Extended Data Fig. 7: Validation of mammalian reconstitution models.

a, Co-immunoprecipitations of Flag-tagged MSL2^WT, MSL2^dCTD or MSL2^dCXC-CTD hybrid knock-in (also see Fig. 3a) and untagged parental control mES cell lines with cropped immunoblots for the MSL complex members MSL1 and MOF. RNA Pol2 serves as an input loading and negative immunoprecipitation control. Immunoprecipitations were conducted from nuclear extracts. Blots were cropped at the vertical dashed line to remove extra lanes. Displayed bands were derived from the same gels. For source data, see Supplementary Fig. 1. b, RT–qPCR analysis of the mouse MSL2 target genes Bex2, Phf8, Zfp185 and Tsix in parental (clone MF, n = 4), Msl2∆ (clone D12, n = 4), MSL2^WT (clone D11, n = 4), MSL2^dCTD (clone F4, n = 4) and MSL2^dCXC-CTD (clone G2, n = 3) mES cells (also see Fig. 3a). The RNA level of each gene was normalized to Hprt and expressed relative to the parental cell line (MF). The barplot represents the mean ± s.e.m. with overlaid data points representing biological replicates. c, RNA-seq was conducted in MSL2^WT (clone D11), knockout (Msl2∆, clone D12) or hybrid (MSL2^dCTD, clone F4; MSL2^dCXC-CTD, clone G2)-expressing mES cells. In the scatterplot, each dot shows the log₂[fold-change] of the RNA expression of a mouse gene in a given MSL-hybrid cell line versus the MSL2^WT mES cells. Differentially expressed (DE) genes were colored in red (upregulation) or blue (downregulation), if the FDR from DESeq2 was <0.05 in any of the MSL2 hybrid cell lines. The Pearson correlation coefficient of the gene expression changes was calculated with the panel.cor function in R. d, RNA-seq as in c. Heatmap and dendrogram displaying the log₂[fold-change] of the significantly (FDR < 0.05) DE genes in any of MSL2 hybrid versus the control (MSL2^WT) mES cells by RNA-seq. The log₂[fold-change] of these genes in Msl2∆ knockout versus control (MSL2^WT) was plotted to compare whether DE genes in MSL2 hybrid expressing cells are also misregulated in the knockout. e, Cropped agarose gel of PCR products across the Zfp185 locus from gDNA of parental and HAS knock-in clones derived from G2 (MSL2^dCXC-CTD hybrid) as parental mES cell line (also see Supplementary Data 4) with primers binding outside both homology arms. This product was then re-amplified in a nested PCR for resolving the size shift introduced by replacing the 2 kb Zfp185 region with 1.5 kb of HAS sequences. The original PCR product was analysed by Sanger sequencing to verify insertion of the HAS and deletion (data not shown). For source data, see Supplementary Fig. 1. f, RT–qPCR analysis of Zfp185 and its two neighbouring genes, Nsdhl and Pnma5, the latter of which is also downregulated in Msl2∆ mES cells. Bex2 and Firre are target genes at a different locus, Rplp0 and Tbp serve as non-target controls. The Zfp185 knock-in cell lines displayed with green bars are all derived from the MSL2^dCXC-CTD hybrid cell line (G2, blue). The clone IDs from top to bottom are D11 (MSL2^WT, black), G2 (MSL2^dCXC-CTD, blue), 444-A11, 444-C2, G2-F4, 376-28, 377-A4 and 377-D6. The RNA level of each gene is normalized to Hprt and expressed relative to the MSL2^WT cell line. The barplot represents the mean ± s.e.m. of at least n = 4 biological replicates. g, as in Fig. 3f, RT–qPCR analysis of the indicated genes in roX2 (exon 3)- or EGFP control-expressing cell lines that were derived from mES cell clones D11 (MSL2^WT), G2 (MSL2^dCXC-CTD), G2-F4 (MSL2^dCXC-CTD, roX1 HAS at Zfp185), 444-C2 (MSL2^dCXC-CTD, socs16D HAS at Zfp185). The RNA level of each gene was normalized to Hprt. The barplot represents the mean with overlaid datapoints of biological replicates (n = 2 independently derived cell lines). h, RT–qPCR analysis of the indicated genes in mES cell clones D11 (MSL2^WT), G2 (MSL2^dCXC-CTD hybrid) or G2-A5 that expresses roX2 (exon 3) from the HBB-y locus (see Fig. 4c and Supplementary Data 4). The RNA level of each gene was normalized to Hprt. The barplot represents the mean ± s.e.m. of at least n = 2 biological replicates. The P-values were obtained by a two-sided Wilcoxon rank-sum test.