Histone synthesis and turnover in alfalfa. Fast loss of highly acetylated replacement histone variant H3.2

Abstract

Histone synthesis in alfalfa tissue culture cells was studied by labeling with tritiated lysine, purification of histone proteins by reversed-phase high pressure liquid chromatography, and fluorography of acid/urea/Triton X-100 polyacrylamide gels. Minor histone variant H3.2 was synthesized twice as fast as major variant H3.1. The predicted difference in histone H3 variant turnover was examined during continued growth. More than 50% of newly synthesized histone H3.2 and 20% of new H3.1 were lost from chromatin over a period of 100 h. This produced a ratio between the stable remaining portions of each new histone H3 variant protein identical to that of the steady-state histone H3 variants. The labile portion of new histone H3.2 (half-life of 20 h) was rapidly lost specifically from transcriptionally active chromatin as judged by the acetylation level of nearly 1.5 acetylated lysines/histone molecule, a level 50% higher than the acetylation in histone H3.2 overall and three times that of histone H3.1. These results and the constitutive level of H3.2 gene expression identify histone H3.2 of alfalfa as a functional replacement histone variant. The extent of its preferential assembly into active chromatin nucleosomes and the rapid rate of its subsequent loss indicate significant dissolution of plant nucleosomes during gene transcription.