Factors binding a non-classical cis-element prevent heterochromatin effects on locus control region activity

A locus control region (LCR) is a cis-acting gene-regulatory element capable of transferring the expression characteristics of its gene locus of origin to a linked transgene. Furthermore, it can do this independently of the site of integration in the genome of transgenic mice. Although most LCRs contain subelements with classical transcriptional enhancer function, key aspects of LCR activity are supported by cis-acting sequences devoid of the ability to act as direct transcriptional enhancers. Very few of these "non-enhancer" LCR components have been characterized. Consequently, the sequence requirements and molecular bases for their functions, as well as their roles in LCR activity, are poorly understood. We have investigated these questions using the LCR from the mouse T cell receptor (TCR) alpha/Dad1 gene locus. Here we focus on DNase hypersensitive site (HS) 6 of the TCRalpha LCR. HS6 does not support classical enhancer activity, yet has gene regulatory activity in an in vivo chromatin context. We have identified three in vivo occupied factor-binding sites within HS6, two of which interact with Runx1 and Elf-1 factors. Deletion of these sites from the LCR impairs its activity in vivo. This mutation renders the transgene locus abnormally inaccessible in chromatin, preventing the normal function of other LCR subelements and reducing transgene mRNA levels. These data show these factor-binding sites are required for preventing heterochromatin formation and indicate that they function to maintain an active TCRalpha LCR assembly in vivo.