A New Element within the T-Cell Receptor α Locus Required for Tissue-Specific Locus Control Region Activity

The locus control region (LCR), first described in the β-globin gene cluster (18, 23), is defined by its ability to impart position-independent and high-level tissue-specific expression of a linked transgene in chromatin (recently reviewed in references 19 and 32). An LCR protects a transgene from “position effect variegation,” which could silence its transcription if it integrates into inactive chromatin (46). LCRs are thought to provide an open chromatin environment, which is necessary for physiological expression of a linked transgene at any integration site (17, 42). LCRs are frequently associated with loci that maintain tissue-restricted expression. Accordingly, it has been observed that the activity of an LCR is restricted to the tissues in which its locus of origin is normally expressed (2, 3, 5, 23, 31, 35, 38), even when it is linked to heterologous transgenes (22, 45, 53). What provides tissue specificity to LCR activity is not completely clear. Mutational analyses of LCRs have led to the conclusion that the chromatin opening activity of an LCR is inherently tissue specific in nature (11, 47, 53). The T-cell receptor α (TCRα) gene is exclusively expressed in T-lineage cells. Prerearranged TCRα transgenes under endogenous controls are expressed only in T-cell-bearing tissues, such as thymus and spleen, but not in other organs, such as liver and heart (9). Mouse TCRα exists in a complex locus on mouse chromosome 14 that also includes the genes for the TCRδ chain and Dad1 (1, 26). TCRα and TCRδ genes are mutually exclusively expressed by the αβ and γδ subpopulations of T cells, respectively. In contrast, the Dad1 gene, which is 3′ of TCRα, is expressed ubiquitously. We have described an LCR in this locus that, in T-cell lines, manifests itself as eight DNase I hypersensitive sites (HS) extending from the TCRα chain constant region exons to 5′ of the Dad1 gene. HS1 maps to the well-characterized TCRα enhancer (25, 36, 58). HS2 to HS6 lie 3′ of HS1 in the locus. HS7 and -8 are 5′ of HS1. Fragments containing HS7 and -8 also contain transcriptional silencers defined in transient transfection assays (59). Additionally, a ninth HS, named HS1′, has been discovered in a 1-kb region of unknown function between HS1 and HS2 (26, 45). The chromatin at the endogenous LCR exists in differential configurations in TCRα-expressing and nonexpressing tissues (26, 45). In normal thymocytes, HS1 and HS6 are the strongest HS in the LCR region. These HS are either weak or are not present in non-T-cell-bearing organs. HS2 to -5 and HS7 and -8, are weak in all organs, while HS1′ is predominantly present in nonlymphoid tissues. The 9-kb region containing all nine HS of the LCR has been shown to direct high-level, position-independent, copy number-dependent, and T-cell compartment-specific expression of a linked TCRα transgene and a heterologous human β-globin transcription unit (9, 45). Our initial characterization of the LCR (45) revealed that it contained an unrestricted chromatin opening activity located in the 3′ HS2 to HS6 region. This fragment drives widespread transgene expression and adopts an abnormal, wide-open chromatin configuration in which all of HS2 to -6 are equally prominent in both lymphoid and nonlymphoid organs. The 5′ LCR region containing HS7, -8, -1, and 1′ confers cell-type specificity to the chromatin opening activity. This is marked by a restoration of T-cell-specific expression and the naturally occurring tissue-differential chromatin structures observed at the TCRα locus. The unique arrangement of a 3′ unrestricted chromatin opening activity and a 5′ T-cell-specificity region in the TCRαLCR is noteworthy. It suggested that, endogenously, the Dad1 gene, residing 3′ of the LCR, and the TCRα gene, localized 5′ of the LCR, may be sharing some of the LCR elements (26). How the regulation of T-cell-specific and ubiquitously expressed genes is coordinated within the same locus is a question of considerable interest. Our previous work showed for the first time that the chromatin-opening and tissue-specific functions of LCRs can, at least in some cases, be separated (45). The presence of a T-cell-specific enhancer and silencers in the 5′ tissue-specificity region would suggest a role for these elements in the restriction of LCR activity. While the 1-kb region containing HS1′ is highly conserved between mouse and human loci (33), to date, no activity has been ascribed to it. To determine the contributions of activities in HS7 and -8, HS1, and HS1′ to tissue-specific LCR function, we report here further deletion analysis of the mouse TCRαLCR. We find that the silencer region containing HS7 and -8 has a very minor inhibitory effect on transgene expression in T-cell-bearing organs and appears dispensable for complete LCR activity. The T-cell-specific enhancer (HS1) increases transcription in thymus and has no other apparent effect on expression in other organs. In addition, HS1 contributes to copy-number-dependent expression of the reporter and is thus an integral part of the LCR. Surprisingly, removal of these silencer- and enhancer-containing regions has no severe effect on the chromatin structure of the remaining LCR sequences. Rather, the HS1′ region appears to be responsible for maintaining the cell-type-specific chromatin structures observed at the 3′ chromatin opening region. Although no transcriptional activity has ever been described in the HS1′ region, we find that this region increases transcription in thymus and spleen and suppresses ectopic expression of our reporter transgene. Thus, this previously undescribed control element plays an important role in tissue-specific functions of the TCRαLCR. The location of this novel activity, which we term the “HS1′ element,” between the T-cell-specific enhancer and the region containing unrestricted chromatin opening activity may also suggest a potential role for it in separating the regulation of the TCRα and Dad1 genes.