Extended Data Fig. 9: Validation of mammalian reconstitution models expressing roX2 from Zfp185.

a, Schematic representation of experimental outline to create mES cells with targeted integrations of roX2 at mammalian loci to study induction of a local dosage compensation-like phenomenon. A scarless CRISPR strategy without integration of a selection cassette was used³⁰. roX2-expressing cells were derived from the MSL2^dCXC-CTD hybrid cell line (clone G2). roX2 or shuffled roX2 expression was driven by the Pgk1 promoter. The roX2 expression cassette is followed by 1.5 kb of Drosophila HAS (roX1, socs16D) or control sequences: odsh (Drosophila non-target) and Bscl2 (Mouse target). All cell lines are reported in Supplementary Data 4. b, Cropped agarose gel of PCR products across the Zfp185 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). The locus was amplified with primers in the Pgk1 promoter and 3′ outside of the homology arm. The PCR product was analysed by Sanger sequencing to verify the insertions. The control PCR shown below each gel amplifies the HBB-y locus. For source data, see Supplementary Fig. 1. c, RNA FISH of roX2 (red), Huwe1 (white) and DAPI (blue) of the mES cells MSL2^dCXC-CTD (G2), Pgk1-roX2-mCTRL (422-C9 (Bscl2)), Pgk1-roX2-socs16D HAS (424-B12). The pictures are single z-planes with scale bars = 5 μm, the hybridization was performed three times with similar results (also see Supplementary Note for technical information on RNA FISH). The co-localization with the X-linked Huwe1 RNA implies that these foci, which occur independently of the presence of HAS, correspond to transcripts at the targeted Zfp185 locus. d, RNA FISH for roX2 upon exposure of mES cells to 3% 1,6-hexanediol for 3 or 10 min. mES cells express roX2 from Zfp185 (cell line 419-D2), the MSL2-CTD is drosophilized (MSL2^dCXC-CTD hybrid). Scale bars = 5 μm, the hybridization was performed once. Right, quantification of the roX2 foci areas (n = 27 (0 min), n = 20 (3 min), n = 59 (10 min)). Barplots show mean ± s.e.m. with overlaid data points representing individual foci. The P-values were obtained by a Kruskal–Wallis test followed by a Dunn’s statistical test for multiple comparisons (see Extended Data Fig. 4a-g for response of the Drosophila MSL territory to 1,6-hexanediol treatment). e, RT–qPCR analysis of the indicated genes. The roX2 Zfp185 knock-in cell lines are displayed with red shaded bars, shuffled roX2 with grey shaded bars, the wild-type mouse MSL2-expressing cell line (D11) in black and the MSL2^dCXC-CTD hybrid cell line (G2) in blue. The RNA level of each gene was normalized to Hprt and expressed relative to the MSL2^WT cell line. The barplot represents the mean ± s.e.m. of at least 3 biological replicates. The roX2 and shuffled roX2 RNA levels are expressed as a geometric mean over all cell lines, as these transcripts are not expressed in the MSL2^WT cell line. All cell lines and RT–qPCR significance testing for Pnma5 are listed in Supplementary Data 4. A subset of the same data (result for MSL2^WT, D11; MSL2^dCXC-CTD, G2; 424-B12 (roX2) and 443-C3 (shuffled roX2) is shown in Fig. 4a. f, H4K16ac ChIP–qPCR analyses from MSL2^WT (D11), MSL2^dCXC-CTD (G2) and Pgk1-roX2-HAS (424-B12, 421-B5, 419-C3)-expressing mES cells. The barplot with overlaid data points shows the mean of n = 2 independent experiments (n = 1 for MSL2^WT) at the indicated positions along the Zfp185 locus. Enrichment values were calculated relative to input and serial dilutions performed to account for primer efficiency. The data are expressed as fold change enrichment over the Intergenic 3 region (see Supplementary Data 4), which resides near the transcriptionally inactive HBB-y gene (for clarity not shown in panel).