(A–C) Binding of CIITA to the RAB4B promoter region in DC. Binding of CIITA in immature DC (iDC) was assessed by ChIP-on-chip experiments as in Figure 2. The control hybridization was done with CIITA-ChIP DNA from mature DC (mDC).

Occupation of the RAB4B promoter by the MHC-II specific regulatory machinery. (A) The association of CIITA, RFX5 and CREB with the HLA–DRA, RAB4B and TBP promoters was assessed by ChIP in wt B cells. Results are expressed as % of occupation observed at the HLA-DRA promoter. (B) Occupancy of the RAB4B promoter by CIITA, RFX5 and CREB was analyzed in wt B cells and in mutant B cells lacking CIITA or RFX5. Results are expressed as % of the occupation observed in the wt cells. (C) Occupancy of the HLA–DRA promoter was analyzed as in (B). (D) Binding of CIITA to the RAB4B promoter was analyzed by ChIP-on-chip. Results are represented as ratios between the signals obtained with CIITA-ChIP DNAs from wt and CIITA-/- B cells (top) or wt and RFX5-/- B cells (bottom). N.D., not done.

Occupation of the RAB4B promoter by the MHC-II regulatory machinery in DC and IFN-γ induced cells. (A) Binding of CIITA to the RAB4B, HLA-DRA and TBP promoters was analyzed by ChIP in DC before (iDC) and after (mDC) maturation with LPS. Results are represented as % of the occupation observed at the HLA-DRA promoter in iDC. The TBP promoter was used as a negative control. (B and C) ChIP was used to measure occupancy of the RAB4B promoter by CIITA (B) and RFX5 (C) in Me67.8 melanoma cells induced with IFN-γ for 0, 2, 4, 6 and 12 h. Results are represented as % of the occupation observed at the HLA–DRA promoter after 12 h of induction.

Expression of RAB4B is controlled by CIITA and is induced by IFN-γ. mRNA levels for the RAB4B, HLA-DRA and RAB4A genes were determined for (A) wt B cells (Raji), CIITA-/- B cells (RJ2.2.5) and CIITA-/- cells complemented with expression vectors encoding the three isoforms of CIITA (CIITA I, III and IV), (B) Me67.8 melanoma cells (Me) induced with IFN-γ for 0, 12, 24 and 48 h and (C) HUVEC cells induced with IFN-γ for 0, 12, 24 and 48 h. Results were normalized using TBP mRNA. Values for RAB4B and RAB4A are expressed as % of the levels found in wt B cells (A) or relative to uninduced cells (B and C). Values for HLA–DRA mRNA are expressed as % of the levels found in wt B cells (A) or cells induced with IFN-γ for 48 h (B and C).

The RAB4B S-Y module functions as a CIITA and RFX-dependent transcriptional enhancer. (A) Transient transfections were performed with luciferase reporter gene constructs containing the HLA–DRA regulatory region or hybrid promoters in which the S-Y module from HLA–DRA was replaced with the RAB4B S-Y sequence in the forward or inverse orientations. A construct containing the basal HLA–DRA promoter lacking the S-Y module (minimal) was used as a negative control. Constructs were transfected into wt, CIITA-/- and RFX5-/- B cells, and into CIITA-/- and RFX-/- cells complemented with CIITA or RFX5 expression vectors, respectively. Results are expressed as % of the activity of the HLA–DRA S-Y construct in wt cells. (B) Mutations were introduced into the S, X, X2 and Y sequences of the luciferase construct containing the RAB4B S-Y module. The wt and mutated S, X, X2 and Y sequences are provided. Activities of the resulting constructs were tested after transfection into wt and CIITA-/- cells. The minimal construct was used as a negative control. Results are expressed as % of the activity of intact RAB4B construct in wt cells.

Binding of CIITA to the RAB4B promoter region in B cells. Binding of CIITA to the RAB4B (A), RAB4A (B) and HLA–DRA, HLA–DPA and HLA–DPB (C) promoter regions was assessed by ChIP-on-chip experiments. The results are represented as log₂ ratios between the hybridization signals obtained for CIITA-ChIP DNAs derived from CIITA-positive B cells (wt) and those obtained with control DNAs. Controls were CIITA-ChIP DNAs from CIITA-/- B cells or input genomic DNA. Arrays were either the standard NimbleGen promoter arrays or a high density custom array. Each bar corresponds to a single oligonucleotide on the array. Grey bars correspond to log₂ ratios lower than zero and reflect stronger signals for the control samples. The latter probably result from sporadic random amplification of short DNA fragments in the control samples. Schematic maps with positions of the genes (open boxes), transcription start sites (arrows) and S-Y modules (grey boxes) are shown.

Identification of an S-Y module in the upstream region of the RAB4B gene. (A) Schematic drawing of the regulatory machinery controlling the transcription of MHC-II genes. The promoters of all MHC-II genes contain a conserved regulatory module (S-X-X2-Y) that is recognized by an enhanceosome complex consisting of RFX, CREB and NF-Y. Transcription is activated by recruitment of the co-activator CIITA to this enhanceosome. (B) The 3 kb upstream regions of all human genes were scanned with the MHC-II S-Y profile. Sequences bearing similarity to S-Y modules are plotted with respect to their position in the genome and the score attributed by the search algorithm. S-Y sequences in the RAB4B, invariant chain (Ii) and MHC-II genes are indicated. (C) Alignment between the human HLA–DRA S-Y module and the S-Y sequences found in the RAB4B genes of the indicated organisms. The most conserved residues are highlighted. A sequence logo representing the consensus MHC-II S-Y motif is shown below; the font size reflects the frequency at which a nucleotide is found at a given position. (D) Sequence conservation in the intergenic region between the RAB4B and MIA genes is represented by a graph derived from the Conservation track available at the UCSC Genome Browser at genome.ucsc.edu. The track shows a measure of the evolutionary conservation between 17 vertebrates. A peak corresponding to the most strongly conserved sequence in the intergenic region overlaps with the X–X2 region of the S-Y module.