Figure 5

Identification of a novel intronic Cd74 promoter. (A) The sequence upstream of primer SR2 from the RLM-RACE - position of which is indicated in Figure 3C - was applied as query sequence (mm9) to retrieve orthologous sequences from the UCSC genome browser for the indicated species assemblies; rat (rn4), human (hg18), marmoset (calJac1), and horse (equCab1). The retrieved sequences were analyzed by Chaos and Dialign software [49] and the part depicted displayed the highest similarity within the query sequences upstream of the transcriptional initiation region identified by RLM-RACE, the start of which is marked by a horizontal arrow above the sequence. The indicated putative binding site core sequences of transcription factors expressed in the immune system were identified by the MatInspector software [50]. The vertical arrows mark the start and the end, respectively, of the promoter fragment tested functionally in luciferase assays. (B) HEK293T cells were transfected with the firefly luciferase reporter pGL3-enhancer (pGL3) or pGL3-derived vectors with the SV40 enhancer in the pGL3-enhancer construct substituted by Cd74 intronic enhancers (pGL3e1 and pGL3e2) or by the U3 region of the provirus in sense (pGL3US) or antisense (pGL3UA) orientation respectively, and with or without the promoter fragment (F1) marked by arrows in (A). Luciferase activity of reporters without an inserted promoter are depicted by light grey bars, whereas the activity of equivalent reporters with the F1 promoter region inserted upstream of firefly luciferase are represented by dark grey bars. A plasmid encoding Renilla luciferase under the control of a CMV promoter was co-transfected in all experiments. Luciferase activity was measured 48 hours post transfection, corrected for differences in transfection efficiencies and normalized to the activity of the individual expression vectors with the indicated enhancer but without the F1 promoter fragment which was set to 1.