The human malaria parasite genome is configured into thousands of coexpressed linear regulatory units

Abstract

The human malaria parasite Plasmodium falciparum thrives in radically different host environments in mosquitoes and humans, with only a limited set of transcription factors. The nature of regulatory elements or their target genes in the P. falciparum genome remains elusive. Here, we found that this eukaryotic parasite uses an efficient way to maximally use genetic and epigenetic regulation to form regulatory units (RUs) during blood infections. Genes located in the same RU tend to have the same pattern of expression over time and are associated with open chromatin along regulatory elements. To precisely define and quantify these RUs, a novel hidden Markov model was developed to capture the regulatory structure in a genome-wide fashion by integrating expression and epigenetic evidence. We successfully identified thousands of RUs and cross-validated with previous findings. We found more genes involved in red blood cell (RBC) invasion located in the same RU as the PfAP2-I (AP2-I) transcription factor, demonstrating that AP2-I is responsible for regulating RBC invasion. Our study has provided a regulatory mechanism for a compact eukaryotic genome and offers new insights into the in vivo transcriptional regulation of the P. falciparum intraerythrocytic stage.

Introduction

Malaria. P. falciparum remains one of the most devastating parasitic human diseases, killing almost a half million people annually (World Malaria Report, 2016). After infection, disease symptoms occur during intraerythrocyte development (IED). A small number of sexual gametocytes are generated during IED. The gametocytes are ingested by the mosquito vector, which is necessary for malaria disease transmission. With the development of next-generation sequencing, a number of studies (Otto et al., 2010; Bulger and Groudine, 2011; López-Barragán et al., 2011) have demonstrated that the transcription of P. falciparum occurs in tightly regulated cascades, during IED and during sexual growth in the mosquito host (Ay et al., 2015). Exploring regulation mechanisms of gene transcription is crucial for understanding the biological processes of Plasmodium because it could provide potential drug targets for malaria treatment.

Over the past few years, accumulating evidence has shown that the transcriptional regulation mechanisms of P. falciparum are largely dependent on a combination of epigenetics and transcription factors (TFs) (Cui and Miao, 2010; Hoeijmakers et al., 2012; Ay et al., 2015). Nearly 50 different histone post-translational modifications have been identified in P. falciparum (Miao et al., 2006; Trelle et al., 2009; Treeck et al., 2011). Compared with other multicellular eukaryotes, a large proportion of P. falciparum genome is observed to be constitutively acetylated (Miao et al., 2006; Lopez-Rubio et al., 2009). Inhibition of histone acetyltransferase and deacetylase activity affects gene expression regulation interfering with parasite growth (Chaal et al., 2010). Compared with the broadly distributed euchromatin marks, the heterochromatin modifications are mostly in repressive clusters containing virulence genes, such as var, pfmc-2tm, rifin, and stevor, (Lopez-Rubio et al. 2007; Jiang et al., 2013). For example, H3K9me3 and heterochromatin protein 1 are only found in subtelomeres and a few genomic regions including the majority of var genes (Lopez-Rubio et al. 2007, 2009; Salcedo-Amaya et al., 2009). H3K36me3 is also mainly present along the entire var gene body, playing a critical role in var gene repression (Jiang et al., 2013).

Broadly distributed activating epigenetic marks along the P. falciparum intergenic regions provide sufficient areas for TF binding. For example, a poised or open chromatin region with the H2A.Z histone marker at the gene 5′ and 3′ ends facilitates the access of TFs (Bártfai et al., 2010). In Plasmodium, TFs have been reported to directly regulate parasite development (Kafsack et al., 2014; Sinha et al., 2014). A notable example is PfAP2-G, a member of the ApiAP2 TF family. The expression levels of PfAP2-G correlate strongly with levels of gametocyte formation in both P. falciparum (Kafsack et al., 2014) and Pberghei (Sinha et al., 2014). Despite the significant importance of TFs in Plasmodium, transcriptional regulation via TFs has not been studied much. Compared with comprehensive histone mark annotation by genome-wide Chromatin Immunoprecipitation Sequencing (ChIP-Seq) experiments (Bártfai et al., 2010; Gupta et al., 2013; Karmodiya et al., 2015), genome-wide TF binding annotation is still limited in Plasmodium genomes. To our knowledge, only AP2-I (Santos et al., 2017) and AP2-O (Kaneko et al., 2015) (an ApiAP2 TF family) binding sites have been detected. Insufficient TF binding annotations have impeded the study of gene regulation networks in Plasmodium. Current understanding of cis-regulatory DNA elements on the whole-genome scale is still mainly based on assigning a gene to its nearest TF-binding motif (Campbell et al., 2010; Kafsack et al., 2014; Wang et al., 2016).

An open or relatively ‘accessible’ chromatin environment is associated with the binding of specific trans-factors. Formaldehyde-assisted isolation of regulatory elements has been implemented to screen the chromatin accessibility in P. falciparum (Ponts et al., 2010). However, these data were not able to provide sufficient resolution to detect the regulatory elements. The assay for transposase-accessible chromatin using sequencing (ATAC-Seq) provides a high signal-to-noise ratio and has been wildly used because of its fast and straightforward experimental protocol (Buenrostro et al., 2013).

The combination of RNA-Seq and ATAC-Seq on eight tightly synchronized stages during P. falciparum IED (Ruiz et al., 2018; Toenhake et al., 2018) provides an ideal data set to profile gene regulatory events. Considering little evidence for enhancers exists for the P. falciparum genome, assigning this regulatory element to the nearest downstream gene ought to be sufficient. If head-to-head genes are located on either side of a regulatory element, the elements may regulate both genes together. Furthermore, multiple adjacent regulatory elements have similar activity during the IED and could result in similar expression patterns for multiple genes.

In this study, we observed that coregulated genes and regulatory elements in P. falciparum tend to form units of variable sizes throughout the genome. Therefore, to obtain a deeper understanding of transcription regulation organization, a novel algorithm was developed to detect these regulation units, defined as regulatory units (RUs). This study provides the first targeted regulation gene identification method for cis-regulatory elements in the P. falciparum genome. This provides an important step toward exploring the transcription regulation mechanisms of this devastating parasite.