CTCF-independent, but not CTCF-dependent, elements significantly contribute to TCR-alpha locus control region activity

Abstract

The mouse TCRalpha/TCRdelta/Dad1 gene locus bears a locus control region (LCR) that drives high-level, position-independent, thymic transgene expression in chromatin. It achieves this through DNA sequences that enhance transcription and protect transgene expression from integration site-dependent position effects. The former activity maps to a classical enhancer region (Ealpha). In contrast, the elements supporting the latter capacity that suppresses position effects are incompletely understood. Such elements likely play important roles in their native locus and may resemble insulator/boundary sequences. Insulators support enhancer blocking and/or chromatin barrier activity. Most vertebrate enhancer-blocking insulators are dependent on the CTCF transcription factor and its cognate DNA binding site. However, studies have also revealed CTCF-independent enhancer blocking and barrier insulator activity in the vertebrate genome. The TCRalpha LCR contains a CTCF-dependent and multiple CTCF-independent enhancer-blocking regions whose roles in LCR activity are unknown. Using randomly integrated reporter transgenes in mice, we find that the CTCF region plays a very minor role in LCR function. In contrast, we report the in vivo function of two additional downstream elements located in the region of the LCR that supports CTCF-independent enhancer-blocking activity in cell culture. Internal deletion of either of these elements significantly impairs LCR activity. These results reveal that the position-effect suppression region of the TCRalpha LCR harbors an array of CTCF-independent, positive-acting gene regulatory elements, some of which share characteristics with barrier-type insulators. These elements may help manage the separate regulatory programs of the TCRalpha and Dad1 genes.