Abstract
Despite histone H2A variants and acetylation of histones occurring in almost every eukaryotic organism, it has been difficult to establish direct functional links between canonical histones or H2A variant acetylation, deposition of H2A variants and transcription. To disentangle these complex interdependent processes, we devised a highly sensitive strategy for quantifying histone acetylation levels at specific genomic loci. Taking advantage of the unusual genome organization in Trypanosoma brucei, we identified 58 histone modifications enriched at transcription start sites (TSSs). Furthermore, we found TSS-associated H4 and H2A.Z acetylation to be mediated by two different histone acetyltransferases, HAT2 and HAT1, respectively. Whereas depletion of HAT2 decreases H2A.Z deposition and shifts the site of transcription initiation, depletion of HAT1 does not affect H2A.Z deposition but reduces total mRNA levels by 50%. Thus, specifically reducing H4 or H2A.Z acetylation levels enabled us to reveal distinct roles for these modifications in H2A.Z deposition and RNA transcription.
Highlights
Despite histone H2A variants and acetylation of histones occurring in almost every eukaryotic organism, it has been difficult to establish direct functional links between canonical histones or H2A variant acetylation, deposition of H2A variants and transcription
To identify all posttranslational modifications (PTMs) enriched at TSSs in an unbiased manner, we sought to isolate nucleosomes from transcription start sites (TSS-nucleosomes) and nucleosomes not located at start sites (Supplementary Fig. 1a) and to compare their PTM patterns
Histone acetylation and variant forms of H2A seem to be present in almost every organism that contains histones and appear to have a highly conserved role in regulating RNA Pol II transcription
Summary
Despite histone H2A variants and acetylation of histones occurring in almost every eukaryotic organism, it has been difficult to establish direct functional links between canonical histones or H2A variant acetylation, deposition of H2A variants and transcription. To disentangle these complex interdependent processes, we devised a highly sensitive strategy for quantifying histone acetylation levels at specific genomic loci. Among the best-characterized histone PTMs are acetylation, phosphorylation, and methylation These PTMs can alter the conformation of nucleosomes or function as specific binding sites for enzymatic complexes, which subsequently alter the chromatin structure
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