Figure 3.
Downregulation of DNMT3A results in superior growth in EPO-rich environments. (A) Schematic representation of single-cell deposition of K562 cells after introduction of the mutations by CRISPR, expansion in vitro followed by colony screening to select the monoclonal colonies harboring specific mutations to perform RNA sequencing (RNA-seq). (B) Previously characterized DNMT3A transcripts and the corresponding ENSEMBL annotation. (C) Heat map of DNMT3A transcripts annotated in ENSEMBL and detected in the bulk RNA-seq. W305∗ mutant K562 show lower levels of reported protein-coding transcripts and increased levels of transcripts annotated to undergo NMD, compared with the other genotypes. (D) Principal component analysis–based clustering of transcriptome profiles of DNMT3A downregulated vs control HUDEP-2 cells, generated as shown in supplemental Figure 5A (n = 9). (E) Gene set enrichment analysis of heme metabolism signature in DNMT3A downregulated vs control HUDEP-2 cells. (F) Normalized expression counts (DESeq2) for indicated genes in DNMT3A downregulated (red) vs control HUDEP-2 (blue) cells (n = 9; adjusted P values for HBA1, HBA2, HBB, and EPOR are 1.39 × 10–15, 1.46 × 10–20, 1.91 × 10–19, and 2.62 × 10–6, respectively). (G) DNMT3A downregulated (BFP+) and HUDEP-2 control (GFP+) cells were cocultured at the indicated ratio in regular and erythroid differentiation media. The ratio between BFP+ and GFP+ cells was analyzed over a period of 8 days and is presented relative to the input (n = 3; 3 independent experiments; measurement in duplicates). P values differentiation vs regular media for time points days 1, 3, 5, and 8 were: P = 0.33; P = 0.19; P = 0.03 (∗); and P = 0.01 (∗), respectively. Diff, differentiation ; FDR, false discovery rate; gRNA, guide RNA; NES, normalized enrichment score; PC, principal component; Reg, regular.

Downregulation of DNMT3A results in superior growth in EPO-rich environments. (A) Schematic representation of single-cell deposition of K562 cells after introduction of the mutations by CRISPR, expansion in vitro followed by colony screening to select the monoclonal colonies harboring specific mutations to perform RNA sequencing (RNA-seq). (B) Previously characterized DNMT3A transcripts and the corresponding ENSEMBL annotation. (C) Heat map of DNMT3A transcripts annotated in ENSEMBL and detected in the bulk RNA-seq. W305∗ mutant K562 show lower levels of reported protein-coding transcripts and increased levels of transcripts annotated to undergo NMD, compared with the other genotypes. (D) Principal component analysis–based clustering of transcriptome profiles of DNMT3A downregulated vs control HUDEP-2 cells, generated as shown in supplemental Figure 5A (n = 9). (E) Gene set enrichment analysis of heme metabolism signature in DNMT3A downregulated vs control HUDEP-2 cells. (F) Normalized expression counts (DESeq2) for indicated genes in DNMT3A downregulated (red) vs control HUDEP-2 (blue) cells (n = 9; adjusted P values for HBA1, HBA2, HBB, and EPOR are 1.39 × 10–15, 1.46 × 10–20, 1.91 × 10–19, and 2.62 × 10–6, respectively). (G) DNMT3A downregulated (BFP+) and HUDEP-2 control (GFP+) cells were cocultured at the indicated ratio in regular and erythroid differentiation media. The ratio between BFP+ and GFP+ cells was analyzed over a period of 8 days and is presented relative to the input (n = 3; 3 independent experiments; measurement in duplicates). P values differentiation vs regular media for time points days 1, 3, 5, and 8 were: P = 0.33; P = 0.19; P = 0.03 (∗); and P = 0.01 (∗), respectively. Diff, differentiation ; FDR, false discovery rate; gRNA, guide RNA; NES, normalized enrichment score; PC, principal component; Reg, regular.

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