Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus.

Abstract

The enhancer elements from either simian virus 40 or murine sarcoma virus activate the expression of a transfected rat insulin 1 (r11) gene when placed within 2.0 kilobases or less of the rl1 gene cap site. Inclusion of 4.0 kilobases of upstream r11 sequence, however, results in a substantial reduction in the enhancer-dependent insulin gene expression. These observations suggested that a negative transcriptional regulatory element was present between 2.0 and 4.0 kilobases of the r11 sequence. To test this notion, we employed a heterologous enhancer-dependent transcription assay in which the simian virus 40 72-base-pair repeat is linked to a human f8-globin gene. Addition of the upstream rI1 element to this system decreased the level of enhancer-dependent fl-globin transcription by a factor of 5 to 15. This rIb "silencer" element functions in a manner relatively independent of position and orientation and requires a cis-dependent relationship to the transcription unit on which it acts. Thus, the silencer sequence seems to have a number of the characteristics of enhancer elements, and we suggest that it may function by the converse of the enhancer mechanism. The rIb silencer sequence was identified as a member of a long interspersed rat repetitive family. Thus, a potential role for certain repetitive sequences interspersed throughout the eukaryotic genome may be to regulate gene expression by retaining transcriptional activity within defined domains.Enhancers are cis-dependent DNA sequences that activate the transcription of viral and cellular genes in a manner relatively independent of position and orientation (1,2). This transcriptional activation can occur over distances as great as 6 kilobases (kb) (3, 4) from either the 5' or 3' end of the gene. What appears to be more important than the distance between an enhancer and the promoter being assayed for transcription are the specific sequences that intervene. Thus, for example, if an enhancer is flanked on both sides by active promoters, its effect on more distal promoter elements is likely to be significantly reduced (4-6).In studying the sequences upstream of the rat insulin 1 (rIb) gene, we have identified an element that appears to suppress enhancer-dependent transcriptional activity by a distinct mechanism. In fact, this negative regulatory element, which we will refer to as a silencer (see ref. 7), appears to have many of the characteristics of enhancers including position-and orientation-independent function on a heterologous gene. The identification of this rI, silencer element as a member of a family of long interspersed rat repetitive sequences (LINES; refs. 8 and 9) suggests a potential role for some of these repetitive elements in gene regulation. MATERIALS AND METHODSPlasmid Constructions. The recombinant plasmids used in this study were constructed according to methods that use standard recombinant DNA technology (10). The parental vector irSVHPA128 (kindly provided by M. Green and T.Maniatis) contains the human P3-globin gene with its prom...

Insulin expressing cells from differentiated embryonic stem cells are not beta cells

“…Surprisingly, activation of the transgenic promoter in two different cell clones was not accompanied by expression of the endogenous Insulin I gene. The explanation for these apparently contradictory results may reside in the fact that the Insulin I gene contains a cis-acting silencer located between −4.0 and −2.0 kb from the transcriptional start site [48] that was not included in the DNA construct used in this study. Since the same promoter region is commonly used for studies on regulation of insulin gene expression, our data caution against interpreting results based only on activation of a similar reporter gene.…”

Sequence conservation in avian CR1: an interspersed repetitive DNA family evolving under functional constraints.

“…The region between 424 and 439 is included in a somewhat larger domain that has been shown to bind a nuclear protein of unknown identity (13). (22). The distribution of transcriptional silencers throughout the genome on the elements of a repeated gene family may play a role in establishing the default mode of chromatin, namely transcriptional inactivity.…”

A developmental-specific factor binds to suppressor sites flanking the immunoglobulin heavy-chain enhancer.

“…In addition, negative regulation of the enhancer is also an important mechanism for the control of heavy-chain expression. Negative-enhancer regulation may be a general mechanism for tissue-specific expression as negativeregulatory elements also have been found that are associated with the insulin 1 gene enhancer (Laimins et al 1986;Nit et al 1986), the B-interferon gene enhancer {Goodbourn et al 1986), and viral enhancers in embryonic stem cells (Gorman et al 1985), and mouse mammary epithelial cells (Langer and Ostrowski 1988).…”