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. 2016 Feb 8;213(2):177–187. doi: 10.1084/jem.20150435

Figure 1.

Figure 1.

Coregulation of BTN2A2 with MHCII genes. (A) Schematic representation of transcription factors (RFX, NF-Y, and CIITA) bound to the SXY module of MHCII promoters. The consensus SXY sequence is shown. TSS, transcription start site. (B) The human genome was scanned for promoters containing SXY motifs. Homology scores are plotted as a function of position in the genome. Dots corresponding to known RFX and CIITA targets (BTN2A2, RAB4B, Ii, and MHCII genes) are labeled. A map of the BTN locus is indicated underneath the graphs. (C) Human BTN2A2 and mouse Btn2a2 SXY modules are aligned with that of HLA-DRA. Conserved positions are in bold. (D) The occupation of HLA-DRA and BTN2A2 promoters by RFX and CIITA was quantified by ChIP in WT (Raji), CIITA−/− (RJ2.2.5), and RFX5−/− (SJO) B cells, or in untreated and IFN-γ–induced Me67.8 cells. Occupancy is expressed relative to WT or induced cells. (E, left) BTN2A2 and HLA-DRA mRNAs were quantified by qRT-PCR in Raji (R), RJ2.2.5 (CIITA−/−), RJ2.2.5 complemented with expression vectors encoding CIITA isoforms I, III, or IV, and RFX-deficient B cell lines BLS-1 (B), Da (D), Ro, Abd (A), and 6.1.6 (6). (Right) BTN2A2 and HLA-DRA mRNAs were quantified by qRT-PCR in untreated and IFN-γ–induced human umbilical vein endothelial cells (WT) and CIITA−/− fibroblasts (BLS3). Results were normalized using TBP mRNA and expressed relative to Raji (left) or uninduced WT cells (right). (F) Btn2a2 and H2-Aa mRNAs were quantified by qRT-PCR in splenic B cells, BMDCs, or untreated and IFN-γ–treated MEFs from WT, CIITA−/−, and RFX5−/− mice. Results were normalized using TBP mRNA or 18S ribosomal RNA and expressed relative to WT. (G) BTN2A2 expression by untreated and LPS-activated splenic CD19+ B cells from WT, Btn2a2−/−, Rfx5−/−, and CIIta−/− mice was analyzed by flow cytometry. (H) Luciferase reporter assays were performed in WT (Raji), CIITA−/− (RJ2.2.5), and RFX−/− (SJO) cells using constructs driven by HLA-DRA (left) or BTN2A2 (middle) promoters, or by a hybrid promoter in which the SXY module of HLA-DRA was replaced with that of BTN2A2 (right). Activity is expressed relative to WT. (I) Luciferase activity was measured in WT (Raji) and CIITA−/− (RJ2.2.5) cells for BTN2A2-DRA constructs containing mutations in the S, X, X2, and Y elements. Results are expressed relative to HLA-DRA in WT. (D–F, H, and I) Results show means and standard deviations derived from three experiments. (F and G) At least three mice per group were analyzed in each experiment.