Figure 1. The L2a element.

(A) Schematic of the murine CD8 locus and intergenic region with expanded details of L2a. Positions of established exons (red boxes), enhancer regions (blue lines) and DNAse 1 hypersensitivity sites (blue arrows) shown to be critical for CD8 transcriptional regulation are indicated. A third cluster of two hypersensitive sites (Cluster I, not shown) resides 3’ to CD8a but is yet to be implicated in its regulation. The L2a element (green) is located ~4.5 kb upstream of the mouse CD8a gene. The 270 bp AccI/SstI fragment spanning L2a is expanded to show the L, S, and INTER-LS regions identified in footprinting studies. A 12 bp palindromic sequence is found located at the end of the INTER-LS region close to the S region. (B) Schematic of the transgenic constructs. Both wildtype (WT) and L2a deleted (L2aD) constructs were derived from a human CD2 reporter gene construct (Sawada et al., 1994) and a CD8 intergenic construct (Tg-a) modified by Ellmeier et al. (1997). For L2aWT, a 4.3 kb HindIII/HindIII DH Cluster II fragment containing L2a was introduced upstream of the CD8α promoter, and a 7.6 kb BamHI/BamHI fragment containing the E8_I enhancer was subcloned upstream of DH cluster II fragment. Components of the constructs are highlighted. The L2aD deletion mutant was generated by deletion of a 200 bp AccI/BstXI fragment spanning L2a. This fragment served as the WT200 L+S probe in EMSA experiments.