Figure 4: Enhanced expression of a large set of genes and transposons in Tctp mutants.

(a–d) Tctp colocalizes with Pol-II in the interbands of squashed SG chromosome stained for Tctp and different CTD forms of the largest Pol-II subunit. (a) Staining with anti-Pol-II for non-phospho and phosphorylated CTD in all transcription steps. (b) anti-Pol-IIa for non-phosphorylated CTD in the promoter-recruited step. (c) anti-Pol-II^Ser5 for Ser-5-phosphorylated CTD in transcription initiation. (d) anti-Pol-II^Ser2 for Ser-2-phosphorylated CTD in transcription elongation. Boxed areas are enlarged as split images. Scale bars, 50 μm. (e) Co-IP using nuclear S2 cell extract transfected with both 2xFLAG-Brm and 2xMYC-Tctp. Tctp forms a large protein complex with Brm and Pol-II. These interactions are resistant to benzonase treatment suggesting direct interaction. The interaction between Tctp and Pol-II or Brm and Pol-II is relatively weak (longer exposures). (f) Effects of Tctp on transcription initiation and elongation in SG. Increased Pol-II^Ser5 and Pol-II^Ser2 levels in Tctp^EY/h59 mutants (white arrows), which are restored by genomic Tctp^Ge30-2 in mutants (Tctp^RES) or by heterozygous brm² mutation (black arrows). Note that the amount of Tctp in brm^2/+, Tctp^EY/h59 is not restored. β-tubulin and Histone 3 (H3) are loading controls. (g,h) Chromosome localization of elongating Pol-II. (g) Pol-II^Ser2 localization before heat-shock (-HS). (Top) Increased transcription elongation levels in Tctp^EY/h59, compared with w¹¹¹⁸ control. (Bottom) Genomic Tctp^Ge30-2or brm^2/+ rescues the high level of Pol-II^Ser2 in Tctp^EY/h59. (h) Elongating Pol-II localization after heat-shock (+HS). (Top) Pol-II^Ser2 in wild-type exclusively localizes to heat-chock loci after HS. Boxed heat-shock loci between 87 A and 95D are enlarged in the right. (Bottom) Substantial levels of Pol-II^Ser2 in Tctp mutants still remain at many euchromatic sites (white arrowheads). Scale bars, 50 μm. (i) QPCR. 34 of 42 genes with high expression levels in Tctp mutants were suppressed by brm²/+ mutation. (j,k) QPCR of transposon expression in Tctp mutants. (j) Retrotransposons like Copia, Blastopia, Gypsy, and R2 are highly expressed in various tissues from Tctp^EY/h59. (k) Additional brm mutation in Tctp^EY/h59 (brm^2/+, Tctp^EY/h59) suppresses the high level expression of transposons in SG (mean±s.d.). Het-A, TART, and Invader4 are located in the telomere region.