Histone H3 trimethylated at lysine 4 is enriched at probable transcription start sites in Trypanosoma brucei

Abstract

Recent studies have identified histone modifications and suggested a role for epigenetic gene regulation in Trypanosoma brucei. The histone modification H4K10ac and histone variants H2AZ and H2BV localize to probable sites of transcription initiation. Although all T. brucei histones have very evolutionarily divergent N-terminal tails, histone H3 shows conservation with other eukaryotic organisms in 6 of 8 amino acids encompassing lysine 4. Tri-methylation of H3K4 is generally associated with transcription. We therefore generated a specific antibody to T. brucei H3K4me3 and performed chromosome immunoprecipitation and high-throughput sequencing. We show that H3K4me3 is enriched at the start of polycistronic transcription units at divergent strand-switch regions and at other sites of RNA polymerase II transcription reinitiation. H3K4me3 largely co-localizes with H4K10ac, but with a skew towards the upstream side of the H4K10ac peak, suggesting that it is a component of specific nucleosomes that play a role in Pol II transcription initiation.

Fig. 1

Genome-wide distribution of H3K4me3. Nucleotide hits from 36-bp sequences were averaged over a 100-bp window. Inset (A) The N-terminal sequence of T. brucei H3 is evolutionarily conserved. Divergent residues are shown in red and the conserved K4 lysine is highlighted. (B) Specificity of H3K4me3 ChIP-grade antibody. The total extract from 2×10⁶ Dot1B null cells was electrophoresed and blotted with purified H3K4me3 antibody that was preincubated for 1 h with 1 µg/µl of the indicated peptides. Peptide sequences are as follows: H3K4me3 RTKETARTC; H3K73me3 VSGAQKEGLRFC; H3K76me2 VSGAQKEGLRFC (provided by Christian Janzen); H3K32me3 ASGVKTAQRC.

Fig. 2

H3K4me3 overlaps with H4K10ac peaks of enrichment. H4K10ac and H3K4me3 hits per 100 base pair windows were normalized to the total number of hits per chromosome. Orange boxes represent ORFs and arrows indicate the direction of transcription. H3K4me3 co-enriches with H4K10 at all peaks, including SSR (bottom left) and tRNA gene regions (bottom right). H3K4me3 shows a narrower range of enrichment, with H4K10ac broadening in the direction of transcription (bottom panels)