Fig 1: Identification of RFX target genes. (A) The pie charts show the fractions (%) of genes that are downregulated (top) or upregulated (bottom) in Rfx2-/- cells (left), Rfx3-/- cells (middle) or both (right) and have ChIP-seq peaks for RFX1, RFX2 and/or RFX3 within their promoters. Approximately one-third of downregulated genes have binding sites for either RFX1 plus RFX3 or all three RFX factors in their promoters. A minority of downregulated genes have binding sites for only RFX1 or RFX3. No downregulated genes are occupied by RFX2 alone. Most genes upregulated in Rfx2-/- or Rfx3-/- cells alone, and all genes upregulated in both Rfx2-/- and Rfx3-/- cells, lack RFX-occupied sites in their promoters. (B) Gene set enrichment analysis (GSEA) demonstrates that genes having RFX1, RFX2 or RFX3 ChIP-seq peaks in their promoters are strongly enriched in genes that are differentially expressed in Rfx2-/- or Rfx3-/- cells. (C, D) Histograms show the fold change (log2 FC) in expression for genes that are downregulated >2 fold in Rfx2-/- (light grey) or Rfx3-/- cells (dark grey). The presence of RFX1, RFX2 and/or RFX3 ChIP-seq peaks in the promoter is indicated for each gene at the right. (D) As in C, focusing on genes that are downregulated in both Rfx2-/- and Rfx3-/- cells, and have promoters occupied by all three RFX factors. All of these genes have at least one X-box motif in their promoter (-1000 to +500 bp), as highlighted by the blue font.
Fig 2: Dependence of a distal Pbx1 enhancer activity on co-binding of Six1 and Rfx1/3 through SIX:RFX motifs. (A) Genomic browser visualization of multiple Six1-bound regions at the Pbx1 and enlarged view of the intronic peak at ~49-kb downstream from the TSS. (B) Pbx1+49 000 contains two SIX-motifs separated by a RFX-motif. ChIP-qPCR using chromatin from E14.5–E15.5 cochlear epithelium shows strong binding with Rfx1 and relative weaker binding with Rfx3. A 510-bp fragment of Pbx1+49000 driving LacZ reporter transgene and two mutant reporter transgenes were generated by introducing mutations into the predicted SIX-motifs or both SIX:RFX-motifs in combination. These reporters were assessed by ChIP-qPCR using chromatin prepared from 293 cells cotransfected with His-Six1 expression plasmid and reporter Pbx1+49 000, Pbx1+4900SIXmt or Pbx1+49000SIX:RFXmt. These mutations abolished Six1 or Rfx3 binding. Transfection was repeated three times and qPCR was performed in triplicates for each independent experiment. Input was used for normalization (see Materials and Methods) and the enrichment of mock IP was considered 1-fold. *P < 0.05, **P < 0.01. (C) G0 transgenic analysis of LacZ transgene driven by a 510-bp of Pbx1+49 000 showing activity in the sensory epithelium and flanking GER and Hensen's (Hen) ells (n = 7/7 transgenic embryos), while Pbx1+49 000SIXmt (n = 3/3 transgenic embryos) or Pbx1+49000SIX:RFXmt (n = 8/8 transgenic embryos). Brackets indicate the organ of Corti. (D) In situ hybridization of E17.5 Eya1CreER or Six1Cko/Cko (Eya1CreER;Six1fl/fl, tamoxifen given at E12.5). Top panels, sections of whole-cochlea shown in bottom panels indicated by dashed lines. Brackets indicate the organ of Corti. (B) Genomic browser visualization of multiple Six1-bound regions at the Pbx1 and enlarged view of the intronic peak at ~49-kb downstream from the TSS. (E) CoIP analysis of nuclear extracts from E14.5 cochleae or 293 cells transfected with Flag-Pbx1/His-Six1. Other abb.: GER, greater epithelial ridge; IHC, inner hair cell; Hen, Hensen's cells; OHC, outer hair cell; SCs, supporting cells. GER, greater epithelial ridge. Scale bars: 30 µm.
Fig 3: Many ciliopathy genes are direct targets of RFX1, RFX2 and/or RFX3. The histograms show the fold change in expression (log2 FC) in Rfx2-/- and Rfx3-/- cells for 187 mouse orthologs of genes that are affected in human ciliopathies. The presence RFX1, RFX2 and/or RFX3 ChIP-seq peaks in their promoters is indicated at the right. Significant reductions in gene expression are highlighted in light (Rfx3-/- cells) or dark (Rfx2-/- cells) grey, whereas non-significant variations are indicated in orange (Rfx3-/- cells). Genes highlighted in red are orthologs of human genes implicated in Primary Ciliary Dyskinesia (PCD). Nearly all of these PCD genes are direct targets of RFX1, RFX2 and/or RFX3, and are downregulated significantly in Rfx2-/- and/or Rfx3-/- cells. All genes have at least one X-box motif in their promoter, except for those indicated in black font.
Fig 4: RFX3 expression in the mouse inner ear.Inner ear sections from a P1 wild-type mouse stained with an antibody for MYO6, which marks the inner ear hair cells (left panel) and RFX3 (right panel), in red, and counterstained with DAPI, in blue. A robust nuclear expression of RFX3 is detected in all HCs with a much weaker expression in ENHCs. Staining of the stereocilia with the RFX3 antibody is non-specific, validated by staining cKO ears in which the nuclear staining is abolished and the stereocilia staining persists (Supplementary Fig. 11). IHC, inner hair cell; OHCs, outer hair cells. Representative images of n>5 experiments. Scale bar, 60 µm.
Fig 5: Analysis of differentially expressed genes in ependymal cells from Rfx1, Rfx2 or Rfx3 deficient mice. (A) The volcano plots represent statistical significance (Y-axis, –log10P-value) and fold change (X-axis, log2 fold change) for alterations in gene expression observed in Rfx1-/-, Rfx2-/- or Rfx3-/- cells relative to WT cells. Whereas only few genes exhibited significantly altered expression in Rfx1-/- cells, numerous genes were differentially expressed in Rfx2-/- and Rfx3-/- cells. (B) The Venn diagrams summarize the overlaps observed between up (left) or down (right) regulated gene sets (fold change >2) in Rfx1-/-, Rfx2-/- or Rfx3-/- cells. Whereas there is little overlap between genes upregulated in Rfx2-/- and Rfx3-/- cells (left), most genes downregulated in Rfx2-/- cells are also downregulated in Rfx3-/- cells (right). There is no overlap between genes that are differentially expressed in Rfx1-/- cells and Rfx2-/- or Rfx3-/- cells. (C) The bar graphs show the fold changes in gene expression (log2 fold change) observed in Rfx2-/- (black bars) and Rfx3-/- (grey bars) cells for the 30 genes that are downregulated most strongly in Rfx2-/- (left) or Rfx3-/- (right) ependymal cells. Whereas nearly all genes that are strongly downregulated in Rfx2-/- cells are also strongly downregulated in Rfx3-/- cells (left), the reverse is not true (right). Genes highlighted in blue contain at least one X-box motif in their promoter region (-1000 to +500 bp).
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