Site‐specific human histone H3 methylation stability: fast K4me3 turnover

Abstract

We employ stable isotope labelling and quantitative mass spectrometry to track histone methylation stability. We show that H3 trimethyl K9 and K27 are slow to be established on new histones and slow to disappear from old histones, with half-lives of multiple cell divisions. By contrast the transcription-associated marks K4me3 and K36me3 turn over far more rapidly, with half-lives of 6.8 h and 57 h, respectively. Inhibition of demethylases increases K9 and K36 methylation, with K9 showing the largest and most robust increase. We interpret different turnover rates in light of genome-wide localization data and transcription-dependent nucleosome rearrangements proximal to the transcription start site.

Unabridged Analysis of Human Histone H3 by Differential Top-Down Mass Spectrometry Reveals Hypermethylated Proteoforms from MMSET/NSD2 Overexpression

“…The precipitate was washed first with 0.1% HCl in acetone and twice with pure acetone. Crude histones were then resuspended in water and subjected to fractionation using reverse phase high pressure liquid chromatography (RP-HPLC) as described previously (51). Briefly, histones were separated using a Jupiter C18 analytical column (Phenomenex, Torrance, CA), 15 cm ϫ 4.6 mm, 5 m diam., 300 Å pores, using a gradient of 30 -57% B in 90 min (Buffer A: 5% ACN, 0.1% TFA; Buffer B: 90% ACN, 0.094% TFA) at a flow rate of 0.8 ml/min using an Agilent 1100 HPLC system (Agilent, Santa Clara, CA) monitored by UV absorbance at 214 nm.…”

An Essential Viral Transcription Activator Modulates Chromatin Dynamics

“…Acetylation of histone tails by histone acetyltransferases generally destabilizes nucleosomes, whereas their methylation by histone methyltransferases destabilizes or stabilizes nucleosomes, depending on the site and the degree of methylation [21–29]. Nucleosomes containing H3.3 are more dynamic than those containing H3.1 [30].…”

Transient pausing by RNA polymerase II

“…At a gene with 1% occupancy, each copy of that gene across the population of cells would be visited 20 times a day, or about once each hour. In contrast, if Pol II pauses for 20 min, most copies of that low-occupancy gene would be visited less than once per day, and this is not often enough to maintain the H3K4me3 mark, which has a half-life of 6.8 h (17).…”