Figure 2.

Eliminating DNMT3A/3B prevents erosion of dosage compensation

(A) Experimental scheme for generation of single DNMT3A, single DNMT3B, and double knockout lines. All the knockout lines were produced by the CRISPR-Cas9 system. The XIST-positive line HUES21 p14-16 was used as the parental line. After genotyping, OCT4/H3K27me3 immunofluorescence was used to determine XCI state in each clone. See also Figure S2B.

(B) Western blotting for DNMT3A and DNMT3B. GAPDH was used as the loading control. In DNMT3B, the upper band is the targeted size.

(C) Experimental scheme for erosion induction and assessment of erosion state.

(D) XIST/XACT RNA-FISH assay. At day 18 after low-density plating, each line was analyzed. The scale bar indicates 10 μm.

(E) Quantification of XIST/XACT expression state by RNA-FISH. n, number of cells analyzed. A detailed classification of the results is shown in Figure S4A.

(F) qPCR analysis of XIST expression level in wild-type (WT) HUES21 p15 and DNMT3A^−/−3B^−/− lines. A t test was used for statistical calculation.

(G) XIST/XACT expression state by RNA-FISH in DNMT3A^−/−3B^−/− at day 30 after low-density plating. Note that the DNMT3A^−/−3B^−/− line was cultured for several months for gene editing before the low-density plating experiments. The scale bar represents 50 μm. The scale bars for images (i)–(iii) represent 10 μm. n, number of cells analyzed. The categories of expression state are the same as in (D). Two independent experiments were conducted.

(H) H3K27me3/OCT4 immunofluorescence analysis in DNMT3A^−/−3B^−/− at day 30. The scale bar represents 10 μm. The quantification results of H3K27me3 foci in OCT4-positive cells are shown as a pie chart. n, number of analyzed cells. Two independent experiments were conducted.

(I) DNA methylation (DNAme) state at XIST promoter regions in DNMT3A^−/−3B^−/− at day 30. BS-1 and BS-2 indicate CpG regions examined by bisulfite sequence.

(J) Experimental scheme of DNMT3B doxycycline (dox)-inducible system in the HUES21 DNMT3A^−/−3B^−/− line from the AAVS locus.

(K) Immunofluorescence analysis of Myc-tag and H3K27me3 at 120 h after dox treatment. Each dot shows the percentage of MYC-positive cells with H3K27me3 signal in observed areas. Two independent experiments were conducted.

(L) Immunofluorescence combined with XIST/XACT RNA-FISH analysis in DNMT3B inducible line after long-term dox treatment. The dox treatment was conducted for 22 days. OCT4 was used as the undifferentiated hPSC and XIST population was determined by only OCT4-positive cells. Two independent experiments were conducted. A detailed classification of the results is shown in Figure S4B. The scale bars represent 10 μm. n, number of cells analyzed.