Fig. 4

Enok associates with chromatin and facilitates trxG by inhibiting PC binding. a–c Polytene chromosomes from third instar larvae expressing Myc-tagged-Enok were stained with anti-Myc (a) and anti-TRX (b) antibodies. Enok and TRX colocalize at several loci (c, arrows). d ChIP with anti-FLAG antibody using stable cells expressing FLAG-tagged-Enok (FH-Enok) showed enrichment of Enok at non-homeotic (psq, pnr, pnt, disco) and homeotic (iab-7, bxd, Dfd) targets when compared with anti-FLAG ChIP from empty vector control cell line. Enok was absent on an Intergenic Region (IR) used as negative control. ChIP from S2 cells using anti-Enok antibody showed strong enrichment of Enok at trxG targets mentioned above. e Effect of enok knockdown on pnr and pnt in D.Mel-2 cell line. Cells treated with dsRNA against enok showed a trend similar to that of trx knockdown, with decreased expression of pnr and pnt when compared with LacZ dsRNA-treated cells (control). f ChIP with anti-PC using enok-depleted cells showed increased enrichment of PC at pnr, pnt, bxd and Dfd when compared to LacZ dsRNA-treated control cells. g ChIP with anti-H2A-ubi from cells treated with dsRNA against enok showed increased enrichment of H2AK118ub1 on pnr, pnt, bxd and Dfd as compared to control cells. h Cells exposed to dsRNA against both enok and E(z) showed a decrease in transcript levels of pnr and pnt in a manner similar to enok knockdown. Cells treated with dsRNA against both Pc and enok showed pnr and pnt expression levels comparable to control cells. i, j ChIP with Pol-II antibody from cells treated with dsRNA against enok alone, enok + E(z) or enok + Pc. Knockdown of enok alone or enok + E(z) showed increased occupancy of Pol-II at TSS (i) and decreased occupancy in gene body (j) of pnt and pnr. In contrast, enok + Pc knockdown revealed Pol-II levels similar to control cells. Knockdown experiments shown in (e, h) were performed in triplicates and ChIP experiments shown in (d, f, g, i and j) were performed in duplicates. Individual Student’s t tests were performed and the p values obtained were marked as ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 or ****p ≤ 0.0001. Error bars represent SEM