Supplementary Figure 6: KDM1A K114me2 controls TMPRSS2-ERG fusion.

(a) Schematic representation of DHT-induced intrachromosomal interaction occurring at the TMPRSS2 locus between the enhancer region and a region where DNA double strand breaks (breakpoint) occur during TMPRSS2-ERG fusion. Androgen-dependent chromosomal loop formation is detected in 3C experiments using the indicated primers. AREs at the enhancer and breakpoint regions are labeled yellow and light blue, respectively. Positions of the NspI sites relevant for performing 3C experiment are indicated. (b) 3C analysis. Samples are DNA from LNCaP cells cultured in absence and presence of DHT that were analyzed for the DHT-dependent spatial chromatin interaction of the TMPRSS2 enhancer region and the breakpoint region during TMPRSS2-ERG fusion. The enhancer-breakpoint 3C PCR product was verified by sequencing. (c) Controls for 3C analysis. (d,e) Anti-Flag (d), anti KDM1A (d) anti-CHD1(e) and anti-tubulin (d,e) Western blots. Samples are lysates of LNCaP cells expressing RNAi-resistant (rr) Flag NLS KDM1A-rr, or Flag NLS KDM1A-rr K114A (d) CHD1-rr or CHD1-rr D425A (e) and treated with siRNA. (f) Model representing how assembly of methylated KDM1A and CHD1 drives AR-dependent transcription. (g) Model representing how assembly of methylated KDM1A and CHD1 drives AR-dependent loop formation and translocation at the TMPRSS2 gene. (b-e) Results are representatives of 3 independent experiments.