DNase I hypersensitive site mapping over the Pax6 promoter regions.
DNaseI HS mapping was carried out in three different mouse cell lines, MV+ (lens derived), N2A (neuroblastoma derived) and RAG (embryonic kidney derived). A) Schematic overview of the Pax6 genomic locus on mouse chromosome 2E3. The extent of the locus examined by HS mapping is indicated by the solid portion on the dashed line. The exons of Pax6 and the adjacent Elp4 gene are shown as black rectangles. Blue and orange ellipses indicate the positions of some known enhancers, and red arrows show the position of previously documented hypersensitive sites in the human PAX6 downstream regulatory region (DRR). Below the proximal Pax6 region is shown enlarged, with the exons indicated by numbered rectangles (open rectangle for non-coding exons, black for coding exons). Positions of the P0, P1 and Pα promoters are shown. The restriction fragments analysed for hypersensitivity and probe positions are indicated. (B) Expression analysis of Pax6 in the cell lines used in this study. rtPCR for Pax6 confirms expression in MV+ and N2A, and absence of Pax6 mRNA in RAG. The ubiquitously expressed Ercc3 was used as control. (C) HS mapping Southern blots of the Pax6 promoter region. Hypersensitive sites are found in the promoter regions of Pax6 in the expressing cell lines MV+ and N2A, but not in the Pax6 negative RAG cells. HS sites are found in the P0, P1 and Pα promoters in the MV+ cell line, while the N2A cell line only shows hypersensitivity in the P1 promoter. Positions of transcription start sites are marked by the side of the blot. (D) Chromatin immuno precipitation for tri-methylated histone H3K4, a mark of active chromatin, demonstrates the three known Pax6 promoters, P0, P1 and Pα are all active in MV+. In N2A only P1 carries the active H3K4me3 mark, while none of the promoters carry the H3K4me3 mark in RAG cells. The ubiquitously expressed reticulocalbin (Rcn1) promoter was used as positive control and an intergenic sequence lacking any known features was used as negative control.