Extended Data Fig. 10: Validation of mES cells expressing roX2 from Bex2, HBB-y and Drosophila tethering experiment.

a, Cropped agarose gel of PCR products across the Bex2 locus from gDNA of parental and Pgk1 promoter-driven roX2 knock-in clones derived from G2 (MSL2^dCXC-CTD) as parental mES cell line (also see Supplementary Data 4). For source data, see Supplementary Fig. 1. b, RT–qPCR analysis of the indicated genes as in Fig. 4b. The RNA level of each gene was normalized to Hprt and expressed relative to the MSL2^WT cell line (arbitrary units for roX2, since it is not expressed in MSL2^WT cells). The barplot represents the mean ± s.e.m. of at least 3 biological replicates. c, as in a, but for cells with roX2 integrated into the HBB-y locus. For source data, see Supplementary Fig. 1. d, RT–qPCR analysis of the indicated genes as in Fig. 4c. The barplot represents the mean ± s.e.m. RNA levels (normalized to Hprt) of at least 3 biological replicates. RNA levels represent arbitrary units. HBB-bs gene could only be detected in RT–qPCR from a mouse control cDNA, but not from the experimental mES cells. roX2 is not expressed in MSL2^WT cells. e, as in b but in Msl3Δ knockout cells derived from MSL2^WT (D11 ± Msl3Δ, black), MSL2^dCXC-CTD (G2 ± Msl3Δ, blue) and MSL2^dCXC-CTD + roX2 (498-B6 ± Msl3Δ red) cells. roX2 in clone 498-B6 (red bars) is expressed from the Bex2 locus (Fig. 4b). f, Cropped immunoblots of total protein extracts of adult male fly heads expressing MS2CP-tagged MSL2 transgenes (also see Fig. 4d) in comparison with wild-type control (msl-2::HA). All transgenes also contain a 2xHA-mCherry tag and were expressed with tub-Gal4 in a heterozygous msl-2^227/kmA/CyO-GFP background. To account for differences in protein stability, the MSL2Δ^MS2CP (86FB) and MSL2^MS2CP (VK33) transgenes are expressed from different landing sites. The star indicates the expected migration of the full-length fusion proteins, whereas the lower products correspond to degradation products. The experiment has been performed once. For source data, see Supplementary Fig. 1. g, Analysis of adult male viability in flies expressing transgenic MS2CP-tagged or untagged transgenes. The genotype was roX1⁺, roX2⁺; msl-2^kmA/msl-2²²⁷; tub-Gal4/UAS-msl-2 or respective heterozygous CyO-GFP control. The data was expressed relative to females obtained from the same cross. The barplot represents the mean ± s.e.m. with overlaid datapoints reflecting the results from each cross/vial (n = 5 for MSL2^MS2CP, n = 7 for MSL2Δ^MS2CP, n = 6 for MSL2^WT). Details on genotypes and nature of transgenes are provided in Methods. h, Transgenic flies expressing MS2CP-tagged MSL2 transgenes are ectopically tethered to MS2-loops tagged roX RNA (also see Fig. 4d–h, MSL2^WT = wild-type D. melanogaster transgene, MSL2^MS2CP = wild-type MS2CP-tagged and MSL2Δ^MS2CP = MSL2(1-520) MS2CP-tagged). The transgenes were expressed as UAS-msl-2 versions in act-Gal4, msl-2^Δ/msl-2²²⁷ transheterozygous null mutant backgrounds, where the X chromosome is roX1⁺ roX2⁺ (no loops) or roX1^6xMS2, roX2^Δ (with tethering, indicated with loops symbol). For cross setup and details see Methods. Immunofluorescence was performed in salivary glands and the msl-2 transgenes were detected with HA antibodies (red), MSL1 is shown in green, DAPI in blue. The pictures are orthogonal projections of an entire stack with scale bars = 50 μm. i, as in h but in imaginal discs. msl-2 transgenes were detected with HA antibodies (yellow), DAPI in blue. The representative pictures are single z-planes with scale bars = 10 μm. j, Top, schematic representation of the strategy to determine the territory enrichment ratio, which is calculated by dividing average intensity of the territory in one nucleus by the average intensity of an equally sized region elsewhere in the same nucleus. Bottom, quantification of h, density plots of the X-chromosome territory enrichment ratio. The number of quantified nuclei is MSL2^WT (no loops n = 22, with loops n = 24), MSL2^MS2CP (no loops n = 45, with loops n = 42) and MSL2Δ^MS2CP (no loops n = 37, with loops n = 37). The P-values were obtained with a two-sided Wilcoxon rank-sum test comparing the MSL2 enrichment in comparison with DAPI. DAPI is equally dispersed within the nucleus and shows a territory enrichment ratio of 1 (= not enriched).