results serve as a basis for further exploration of the factors involved in the developmental and selective high level tissue expression of the murine adenosine deaminase gene.
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PMID:The murine adenosine deaminase promoter requires an atypical TATA box which binds transcription factor IID and transcriptional activity is stimulated by multiple upstream Sp1 binding sites. 193 99

Using transgenic mice, we have defined novel gene regulatory elements, termed "facilitators." These elements bilaterally flank, by up to 1 kb, a 200-bp T-cell-specific enhancer domain in the human adenosine deaminase (ADA) gene. Facilitators were essential for gene copy-proportional and integration site-independent reporter expression in transgenic thymocytes, but they had no effect on the enhancer in transfected T cells. Both segments were required. Individual segments had no activity. A lack of facilitator function caused positional susceptibility and prevented DNase I-hypersensitive site formation at the enhancer. The segments were required to be at opposed ends of the enhancer, and they could not be grouped together. Reversing the orientation of a facilitator segment caused a partial loss of function, suggesting involvement of a stereospecific chromatin structure. trans-acting factor access to enhancer elements was modeled by exposing nuclei to a restriction endonuclease. The enhancer domain was accessible to the 4-cutter DpnII in a tissue- and cell-type-specific fashion. However, unlike DNase I hypersensitivity and gene expression, accessibility to the endonuclease could occur without the facilitator segments, suggesting that an accessible chromatin domain is an intermediate state in the activational pathway. These results suggest that facilitators (i) are distinct from yet positionally constrained to the enhancer, (ii) participate in a chromatin structure transition that is necessary for the DNase I hypersensitivity and the transcriptional activating function of the enhancer, and (iii) act after cell-type-specific accessibility to the enhancer sequences is established by factors that do not require the facilitators to be present.
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PMID:Dissecting a locus control region: facilitation of enhancer function by extended enhancer-flanking sequences. 782 28

Tissue-specific expression of the human adenosine deaminase (ADA) gene is mediated by transcriptional activation over a thousand-fold range. Cis-regulatory regions responsible for high and basal levels of activation include an enhancer and the proximal promoter region. While analyses of the T-cell specific enhancer have been carried out, detailed studies of the the promoter region or promoter-enhancer interactions have not. Examination of the promoter region by homology searches revealed six putative Sp1 binding sites. DNase I footprinting showed that Sp1 is able to bind these sites. Deletion analysis indicated that the proximal Sp1 site is required for activation of a reporter gene to detectable levels and that the more distal Sp1 sites further activate the level of expression. Inclusion of an ADA enhancer-containing fragment in these deletion constructions demonstrated that Sp1 sites are also essential for enhancer function. Apparently Sp1 controls not only low level expression but is also an integral part of the mechanism by which the enhancer achieves high level ADA expression. Mutagenesis of a potential TBP binding site at base pairs -21 to -26 decreased activity only two-fold indicating that it is not essential for transcriptional activation or enhancement.
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PMID:Sp1 is essential for both enhancer-mediated and basal activation of the TATA-less human adenosine deaminase promoter. 812 16

Murine adenosine deaminase (ADA) is a ubiquitous purine catabolic enzyme whose expression is subject to developmental and tissue-specific regulation. ADA is enriched in trophoblast cells of the chorioallantoic placenta and is essential for embryonic and fetal development. To begin to understand the genetic pathway controlling Ada gene expression in the placenta, we have identified and characterized a 770-base pair fragment located 5.4 kilobase pairs upstream of the Ada transcription initiation site, which directs reporter gene expression to the placenta of transgenic mice. The expression pattern of the reporter gene reflected that of the endogenous Ada gene in the placenta. Sequence analysis revealed potential binding sites for bHLH and GATA transcription factors. DNase I footprinting defined three protein binding regions, one of which was placenta-specific. Mutations in the potential protein binding sites and footprinting regions resulted in loss of placental expression in transgenic mice. These findings indicate that multiple protein binding motifs are necessary for Ada expression in the placenta.
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PMID:Diverse genetic regulatory motifs required for murine adenosine deaminase gene expression in the placenta. 899 42

Ubiquitously expressed genes contain regulatory features which allow expression in virtually all cell types. In an effort to understand the molecular basis for this regulatory feature, the chromatin structure of the murine adenosine deaminase gene was examined by DNase I digestion in nuclei of several tissues. The promoter contained a strong hypersensitive site in all tissues examined, including those with very high and very low levels of ADA expression. Transgenic mouse studies revealed that a 3.3 kb EcoRI (3.3EE) fragment from intron I was required to generate a strong promoter DNase I hypersensitive site, and to produce ubiquitous expression. The 3.3EE fragment also contained a thymic enhancer activity which mapped to sequences conserved with the human ADA gene T-lymphocyte enhancer. Mutational analysis indicated that ubiquitous expression was not dependent on the presence of a functional thymic enhancer. Both the thymic enhancer and the ubiquitous activator within the 3.3EE fragment functioned with heterologous promoters in transgenic mice.
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PMID:An intron 1 regulatory region from the murine adenosine deaminase gene can activate heterologous promoters for ubiquitous expression in transgenic mice. 900 Jan 71

Transcriptional activation of eukaryotic genes involves assembly of specific multiprotein complexes on the promoters and enhancers of the genes. Recently, it has been proposed that the role of some of the proteins in the complex may be architectural, involving DNA bending, orchestration of protein-protein interaction and modulation of nucleosome structure. This role has been proposed for the HMG proteins LEF-1 and TCF-1. We examined the role of a LEF-1/TCF-1 binding site in the human adenosine deaminase (ADA) thymic enhancer. Mutational analysis demonstrated that a functional LEF-1/TCF-1 binding site is not required for enhancer-mediated transcriptional activation in transient transfection studies, but is essential for enhancer function in the in vivo chromatin context of transgenic mice. Mutation of the LEF-1/TCF-1 site destroyed the ability of the ADA enhancer/locus control region to specify high level, insertion site-independent transgene expression in thymus. DNase I and DpnII accessibility experiments indicated dramatic changes in the chromatin organization of the ADA enhancer in transgenic mice with a mutated LEF-1/TCF-1 site. This supports the hypothesis that factors binding the LEF-1/TCF-1 site play an architectural role during the in vivo activation of the ADA enhancer, possibly involving chromatin modification.
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PMID:An enhancer LEF-1/TCF-1 site is essential for insertion site-independent transgene expression in thymus. 901 77

Trophoblast cells are specialized extra-embryonic cells present only in eutherian mammals. They play a major role in the implantation and placentation processes. To understand better the molecular mechanisms that control the development and function of trophoblast cells, we sought to identify the transcription factors that regulate murine adenosine deaminase (ADA) gene expression in the placenta. Here we report a detailed characterization of a placenta-specific footprinting region (FP1) in the Ada placental regulatory element. The sequence of FP1 was mapped by DNase I footprinting and was found to match a consensus AP-2 transcription factor-binding site. Electrophoretic mobility shift assays demonstrated that FP1 interacted with AP-2-like proteins. Further analysis using AP-2 antibody confirmed that AP-2 protein was indeed present in the placenta and bound to FP1. Mutation at the AP-2 site in FP1 abolished the ability of the Ada placental regulatory element to bind AP-2 proteins and failed to target chloramphenicol acetyltransferase reporter gene expression to placentas in transgenic mice, indicating that AP-2 is required for Ada expression in the placenta. In addition, RNase protection assays demonstrated that AP-2gamma was the predominant AP-2 family member expressed in the placenta. In situ hybridization analysis revealed that AP-2gamma expression was enriched in the trophoblast lineage throughout development, suggesting that AP-2gamma may be critical for trophoblast development and differentiation.
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PMID:Transcription factor AP-2gamma regulates murine adenosine deaminase gene expression during placental development. 976 60

An intestine-specific gene regulatory region was previously identified near the second exon of the human adenosine deaminase (ADA) gene. In mammalian intestine, ADA is expressed at high levels only along the villi of the duodenal epithelium, principally if not exclusively in enterocytes. Within the ADA intestinal regulatory region, a potent duodenum-specific enhancer was identified that controls this pattern of expression. This enhancer has been shown to rely on PDX-1, GATA factors, and Cdx factors for its function. Upstream of the enhancer, a separate temporal regulatory region was identified that has no independent enhancer capability but controls the timing of enhancer activation. DNase I footprinting and electrophoretic mobility shift assays were used to begin to characterize temporal region function at the molecular level. In this manner, binding sites for the Onecut (OC) family of factors, YY1, and NFI family members were identified. Identification of the OC site was especially interesting, because almost nothing is known about the function of OC factors in intestine. In transgenic mice, mutation of the OC site to ablate binding resulted in a delay of 2-3 weeks in enhancer activation in the developing postnatal intestine, a result very similar to that observed previously when the entire temporal region was deleted. In mammals, the OC family is comprised of OC-1/HNF-6, OC-2, and OC-3. An examination of intestinal expression patterns showed that all three OC factors are expressed at detectable levels in adult mouse duodenum, with OC-2 predominant. In postnatal day 2 mice only OC-2 and OC-3 were detected in intestine, with OC-2 again predominant.
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PMID:Temporal regulation of enhancer function in intestinal epithelium: a role for Onecut factors. 1695 Jul 65