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

Abstract

Locus control regions (LCRs) refer to cis-acting elements composed of several DNase I hypersensitive sites, which synergize to protect transgenes from integration-site dependent effects in a tissue-specific manner. LCRs have been identified in many immunologically important gene loci, including one between the TCRδ/TCRα gene segments and the ubiquitously expressed Dad1 gene. Expression of a transgene under the control of all the LCR elements is T cell specific. However, a subfragment of this LCR is functional in a wide variety of tissues. How a ubiquitously active element can participate in tissue-restricted LCR activity is not clear. In this study, we localize the ubiquitously active sequences of the TCR-α LCR to an 800-bp region containing a prominent DNase hypersensitive site. In isolation, the activity in this region suppresses position effect transgene silencing in many tissues. A combination of in vivo footprint examination of this element in widely active transgene and EMSAs revealed tissue-unrestricted factor occupancy patterns and binding of several ubiquitously expressed transcription factors. In contrast, tissue-specific, differential protein occupancies at this element were observed in the endogenous locus or full-length LCR transgene. We identified tissue-restricted AML-1 and Elf-1 as proteins that potentially act via this element. These data demonstrate that a widely active LCR module can synergize with other LCR components to produce tissue-specific LCR activity through differential protein occupancy and function and provide evidence to support a role for this LCR module in the regulation of both TCR and Dad1 genes.

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

“…Transgenes in Mice-For many years we (11,12,21) and others (1,2,23,24) have used a 4.9-kb fragment of the human ␤-globin locus as a reporter gene for testing LCR activity in transgenic mouse studies. This fragment, which contains the ␤-globin promoter, exons, introns, and a 3Ј-enhancer, is highly susceptible to integration site-dependent position effects in the absence of additional control elements (25,26).…”

“…TF1 binds an as yet unidentified tissue-unrestricted factor, whereas TF2 and -3 bind complexes that contained the tissuerestricted proteins AML-1/Runx1 and Elf-1, respectively. TF2 and -3 are occupied only when the tissue specificity elements of the LCR are linked in cis, suggesting that factors binding to these sites are important for the ability of HS6 to contribute to LCR activity (12). However, the roles of the TF1-2-3 sequences in the functions of HS6 and the TCR␣ LCR are unknown.…”

“…When a 1.8-kb fragment containing HS6 is linked alone to a reporter transgene, its position-effect-suppressing activity is evident in all tissues of transgenic mice examined (12). However, in the context of the full LCR, HS6 appears to be active only in lymphoid organs (11,21).…”

“…When these sites are removed from the LCR, the remaining HS (HS2-6) drive widely active integration site-independent transgene expression (11,21). HS6 was subsequently found to contain the bulk of this widespread activity (12).…”

“…This is partially because of the low number of complete bona fide LCRs identified and the even lower number of such LCRs that have been studied in mechanistic detail. This latter subset includes the LCRs of the human ␤-globin (13), human CD2 (14), human growth hormone (15), and the mouse T cell receptor (TCR) ␣ encoding gene loci (11,12).…”