(A) Representative CIITA binding profiles obtained for the new target genes in ChIP-chip experiments comparing Raji and SJO cells. Results are represented as in Figure 2. (B) Binding of CIITA to the new target genes was assessed by quantitative ChIP experiments performed with Raji, SJO and BLS1 cells. Results are expressed relative to binding of CIITA at HLA-DRA in Raji, and show the mean and SD of 3 independent experiments (each performed with triplicate PCR measurements). (C) Binding of RFX to the new target genes was assessed by quantitative ChIP experiments performed with Raji, RJ2.2.5 and SJO cells. Results are expressed relative to binding of RFX at HLA-DRA in Raji, and show the mean and SD of 3 independent experiments (each performed with triplicate PCR measurements). (D) Binding of RFX to the new target genes was assessed by quantitative ChIP experiments performed with iDC. Results are expressed relative to binding of RFX at HLA-DRA, and show the mean and SD of 3 independent experiments (each performed with triplicate PCR measurements). nd, not done.

(A) Representative CIITA-binding profiles obtained for the new target genes in ChIP-chip experiments performed with DC. Results are represented as in Figure 2 for ChIP-chip experiments comparing iDC and mDC. (B) Binding of CIITA to the new target genes was confirmed by quantitative ChIP experiments performed with iDC and mDC. Results are expressed relative to binding of CIITA at HLA-DRA in iDC, and show the mean and SD of 3 independent experiments (each performed with triplicate PCR measurements). (C) Binding of CIITA to the new target genes was confirmed by quantitative ChIP experiments performed with Me67.8 melanoma cells stimulated for 0, 6 or 12 hours with IFNγ. Results are expressed relative to the plateau level obtained at HLA-DRA in cells induced for 12 hours, and show the mean and SD of 3 independent experiments (each performed with triplicate PCR measurements). Positive (HLA-DRA, Ii) and negative (background) controls were as in Figure 3.

Binding of CIITA to the HLA-DRA, HLA-DRB1, HLA-DMA, HLA-DOA, Ii and HLA-C genes is evident in the ChIP-chip experiments performed with Raji B cells and iDC. The RAB4A gene was used as negative control. Results are represented as log₂ ratios between hybridization signals obtained with CIITA-ChIP probes from Raji and RJ2.2.5 (left column) or iDC and mDC (right column). Each dot corresponds to a single oligonucleotide on the array. The dotted line in the right HLA-C profile indicates a peak that was not reproduced in all experiments. The schematic maps show positions of the transcription start sites (arrows) and S-Y enhancers (grey boxes). The scale in Kb relative to the start site is provided below.

(A) mRNA levels for the new target genes were compared between RJ2.2.5 cells (open bars) and RJ2.2.5 cells complemented with a CIITA expression vector (black bars). Results are represented relative to the expression found in the complemented cells, and show the mean and SD of 3 independent experiments (each performed in triplicate PCR measurements). (B) mRNA levels for the indicated target genes were measured in Me67.8 cells induced with IFNγ for 0, 12, 24 and 48 hours. Results are represented as fold induction relative to uninduced cells, and show the mean and SD of 3 independent experiments (each performed in triplicate PCR measurements). HLA-DRA and HLA-C were included as control CIITA-regulated genes.

(A) Binding of CIITA to the indicated genes was assessed in Raji B cells by quantitative ChIP. The HLA-DRA and Ii genes were used as positive controls. As negative control we used a sequence exhibiting only nonspecific CIITA association (background). Scores assigned to the candidate target genes (see Materials and Methods, Tables S1 and S2) are indicated above. Results are expressed relative to binding of CIITA at HLA-DRA, and show the mean and SD of 3 independent experiments (each performed with triplicate PCR measurements). Binding was deemed significant if it was greater than 2-fold above background. (B) Representative CIITA-binding profiles obtained in ChIP-chip experiments are provided for the nine new target genes validated in panel A. Results are represented as in Figure 2 for ChIP-chip experiments comparing Raji and RJ2.2.5 cells. (C) Binding of CIITA to the new target genes was confirmed by quantitative ChIP experiments performed with Raji and RJ2.2.5 cells. Positive (HLA-DRA, Ii) and negative (background) controls were as in panel A. Results are expressed relative to binding of CIITA at HLA-DRA in Raji, and show the mean and SD of 3 independent experiments (each performed with triplicate PCR measurements).

(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).

(A) Schematic representation of the expression and function of CIITA. Expression of the CIITA gene is regulated by three independent promoters – pI, pIII and pIV – that are activated in iDC, B cells and IFNγ induced cells, respectively. pI is silenced after maturation of DC (mDC). CIITA is a non-DNA-binding co-activator that activates expression of its known target genes (MHC-II, MHC-I, Ii) by binding to a transcription factor complex that assembles on an enhancer consisting of S, X, X2 and Y sequences. Known CIITA target genes contain an S-Y enhancer typically situated less than 200 bp upstream of the transcription initiation site. The X-box-binding factor RFX is essential for CIITA recruitment. (B) Four ChIP-chip strategies were developed for identifying target genes of CIITA in B cells and iDC. Test probes generated from CIITA-ChIP samples obtained from Raji B cells and iDC were hybridized to NimbleGen promoter arrays. Control probes were prepared from input DNA or CIITA-ChIP samples derived from RJ2.2.5 cells, SJO cells or mDC. (C) Enrichment of known CIITA target sequences in CIITA-ChIP probes. Quantitative PCR was used to verify enrichment of the HLA-DRA promoter in the indicated test samples (black bars) relative to control samples (open bars). The approximate fold-enrichment is indicated. To estimate enrichment relative to input DNA we used a control sequence exhibiting only nonspecific CIITA association (background). The results show the mean and SD of 3 independent experiments (each performed with triplicate PCR measurements).