Adjacent DNA elements dominantly restrict the ubiquitous activity of a novel chromatin-opening region to specific tissues

Abstract

Locus control regions (LCRs) are thought to provide a dominant tissue‐specific open chromatin domain that allows for proper gene regulation by enhancers/silencers and their associated transcription factors. Expression of the T‐cell receptor alpha (TCRα) gene is limited to T cells and its locus exists in different chromatin configurations in expressing and non‐expressing cell types. Here we show that eight DNase I‐hypersensitive sites in the TCRα locus comprise an LCR that confers T‐cell compartment‐specific expression upon a linked heterologous transgene. Removal of the three 5′‐most hypersensitive sites of this LCR, containing TCRα enhancers/silencers, abolishes tissue‐differential chromatin structure and results in transgene expression in all tissues examined. The remaining five DNase I‐hypersensitive sites therefore constitute a novel control element possessing a widely active chromatin‐opening function that allows for ubiquitous expression of a linked transgene in all transgenic founder mice. Furthermore, these data show that cis‐acting elements without inherent LCR activity can dominantly modulate chromatin structure to determine tissue‐specific gene expression in vivo.

Function and Factor Interactions of a Locus Control Region Element in the Mouse T Cell Receptor-α/Dad1 Gene Locus

“…The construction of the 9-kb HS1-8 fragment, 5.9-kb HS2-6 fragment, and the ␤:1-8 and ␤:2-6 transgenes have all been previously described (14). For the ␤:6 construct, a 1.6-kb MfeI/SacI fragment of the HS1-8 region was excised from the pSP72:HS1-8 vector.…”

“…Four independent lines of ␤:6 and five lines of ␤:⌬6 transgenic mice were generated and analyzed. These lines were compared with lines of ␤:2-6 mice that were generated and described previously (14) to determine the sequences contributing to the activity of the HS2-6 region.…”

“…HS7 and HS8 are located 5Ј of the enhancer, whereas HS2-6 are 3Ј of the TCR␣ enhancer. The nine HS of the LCR confer high-level, positionindependent, copy number-dependent, and tissue-specific expression upon a linked transgene in chromatin of mice (14). HS7 and HS8 appear to be dispensable for complete LCR activity.…”

“…HS2 through HS6 are absolutely required for complete, T cell-specific LCR activity (11). In isolation, this TCR␣ distal portion of the LCR also contains an unusual activity that drives widespread transgene expression in mice (14,15). This expression is roughly, but not strictly, related to copy number indicating the presence of elements in this region that counteract integration-site "position-effects" in many tissues.…”

Ectopic T cell receptor expression causes B cell immunodeficiency in transgenic mice

“…The TCR § chain control elements have been well defined, and the pT § cassette contains the 3' TCR § enhancer and most of the TCR § LCR (locus control region), including DNase hypersensitivity sites (HS) 7, 8, 1, 1', 2, and 3 (in 5'-3' order), but lacks HS4, 5 and 6 [22,23]. Deletional analysis of TCR § LCR demonstrated that HS1, which overlaps with the TCR § enhancer, was important for high-level expression of a reporter gene in thymus, but not in the spleen, and that it was not critical for tissue specific chromatin opening [24,25]. Further deletion of HS1' diminished tissue-specific control of chromatin-opening gene expression of genes proximal to TCR § LCR.…”