SAMD1 alters the chromatin landscape of erythroid progenitors. (A) Western blot of histone 3 methylation marks and total histone 3 at differentiation day 12. (B) Densitometry analysis of the histone 3 methylation marks. Relative to total histone 3 (n = 8). (C) Western blot of SAMD1, LSD1, and β-actin in HUDEP-2 cells. (D) Representative flow cytometry plot of anti-CD71 PE and anti-CD235a APC of sgControl sgSAMD1 HUDEP-2 cells. (E) Violin plot of all H3K4me2 peak signals in scramble (sgControl) and SAMD1 knockout (sgSAMD1) HUDEP-2 cells (2-tailed unpaired Student t test). (F) Scatterplot of H3K4me2 peak signal at CpG islands overlapping with SAMD1 or not. (G) Scatter plot of peak signal at overlapping H3K4me2 peaks in sgControl and sgSAMD1 HUDEP-2 cells divided into overlapping with a SAMD1 peak or not. Significantly changing peaks calculated by manorm using H3K4me2 read density at peak.38 (H) GSEA of annotated significantly changing (P < .05) H3K4me2 peaks overlapping with SAMD1. (I) Chromatin occupancy profile and normalized scores from CUT&RUN of SAMD1, LSD1, and H3K4me2 at the SLC4A1, SP3, and BCL11A loci in sgControl and sgSAMD1 HUDEP-2 cells. Δ(sgSAMD1 – sgControl) track indicates the change in H3K4me2 after SAMD1 knockout. Red box indicates the normalized score region (n = 2; 2-tailed paired Student t test). (J) Normalized RNA-seq of sgControl and sgSAMD1 primary erythroid progenitors at day 12 of erythroid differentiation (n = 4). RNA-seq counts were normalized based on reads per kilobase per million mapped reads (TPM). (K) Enrichr analysis of annotated H3K4me2 peaks increasing upon SAMD1 knockout, using data from the GWAS catalog. Error bars represent SD. ∗∗∗P < .001; ∗∗∗∗P < .0001. GSEA, gene set enrichment analysis; GWAS, genome-wide association study; NES, normalized enrichment score; ns, not significant.
