H2BE's post-translational modifications (PTMs) differ from those of canonical H2B. (A,B,D,E) Representative images of H2B-Lys5-Me (A, B, D, and E) and FLAG-H2BE (A, B, and E) staining in the MOE of Flag-H2be (A and B), H2be-KO (D) or H2be-GF (E) mice. (B) High-magnification image of FLAG-H2BE and H2B-Lys5-Me colocalization shows that H2BE is depleted in nuclear regions enriched for H2B-Lys5-Me (arrowheads). Mouse ages: (A), (B), and (E), 10 weeks; (D), 34 weeks. (C) Confirmation of reactivity of the anti-H2B-Lys5-Me1 polyclonal antibody with the Lys5-Me PTM in the context of the H2BE protein sequence. Image (top) and quantification (bottom) of an ELISA assay for peptides corresponding to canonical H2B or H2BE and containing the Lys5-Me PTM. (F) Two-color fluorescent western analysis of Lys5-Me modification of FLAG-H2BE in MOE lysates from WT and Flag-H2be (F-H) mice. No detectable H2B-Lys5-Me staining of the FLAG-H2BE bands (red; arrowheads) is observed. Approximate molecular weights (kDa) are indicated (left). The bands observed at approximately 30–35 kDa likely correspond to histone dimers. (G–I) Age-dependence of H2BE accumulation. (G and H) Images (left) and quantification (right) of FLAG-H2BE and H2B-Lys5-Me co-localization in 3- (G) and 34-week old (H) mice. Red lines, best fits. (I) Quantification of H2B-Lys5-Me levels in high-H2BE neurons (relative to apical sustentacular cells; n = 20 nuclei from two images per timepoint). (J) Two-color fluorescent western analysis (left) and quantification (right) of FLAG-H2BE relative to total H2B as a function of age in MOE lysates from WT and Flag-H2be (F-H) mice. Approximate molecular weights (kDa) are indicated (left). The bands observed at approximately 30–35 kDa likely correspond to histone dimers. **p<0.01; ****p<0.0001; n.s., not significant. Scale bar for (A), (D), (E), (G), and (H), 20 µm; (B), 5 µm.