CTCF‐independent, but not CTCF‐dependent, elements significantly contribute to the insulation capacity of a locus control region

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Locus control regions (LCR) provide high-level, position-independent expression to a transgene in chromatin. They achieve this through DNA sequences supporting activities that enhance transcription and somehow protect transgene expression from integration site-dependent position effects. The former activity has been mapped to classical enhancer DNA sequences of most LCRs. In contrast, the elements supporting the latter capacity that suppresses position effects are less well understood. Insulator/Boundary sequences are thought to play a role in separating regulatory influence in the genome and support enhancer blocking and/or chromatin barrier activity. The LCR in the mouse T cell receptor (TCR)- 1 □/Dad1 gene locus 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 CTCF-independent elements within the LCR. Internal deletion of either of these elements significantly impairs LCR activity. Our results reveal that the position effect suppression capacity of an LCR arises largely from an array of CTCF-independent elements, some of which share characteristics with barrier-type insulators. Support: NIH #AI-053050 and NSF #MCB-0236964