Figure 3. MSL complex binding to X-chromosome is not dependent on actively elongating RNA polymerase.

(A) Actively elongating RNA polymerase is found on all chromosomes and largely colocalizes with MSL complex on the male X. Flavopiridol treatment removes elongating polymerase but has no effect on MSL localization. (B) SPT5 decorates all chromosome arms and largely colocalizes with MSL complex on male X. The SPT5 pattern in B is shown more clearly in separate color channels in Figure S5. Flavopiridol treatment removes the bulk of SPT5 from all chromosomes. (C) Flavopiridol treatment has no effect on polymerase paused at the TSS. Chromosomes were stained with indicated antibodies against MSL1, Ser2 phopshorylated RNAP, Ser5 phosphorylated RNAP, and/or SPT5. (D) ChIP analysis of male S2 cells immunoprecipitated with anti-MSL1 antibodies. MSL1 is enriched near the 3′ ends of known MSL1 targets CG13316 and CG32767 with comparatively less MSL complex found near the 5′ TSS measured by quantitative real time PCR relative to the autosomal PKA gene. Blocking elongation with flavopiridol does not alter the MSL1 distribution. MSL1 binding to the roX2 DHS control region occurs by a sequence-dependent mechanism and does not require transcription [62].