Figure 1: H4K91 mutants enhance association with the histone variant H3.3 and form distinct nuclear puncta.
(A) Both H4K91 mutants display a puncta pattern. Immunofluorescence images of mESCs that express HA-tagged H4WT or H4K91 mutants (K91R or K91Q) with HA antibody. Images are Z-stack projections. Scale bar: 2μm. (B) Quantification of in-puncta fluorescence intensity in the nucleus. Fluorescence intensity within the puncta is divided by the whole nucleus intensity and displayed as a box plot. The box plot shows all data points with the median, and the boundaries indicate the 25th and the 75th percentiles. Each dot represents one cell. Statistical significance was calculated using an unpaired t-test. **** p<0.0001. (C) Schematic of mononucleosome IP method. Chromatin from 293T cells expressing WT or H4K91 mutants are first subject to MNase digestion. Then, mononucleosomes containing HA-tagged H4WT or H4K91 mutants are pulled down with HA antibody and analyzed by mass spectrometry (MS) analysis or western blot (WB). (D) H4K91 mutants display increased binding with the histone variant H3.3. A plot of the H3.3/H3 ratio based on MS analysis of the pulled-down nucleosomes. Three independent replicates were done. Error bars represent mean ± s.e.m, and statistical significance was calculated using an unpaired t-test. * p<0.05, ** p<0.005. (E) WB of the pulled-down mononucleosomes with the following antibodies: HA, H3.3 specific antibody, and H3 (which recognizes both canonical histone H3 and variant H3.3). (F) H4K91 mutant cells staining with the H3.3-specific chaperones DAXX and ATRX. Immunofluorescence images of mESCs expressing a HA-tagged H4K91 mutant. The following antibodies were used: HA, DAXX, and ATRX. Arrowheads indicate colocalization. A single Z stack image is shown. Scale bar: 2μm. (G) Immunofluorescence images of H3.3−/−, DAXX −/− and Hira−/− mESCs that express HA-tagged H4K91Q. Scale bar: 2μm. (H) Quantification of in-puncta HA fluorescence intensity in different mESCs. Each dot represents one cell. Statistical significance was calculated using an unpaired t-test. * p<0.05. **** p<0.0001. See also Figure S1.