Fig. 4

UBN1 and UBN2 cooperatively deposit H3.3 at cis-regulatory elements in mESC. a Heat map shows that H3.3, HIRA, UBN1, and UBN2 are well co-localized at H3.3 peaks. H3.3 peaks overlapping with UBN1 or UBN2 are sorted descendingly according to the reads density of H3.3. Read density of H3.3, UBN1, UBN2, and HIRA are counted around 5 kb of H3.3 peak center. b UBN1 and UBN2 do not interact with each other. The endogenous interaction between UBN1 and UBN2 is analyzed by Western blot analysis of anti-UBN1 and anti-UBN2 immunoprecipitates. c Western blot shows the proteins levels of HIRA, UBN1, UBN2, and H3.3 in HIRA KO, UBN1 KO, and UBN2 KO cell lines. H3.3 antibody recognizes both 3XFlag-HA knock-in H3.3 and none tagged H3.3. HA antibody recognizes 3XFlag-HA knock-in H3.3. d Heat map shows H3.3 reads density in WT, HIRA KO, UBN1 KO, UBN2 KO, and UBN2 KO-siUBN1 cells. e Venn diagram shows the overlapping among HIRA-H3.3, UBN1-H3.3, and UBN2-H3.3. f Venn diagram shows the overlapping among UBN-H3.3, UBN1-H3.3, and UBN2-H3.3. g, h UBN1 and UBN2 are required for H3.3 deposition at promoters and enhancers. Meta-analysis of dynamic changes of H3.3 deposition at promoters (g) and enhancers (h) after HIRA, UBN1, or UBN2 knockout and double depletion. Reads were normalized to 10 million in each data set. i, j UBN-H3.3 marks more active promoters. Boxplots show the H3K27ac level (I) or gene expression (j) of UBN-H3.3 marked promoters. **: p < 0.01. (k, l) UBN-H3.3 marks more active enhancers. Boxplots show H3K27ac level (K) of UBN-H3.3 marked enhancers and expression level of the regulated genes (l). **: p < 0.01