Fig. 3: UBP5 acts as a H2A deubiquitinase.

a Western blot analysis of H2Aub and H2A in Col-0 and ubp5 seedlings. Histone H3 is used as a loading control. Numbers above blots denote relative H2Aub levels determined by ImageJ. Three independent experiments yielded consistent results. b Venn diagram illustrates overlap between H2Aub marked genes in Col-0 and ubp5, n = no. of genes. The genes are considered as marked when an overlapping H2Aub peak is present in at least two biological replicates based on MACS3 peak calling (q < 0.05 and score >30). Super exact test was conducted. c H2Aub levels in genes overlapping between Col-0 and ubp5 (n = 14816) from Fig. 2B were categorised into hyper/hypo-marked and unchanged. Violin plots overlaid with boxplots show the distribution pattern of data. Displaying median (middle line), first and third quartiles (Q1 and Q3), with whiskers indicating data spread (1.5*IQR from Q1 and Q3). Statistical significance was tested using the Kruskal-Wallis test followed by Dunn’s post hoc analysis. d Bar chart shows the number of H2Aub hyper-, hypo-, de-novo- and unchanged-H2Aub marked genes represented within UBP5 target genes. Significance tested using Super Exact test. e IGV browser views of representative UBP5 target loci of (i) de-novo marked genes in ubp5 and (ii) H2Aub hyper-marked genes in ubp5. f Heatmaps showing H2Aub distribution on genomic sequences targeted by UBP5, clustered based on higher to lower enrichment (top to bottom). g Average signal of H2Aub levels at non-UBP5 targets in Col-0 and ubp5. The violin cum box plots display median (middle line), first and third quartiles (Q1 and Q3), with whiskers indicating data spread (1.5*IQR from Q1 and Q3), two-sided Wilcoxon rank-sum test was performed. h H2A deubiquitination activity of UBP5. Transient expression of i35S::UBP5-GFP and i35S::GFP empty in N. benthamiana leaves. H2A deubiquitination was assessed by immunoblotting with α-H2Aub antibody; controls: α-H2A and α-H3 antibodies. Bar graph representing the relative H2Aub levels derived from band intensity. Error bars indicate SD between two biological replicates with two-tailed unpaired t-test, p values indicated above plot.