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. Author manuscript; available in PMC: 2014 Dec 18.
Published in final edited form as: Nat Genet. 2014 Aug 10;46(9):973–981. doi: 10.1038/ng.3058

Figure 6. The reverse strand PRE/TRE transcript binds specifically to E(Z) in vivo and removes E(Z) from the locus from which it is transcribed.

Figure 6

(a-f) The PcG protein E(Z) binds specifically to the PRE/TRE reverse strand in S2 cells. (a) Constructs containing the PRE/TRE in each orientation under control of the hsp70 promoter (pKC27vg.fwd or pKC27vg.rev) were transfected separately into Drosophila S2 cells. (b) Plasmids expressing either GFP::EZ or GFP were cotransfected separately with each of the constructs in (a). GFP- trap was used to IP the protein and associated RNAs from whole cell extracts without crosslinking. (c) Strand-specific cDNA synthesis and RT-qPCR shows that each of the transfected plasmids predominantly transcribes one strand (data normalised to highest expressed strand, typically between 200 and 1000 % of TBP). The forward strand was transcribed typically 3-20 fold higher in the forward plasmid transfection than the reverse strand in the reverse plasmid transfection (data not shown). Mean and s.e.m. of at least three independent IP experiments are shown. (d) Western blot using αGFP and αTubulin, showing GFP (top) and GFP::E(Z) (bottom) present in input (I), not in unbound (U) and enriched after GFP trap (IP). (e) Enrichment of PRE/TRE RNA after GFP trap on cells transfected with either plasmid as indicated, and GFP or GFP::E(Z). IP levels were normalised to input and further to GFP mRNA levels, and were controlled to exclude DNA contamination. Mean and s.e.m. of at least three independent IP experiments are shown. cDNA synthesis was performed by random priming. Whereas RNA did not coIP with GFP::E(Z) after transfection of the forward construct, GFP::E(Z) cotransfected with the reverse plasmid, which transcribes predominantly reverse strand (c), shows PRE/TRE RNA enrichment of over 100 fold after IP (4th column). (f) This IP material was subjected to strand-specific cDNA analysis, showing that only the reverse strand is detectable. Mean and SD of two experiments is shown; scale shows IP as % of TBP in input. (g) ChIP-seq analysis of E(Z) and H3K27me3 in 0-16h embryos over-expressing forward (top; pKC27vg.fwd') or reverse (bottom; pKC27vg.rev') strand ncRNAs. Left: transgenic locus. Black arrow indicates position and orientation of 1.6 kb PRE/TRE. Grey box: 1.6 kb PRE/TRE sequence identical between transgene and endogenous vg locus. White area: sequence unique to transgenic locus, corresponding to the ampicillin resistance gene of pKC27. Asterisk and arrowhead indicate differences between forward and reverse over-expression. Data are shown as ChIP/input to correct for different copy numbers of the PRE/TRE and the flank. Right: no difference was detectable at the endogenous vg locus (Chr 2R: 8,791,079- 8,794,193; see also Supplementary Fig. 10) or at the Fab-7 PRE/TRE (Chr 3R: 12,723,848- 12,728,057). Thick lines indicate position of PRE/TRE. RPM, reads per million. The experiment was repeated three times for H3K27me3 on pKC27vg.fwd’, once for H3K27me3 on pKC27vg.rev’ and once for E(Z) on both pKC27vg.fwd’ and pKC27vg.rev’. Tracks from one representative ChIP experiment are shown.