Factors Binding a Non-classical Cis-element Prevent Heterochromatin Effects on Locus Control Region Activity

Abstract

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) ␣/Dad1 gene locus. Here we focus on DNase hypersensitive site (HS) 6 of the TCR␣ 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 TCR␣ LCR assembly in vivo.

CTCF-Independent, but Not CTCF-Dependent, Elements Significantly Contribute to TCR-α Locus Control Region Activity

“…In the present study, we focus our search for DNA elements participating in position effect suppression to these three regions of the TCR␣ LCR. We previously reported one such element, a 238-bp sequence within HS6 whose deletion significantly impairs, but does not completely abolish, the LCR's position effect suppression capacity (26). This information, along with the prior enhancer-blocking data (24,25), clearly suggests that additional elements downstream of E␣ contribute to LCR activity.…”

“…Assays in cultured cell lines have identified a broad region of TCR␣ LCR DNA downstream of E␣ that supports enhancer-blocking activity (24,25). In this area, HS1Ј, HS4, and HS6 are the most prominent HS in both transgenic and native TCR␣ LCR loci of thymocytes (15,26). A CTCF site with enhancer-blocking activity has been identified in HS1Ј (25).…”

“…This information, along with the prior enhancer-blocking data (24,25), clearly suggests that additional elements downstream of E␣ contribute to LCR activity. Primary candidates for such elements include the identified CTCF binding site in HS1Ј with enhancer-blocking activity (25), the tissue specifically methylated area of HS4 DNA, the function of which is still unknown (27), and a 316-bp segment of the HS6 region at the very 3Ј end of the LCR (named HS6-316) whose activity was revealed in cell culture (26). In this study, we describe the functional contributions of these three candidate position-effect suppression elements to TCR␣ LCR activity in vivo using an integration site-independent transgenic reporter system in mice.…”

The TCRα Locus Control Region Specifies Thymic, But Not Peripheral, Patterns of TCRα Gene Expression

“…Previously, in vivo studies of TCR␣ LCR activity made use of transgenic mouse models in which a 4.9-kb BglII genomic fragment of human ␤-globin was used as a reporter (13)(14)(15)(16). This fragment, when linked to the wild-type LCR, was expressed lymphoid specifically in thymus and spleen.…”

Chromatin Insulator Elements: Establishing Barriers to Set Heterochromatin Boundaries

“…However, these elements alone are not sufficient to suppress position effect silencing in transgenic mice. Elements located within HS4 and HS6 are required to act as barrier-like elements, while the CTCF element HS1´ is largely dispensable [108][109][110]. The factors and mechanisms employed at these barrier elements remain to be determined.…”