Fig. 4
CBX5 intron 1 generates a novel transcript STET. a Schematized view of CBX5 intron 1. Arrows indicate direction of transcription and rectangles indicate exon sequences. The HP1α coding region is indicated by black colouring. pA indicates the localization of poly-A signals. E1 is the first exon for HP1α-V3, E1A is the first exon for HP1α-V1, E1B is the first exon for HP1α-V2, and E2A the terminal composite exon for the STET transcript. The two different STET transcripts, STET1 and STET2, using different splice sites are also indicated. Below the intron drawing is indicated location of amplicons used to detect transcription throughout the CBX5 intron 1. b Scanning of CBX5 intron 1 5'-region for expressed transcripts. Primer localizations for the various amplicons is shown in A. STET-pan indicates the measurement of the spliced STET transcript. Relative expression was calculated from RT-qPCR using GAPDH expression for normalization. –RT threshold line in red shows the level of background calculated from paired samples with a cDNA syntheses reaction without reverse transcriptase to account for the detection of contaminating DNA. c Nucleotide sequence of the STET E2A and surrounding sequences. The exon sequence extension in STET1 is enclosed by rectangle. pA signal is underlined and in bold. Different ORF’s are shown with the peptide sequences in color. d Expression analysis of STET transcripts in MCF7 and MDA-MB-231 cells. Relative expression was calculated from RT-qPCR using GAPDH expression for normalization. e Expression analysis of STET-pan in HMEC, MCF7, MDA-MB-231, and HeLa cells. Relative expression was calculated from RT-qPCR using GAPDH expression for normalization. The calculated semi-quantitative expression ratio between HP1α-V3 and STET is shown to the right. f TSA mediates STET transcript down-regulation. Relative expression of STET-pan mRNA in MCF7 and MDA-MB-231 cells after 24 h TSA or DMSO control treatment. Relative expression was calculated from RT-qPCR using GAPDH expression for normalization. For all panels, bars represent mean values with standard deviations