Characterization of the accessible genome in the human malaria parasite Plasmodium falciparum

Human malaria is a devastating disease and a major cause of poverty in resource-limited countries. To develop and adapt within hosts Plasmodium falciparum undergoes drastic switches in gene expression. To identify regulatory regions in the parasite genome, we performed genome-wide profiling of chromatin accessibility in two culture-adapted isogenic subclones at four developmental stages during the intraerythrocytic cycle by using the Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq). Tn5 transposase hypersensitivity sites (THSSs) localize preferentially at transcriptional start sites (TSSs). Chromatin accessibility by ATAC-seq is predictive of active transcription and of the levels of histone marks H3K9ac and H3K4me3. Our assay allows the identification of novel regulatory regions including TSS and enhancer-like elements. We show that the dynamics in the accessible chromatin profile matches temporal transcription during development. Motif analysis of stage-specific ATAC-seq sites predicts the in vivo binding sites and function of multiple ApiAP2 transcription factors. At last, the alternative expression states of some clonally variant genes (CVGs), including eba, phist, var and clag genes, associate with a differential ATAC-seq signal at their promoters. Altogether, this study identifies genome-wide regulatory regions likely to play an essential function in the developmental transitions and in CVG expression in P. falciparum

Genome-wide epigenetic cross-talk between DNA methylation and H3K27me3 in zebrafish embryos

AbstractDNA methylation and histone modifications are epigenetic marks implicated in the complex regulation of vertebrate embryogenesis. The cross-talk between DNA methylation and Polycomb-dependent H3K27me3 histone mark has been reported in a number of organisms [1-7] and both marks are known to be required for proper developmental progression. Here we provide genome-wide DNA methylation (MethylCap-seq) and H3K27me3 (ChIP-seq) maps for three stages (dome, 24hpf and 48hpf) of zebrafish (Danio rerio) embryogenesis, as well as all analytical and methodological details associated with the generation of this dataset. We observe a strong antagonism between the two epigenetic marks present in CpG islands and their compatibility throughout the bulk of the genome, as previously reported in mammalian ESC lines (Brinkman et al., 2012). Next generation sequencing data linked to this project have been deposited in the Gene Expression Omnibus (GEO) database under accession numbers GSE35050 and GSE70847

A common copy-number variant within SIRPB1 correlates with human Out-of-Africa migration after genetic drift correction

Previous reports have proposed that personality may have played a role on human Out-Of- Africa migration, pinpointing some genetic variants that were positively selected in the migrating populations. In this work, we discuss the role of a common copy-number variant within the SIRPB1 gene, recently associated with impulsive behavior, in the human Out-Of-Africa migration. With the analysis of the variant distribution across forty-two different populations, we found that the SIRPB1 haplotype containing duplicated allele significantly correlated with human migratory distance, being one of the few examples of positively selected loci found across the human world colonization. Circular Chromosome Conformation Capture (4C-seq) experiments from the SIRPB1 promoter revealed important 3D modifications in the locus depending on the presence or absence of the duplication variant. In addition, a 3' enhancer showed neural activity in transgenic models, suggesting that the presence of the CNV may compromise the expression of SIRPB1 in the central nervous system, paving the way to construct a molecular explanation of the SIRPB1 variants role in human migration

Ancient Genomic Regulatory Blocks Are a Source for Regulatory Gene Deserts in Vertebrates after Whole-Genome Duplications

We investigated how the two rounds of whole genome duplication that occurred at the base of the vertebrate lineage have impacted ancient microsyntenic associations involving developmental regulators (known as genomic regulatory blocks, GRBs). We showed that the majority of GRBs identified in the last common ancestor of chordates have been maintained as a single copy in humans. We found evidence that dismantling of the duplicated GRB copies occurred early in vertebrate evolution often through the differential retention of the regulatory gene but loss of the bystander gene's exonic sequences. Despite the large evolutionary scale, the presence of duplicated highly conserved non-coding regions provided unambiguous proof for this scenario for multiple ancient GRBs. Remarkably, the dismantling of ancient GRB duplicates has contributed to the creation of large gene deserts associated with regulatory genes in vertebrates, providing a potentially widespread mechanism for the origin of these enigmatic genomic traits

Early evolution of the T-box transcription factor family

Deèelopmental transcription factors are key players in animal multicellularity, being members of the T-box family that are among the most important. Until recently, T-box transcription factors were thought to be exclusièely present in metazoans. Here, we report the presence of T-box genes in seèeral nonmetazoan lineages, including ichthyosporeans, filastereans, and fungi. Our data confirm that Brachyury is the most ancient member of the T-box family and establish that the T-box family dièersified at the onset of Metazoa. Moreoèer, we demonstrate functional conserèation of a homolog of Brachyury of the protist Capsaspora owczarzaki in Xenopus laeèis. By comparing the molecular phenotype of C. owczarzaki Brachyury with that of homologs of early branching metazoans, we define a clear difference between unicellular holozoan and metazoan Brachyury homologs, suggesting that the specificity of Brachyury emerged at the origin of Metazoa. Experimental determination of the binding preferences of the C. owczarzaki Brachyury results in a similar motif to that of metazoan Brachyury and other T-box classes. This finding suggests that functional specificity between different T-box classes is likely achieèed by interaction with alternatièe cofactors, as opposed to differences in binding specificity

Active DNA demethylation of developmental cis-regulatory regions predates vertebrate origins

DNA methylation [5-methylcytosine (5mC)] is a repressive gene-regulatory mark required for vertebrate embryogenesis. Genomic 5mC is tightly regulated through the action of DNA methyltransferases, which deposit 5mC, and ten-eleven translocation (TET) enzymes, which participate in its active removal through the formation of 5-hydroxymethylcytosine (5hmC). TET enzymes are essential for mammalian gastrulation and activation of vertebrate developmental enhancers; however, to date, a clear picture of 5hmC function, abundance, and genomic distribution in nonvertebrate lineages is lacking. By using base-resolution 5mC and 5hmC quantification during sea urchin and lancelet embryogenesis, we shed light on the roles of nonvertebrate 5hmC and TET enzymes. We find that these invertebrate deuterostomes use TET enzymes for targeted demethylation of regulatory regions associated with developmental genes and show that the complement of identified 5hmC-regulated genes is conserved to vertebrates. This work demonstrates that active 5mC removal from regulatory regions is a common feature of deuterostome embryogenesis suggestive of an unexpected deep conservation of a major gene-regulatory module

A role for insulator elements in the regulation of gene expression response to hypoxia

Hypoxia inducible factor (HIF) up-regulates the transcription of a few hundred genes required for the adaptation to hypoxia. This restricted set of targets is in sharp contrast with the widespread distribution of the HIF binding motif throughout the genome. Here, we investigated the transcriptional response of GYS1 and RUVBL2 genes to hypoxia to understand the mechanisms that restrict HIF activity toward specific genes. GYS1 and RUVBL2 genes are encoded by opposite DNA strands and separated by a short intergenic region (~1 kb) that contains a functional hypoxia response element equidistant to both genes. However, hypoxia induced the expression of GYS1 gene only. Analysis of the transcriptional response of chimeric constructs derived from the intergenic region revealed an inhibitory sequence whose deletion allowed RUVBL2 induction by HIF. Enhancer blocking assays, performed in cell culture and transgenic zebrafish, confirmed the existence of an insulator element within this inhibitory region that could explain the differential regulation of GYS1 and RUVBL2 by hypoxia. Hence, in this model, the selective response to HIF is achieved with the aid of insulator elements. This is the first report suggesting a role for insulators in the regulation of differential gene expression in response to environmental signals

Amphioxus functional genomics and the origins of vertebrate gene regulation.

Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus (Branchiostoma lanceolatum) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis-regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that-in vertebrates-over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations

Characterization of the accessible genome in the human malaria parasite Plasmodium falciparum

Human malaria is a devastating disease and a major cause of poverty in resource-limited countries. To develop and adapt within hosts Plasmodium falciparum undergoes drastic switches in gene expression. To identify regulatory regions in the parasite genome, we performed genome-wide profiling of chromatin accessibility in two culture-adapted isogenic subclones at four developmental stages during the intraerythrocytic cycle by using the Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq). Tn5 transposase hypersensitivity sites (THSSs) localize preferentially at transcriptional start sites (TSSs). Chromatin accessibility by ATAC-seq is predictive of active transcription and of the levels of histone marks H3K9ac and H3K4me3. Our assay allows the identification of novel regulatory regions including TSS and enhancer-like elements. We show that the dynamics in the accessible chromatin profile matches temporal transcription during development. Motif analysis of stage-specific ATAC-seq sites predicts the in vivo binding sites and function of multiple ApiAP2 transcription factors. At last, the alternative expression states of some clonally variant genes (CVGs), including eba, phist, var and clag genes, associate with a differential ATAC-seq signal at their promoters. Altogether, this study identifies genome-wide regulatory regions likely to play an essential function in the developmental transitions and in CVG expression in P. falciparum

Comparative genomics of Hedgehog loci in chordates and the origin of Shh regulatory novelties

The origin and evolution of the complex regulatory landscapes of some vertebrate developmental genes, often spanning hundreds of Kbp and including neighboring genes, remain poorly understood. The Sonic Hedgehog (Shh) genomic regulatory block (GRB) is one of the best functionally characterized examples, with several discrete enhancers reported within its introns, vast upstream gene-free region and neighboring genes (Lmbr1 and Rnf32). To investigate the origin and evolution of this GRB, we sequenced and characterized the Hedgehog (Hh) loci from three invertebrate chordate amphioxus species, which share several early expression domains with Shh. Using phylogenetic footprinting within and between chordate lineages, and reporter assays in zebrafish probing >30 Kbp of amphioxus Hh, we report large sequence and functional divergence between both groups. In addition, we show that the linkage of Shh to Lmbr1 and Rnf32, necessary for the unique gnatostomate-specific Shh limb expression, is a vertebrate novelty occurred between the two whole-genome duplications