H2A.Z Demarcates Intergenic Regions of the Plasmodium falciparum Epigenome That Are Dynamically Marked by H3K9ac and H3K4me3

Richárd Bártfai,Arne H Smits,Anita Kaan,Hendrik G Stunnenberg,Kees-Jan Françoijs,Eva Janssen-Megens,Wieteke A M Hoeijmakers,Adriana M Salcedo-Amaya,Moritz Treeck,Tim-Wolf Gilberger

doi:10.1371/journal.ppat.1001223

Abstract

Epigenetic regulatory mechanisms and their enzymes are promising targets for malaria therapeutic intervention; however, the epigenetic component of gene expression in P. falciparum is poorly understood. Dynamic or stable association of epigenetic marks with genomic features provides important clues about their function and helps to understand how histone variants/modifications are used for indexing the Plasmodium epigenome. We describe a novel, linear amplification method for next-generation sequencing (NGS) that allows unbiased analysis of the extremely AT-rich Plasmodium genome. We used this method for high resolution, genome-wide analysis of a histone H2A variant, H2A.Z and two histone H3 marks throughout parasite intraerythrocytic development. Unlike in other organisms, H2A.Z is a constant, ubiquitous feature of euchromatic intergenic regions throughout the intraerythrocytic cycle. The almost perfect colocalisation of H2A.Z with H3K9ac and H3K4me3 suggests that these marks are preferentially deposited on H2A.Z-containing nucleosomes. By performing RNA-seq on 8 time-points, we show that acetylation of H3K9 at promoter regions correlates very well with the transcriptional status whereas H3K4me3 appears to have stage-specific regulation, being low at early stages, peaking at trophozoite stage, but does not closely follow changes in gene expression. Our improved NGS library preparation procedure provides a foundation to exploit the malaria epigenome in detail. Furthermore, our findings place H2A.Z at the cradle of P. falciparum epigenetic regulation by stably defining intergenic regions and providing a platform for dynamic assembly of epigenetic and other transcription related complexes.

Highlights

Plasmodium falciparum, the deadly protozoan parasite, causes malaria via the invasion of erythrocytes
We find that all three epigenetic features are predominantly present in intergenic regions of the P. falciparum genome, suggesting an interconnecting role in regulation of gene expression
H2A.Z levels appear to be largely invariable throughout intraerythrocytic development while placement/removal of the histone marks is dynamic with H3K9ac and H3K4me3 being transcriptioncoupled and stage-specific, respectively

Summary

Introduction

Plasmodium falciparum, the deadly protozoan parasite, causes malaria via the invasion of erythrocytes. During its ,48 h asexual reproductive cycle within human red blood cells (RBC), the parasite exploits and remodels the host cell, multiplies and ruptures the RBC to invade fresh erythrocytes. This process demands the timely expression of distinct sets of proteins [1]. Putative functions of AP2-type DNA binding proteins in a simple cascade of gene activation/repression have been postulated [11] and recently two AP2 proteins were shown to act as essential transcription factors in P. berghei ookinete [12] and sporozoite [13] development. The hypothesis that a cascade of AP2 factors would regulate the waves of gene expression typical to intraerythrocytic development of P. falciparum [11] still needs experimental verification

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