Extended Data Fig. 7: Lysine 4 residue is required for histone H3 protein stability in HEK293T cells.
a, Normalized RNA-seq counts representing H3.3B gene expression in control, K36A, K4A mutant clonal lines, and wild type (WT) in ESCs and neurons. P value: Wald test and adjustment using Benjamini Hochberg’s method in DESeq2, n.s: P adj > 0.05; n = 2 (control), n = 3 (K4A/K36A) or n = 4 (WT) independent biological replicates. Significantly lower H3.3B gene expression in K36A cells is partially due to difficulties in mapping (5 nucleotide changes introduced). b-e, Stability analysis of H3.3 mutants in HEK293T cells. H3.3/H3.1 construct fused to HA-FLAG-tag for detection and co-transcribed with P2A-GFP generates two proteins, mostly nuclear H3.3-HA-FLAG and mostly cytosolic P2A-GFP (expression control). Immunoblots of cellular fractions of HEK293T transfected with WT or K4A/R/Q or K36A/R/Q mutant H3.3 (Q, glutamine and R, arginine) (b) and transfected with WT or K4A/K36A mutant H3.3 (c). Immunoblots of whole-cell lysates of HEK293T transfected with WT and K4A mutant H3.1/H3.3 (d) and chromatin fraction of HEK293T untreated or treated with the proteasome inhibitor MG-132 (5 μM for 5 hours prior to harvesting) (e). Suz12 and endogenous H3.3 serve as a loading control. Experiments in b-e were repeated three times independently with similar results. f, H3.3 ChIP-qPCR results in control and K4A mutant ESCs untreated or treated with the proteasome inhibitor MG-132 (5 μM for 5 hours). Relative enrichment of H3.3 wild type or H3.3K4A over input was measured at TSS or TES of indicated genes, or a gene-free region on chromosome 6 (neg. control). The data are represented as the mean of two independent replicates (n = 2) Source data.
