Figure 6. The new target genes contain RFX and CIITA dependent S-Y enhancers.

(A) Alignment between the S-Y motif of the HLA-DRA gene and sequences found within the regions to which CIITA is recruited at the new target genes. Sequences corresponding to the S, X, X2 and Y elements are boxed. The dashed box indicates a pyrimidine (py) rich region generally found at the 5′ end of the X box. Numbers indicate spacing in nucleotides between the S and py or X2 and Y sequences. S and Y sequences are only shown if homology is evident. (B) Transient transfections were performed with luciferase reporter gene constructs containing the HLA-DRA regulatory region or hybrid promoters in which the S-Y motif of HLA-DRA was replaced with the S-Y sequences from the indicated new target genes. A construct containing the basal HLA-DRA promoter lacking the S-Y module (min) was used as negative control. Constructs were transfected into Raji, RJ2.2.5 and SJO cells. Results are expressed relative to the activity of the HLA-DRA construct in Raji, and show the mean and SD of 3 independent experiments (each performed with triplicate luciferase measurements). (C) A mutation disrupting the X box was introduced into the constructs containing the new S-Y enhancers. Activities of the wild type (wt) and mutated (Xm) constructs were compared in Raji cells. Results are expressed relative to the activity of wt construct, and show the mean and SD of 3 independent experiments (each performed with triplicate luciferase measurements).