Enhancer and silencer elements within the first intron mediate the transcriptional regulation of the β3 tubulin gene by 20-hydroxyecdysone in Drosphila Kc cells

Corepressors are involved in gene silencing by various transcriptional repressor proteins such as MAD/MAX and MxiI (2), YY1 (74), KRAB domain proteins (27), NGF1-A, KROX 20 (59, 63) and some members of the nuclear hormone receptor (NHR) superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR) (8,19,38,50). Both TR and RAR repress gene activity in the absence of hormone in vivo (3, 4, 21, 73) and in vitro (28, 67, 68). This repression is mediated by a silencing domain in the carboxy terminus, encompassing about 250 amino acids (aa) (3,33,46,58). In addition to the silencing function, TR and RAR harbor several other functions C-terminal to their DNA binding domain (DBD) including dimerization, hormone binding and hormone-dependent transactivation. These activities can be transferred to heterologous proteins and therefore represent functional domains (for reviews, see references 6, 50, and 65).Gene silencing by NHRs is relieved by addition of the cognate ligand, which induces a conformational change and transforms the receptor into a transcriptional activator. In this way, both hormone binding and the small conserved receptor activation domain, AF2/AF2-AD/4/c (8, 11, 12, 22, 49, 50), representing helix 12 (14, 38, 55, 57, 71), are required to dissociate corepressors from the receptors (8,9,19,38). It is yet unknown why so many different coactivators are involved in transcriptional activation by NHRs. Gene silencing by TR, RAR, Rev-erbA␣, and COUP-TF is mediated, at least in part, by corepressors in vivo (8, 10, 19, 25, 38, 61) and in vitro (68), which bind to the unliganded (apo) receptors. Only one class of nuclear receptor corepressors has been identified, which exhibit hormone-sensitive interaction. This class contains two related members, 38). These corepressors were isolated by the yeast twohybrid system and bind to the silencing domains of TR and RAR only in the absence of ligand. Hormone binding by the receptor leads to dissociation of these corepressors. Furthermore, SMRT and N-CoR are localized in the cell nucleus and harbor an autonomous silencing function when bound to DNA (19,38). The mechanism of repression by the SMRT/N-CoR class involves interaction with SIN3 and a histone deacetylase function (1,35,52).Here, we describe a novel corepressor, Alien, which is unrelated to SMRT and N-CoR and is highly conserved from humans to Drosophila. Conserved sequences are even found in Ricinus communis and Caenorhabditis elegans. Alien interacts with TR only in the absence of hormone and does not interact with RAR, retinoid X receptor (RXR), or glucocorticoid

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“…Furthermore, Alien interacts with the Drosophila EcR (Fig. 3B), which is known to repress gene transcription (24,70) …”

Section: Resultsmentioning

confidence: 99%

Alien, a Highly Conserved Protein with Characteristics of a Corepressor for Members of the Nuclear Hormone Receptor Superfamily

Dressel

Thormeyer

Altincicek

et al. 1999

Molecular and Cellular Biology

183

145

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“…Several Drosophila introns have been described that contain complex transcriptional regulatory sequences. (85)(86)(87)(88) The placement of such an intron within the EIR construct could drive expression of the EIR in an unintended tissue or cell type.…”

Section: Reduction-of-function Transgenicsmentioning

confidence: 99%

The genetics of Drosophila transgenics

Roman¹

2004

BioEssays

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In Drosophila, the genetic approach is still the method of choice for answering fundamental questions on cell biology, signal transduction, development, physiology and behavior. In this approach, a gene's function is ascertained by altering either the amount or quality of the gene product, and then observing the consequences. The genetic approach is itself polymorphous, encompassing new and more complex techniques that typically employ the growing collections of transgenes. The keystone of these modern Drosophila transgenic techniques has been the Gal4 binary system. Recently, several new techniques have modified this binary system to offer greater control over the timing, tissue specificity and magnitude of gene expression. Additionally, the advances in post-transcriptional gene silencing, or RNAi, have greatly expanded the ability to knockdown almost any gene's function. Regardless of the growing experimental intricacy, the application of these advances to modify gene activity still obeys the fundamental principles of genetic analysis. Several of these transgenic techniques, which offer more precise control over a gene's activity, will be reviewed here with a discussion on how they may be used for determining a gene's function.

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“…Although intronic enhancer elements have been described previously (32)(33)(34)(35)(36)(37)(38)(39), this is the first evidence that this mechanism exists within the mammalian ribonuclease gene family. This finding takes on considerable significance because the noncoding exon/single intron/coding exon gene structure is shared by all members of this gene family whose gene structures have been determined (9 -13).…”

Section: Discussionmentioning

confidence: 87%

Enhanced Expression of the Eosinophil-derived Neurotoxin Ribonuclease (RNS2) Gene Requires Interaction between the Promoter and Intron

Tiffany

Handen

Rosenberg

1996

Journal of Biological Chemistry

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The eosinophil-derived neurotoxin (EDN/RNS2) is a member of the mammalian ribonuclease gene family and is one of four proteins found in the large specific granules of human eosinophilic leukocytes. The gene encoding EDN consists of two exons, including a noncoding exon 1, separated by a single intron from the coding sequence in exon 2. We have identified a functional promoter of the EDN gene and shown that optimal expression depends on interaction between the promoter and one or more sequence elements found in the single intron. Cells of the clone 15 eosinophilic variant of the human promyelocytic HL-60 cell line were transfected with constructs that included the promoter region of the EDN gene alone, promoter with exon 1, and promoter with both exon 1 and the intron positioned 5 to the chloramphenicol acetyltransferase (CAT) reporter gene (constructs referred to as PrCAT, PrExCAT, and PrExInCAT, respectively). Although reporter gene activity from either PrCAT or PrExCAT was only 2-3 fold higher than baseline (CAT alone), inclusion of the single intron (PrExInCAT) resulted in a 28-fold increase in reporter gene activity in uninduced clone 15 cells, and an 80-fold in activity when clone 15 cells were induced to differentiate toward eosinophils with butyric acid. The intron-mediated enhancer activity was reproduced in other human hematopoietic cell lines (K562, Jurkat, U937, and HL-60), but was not found in human 293 kidney cells, suggesting that the function of the enhancer element(s) may be tissue-specific. A significant portion of the observed enhancer activity resides in the first 60 base pairs the the intron, which includes consensus binding sites for both AP-1 and NF-ATp transcription factors, and a 15-base pair segment that is identical to a sequence found in the promoter of the gene encoding the neutrophil granule protein, lactoferrin. The noncoding exon 1/single intron/coding exon 2 genomic structure is a common feature among the mammalian ribonucleases; this finding suggests the possibility of a conserved mechanism of regulation in this gene family.

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Enhancer and silencer elements within the first intron mediate the transcriptional regulation of the β3 tubulin gene by 20-hydroxyecdysone in Drosphila Kc cells

Cited by 32 publications

References 28 publications

Alien, a Highly Conserved Protein with Characteristics of a Corepressor for Members of the Nuclear Hormone Receptor Superfamily

Alien, a Highly Conserved Protein with Characteristics of a Corepressor for Members of the Nuclear Hormone Receptor Superfamily

The genetics of Drosophila transgenics

Enhanced Expression of the Eosinophil-derived Neurotoxin Ribonuclease (RNS2) Gene Requires Interaction between the Promoter and Intron

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