Testing functional activity of MSL2 mutants in males. (A) Viability (as a relative percentage) of male adult flies after expression of MSL2 derivatives on the msl2^γ227/msl2^γ227 (msl2-null) background. Flies expressing corresponding MSL2 derivatives in heterozygous msl2^γ227/CyO flies were used as internal controls with 100% viability. Results are expressed as mean±s.d. of three independent crosses. (B) MSL1 and MSL2 localization on the polytene chromosomes from 3rd day male larvae on the msl2-null background expressing different FLAG-tagged variants of MSL2 protein. Panels show immunostaining of 3×FLAG-MSL2 protein with mouse anti-FLAG antibody (green) and MSL1 protein with corresponding rabbit antibody (magenta). DNA was stained with DAPI (blue). (C) Comparison of binding of MSL1, MSL2 and CLAMP at different CES and PionX (marked with red text) regions in the MSL2-expressing flies on the msl2^γ227 background. Histograms show ChIP enrichments at the CES regions on chromatin isolated from male flies expressing different MSL2 variants: MS2^wt(wt), MSL2^Δ13d(Δ13d), MSL2^R*(R*), MSL2^R*Δ13d(R*Δ13d). The results are presented as a percentage of input genomic DNA normalized to corresponding positive autosomal genomic regions for MSL1 (26E3) and MSL2 (25A3), and compared with binding levels in the flies expressing wild-type MSL2 protein (corresponding to the ‘1’ on the scale). Error bars show s.d. of quadruplicate PCR measurements for three independent experiments. *P<0.05, **P<0.01.

Testing functional activity of MSL2 mutants in females. (A) Viability (as a relative percentage) of females to male adult flies after expression of MSL2 derivatives (wt, Δ13d, R*, R*Δ13d). Results are expressed as mean±s.d. of three independent crosses in homozygous (black) and heterozygous (gray) lines. (B) Expression levels of the roX1 and roX2 RNAs in male and female larvae in the y¹w¹ flies (wild-type background) and MSL2-expressing flies (wt, Δ13d, R*, R*Δ13d) on the msl2^γ227 background. Individual transcript levels were determined by RT-qPCR with corresponding primers normalized relative to RpL32 for the amount of input cDNA. Histogram shows the changes of mRNAs for the tested roX genes compared with expression levels in wild-type flies (y¹w¹, ‘1’ on the scale). Error bars show s.d. (n=3). (C) Comparison of binding of MSL1 and MSL2 in the roX1 and roX2 CES regions in the MSL2-expressing flies on the msl2^γ227 background. (D) Distribution of the MSL complex on the polytene chromosomes from 3rd day female larvae expressing different FLAG-tagged variants of MSL2 protein. Other designations are as in Fig. 2.