In eukaryotic cells, chromatin reorganizes within promoters of active genes to allow the transcription machinery and various transcription factors to access DNA. In this model, promoter-specific transcription factors bind DNA to initiate the production of mRNA in a tightly regulated manner. In the case of the human malaria parasite, Plasmodium falciparum, specific transcription factors are apparently underrepresented with regards to the size of the genome, and mechanisms underlying transcriptional regulation are controversial. Here, we investigate the modulation of DNA accessibility by chromatin remodeling during the parasite infection cycle. We have generated genome-wide maps of nucleosome occupancy across the parasite erythrocytic cycle using two complementary assays-the formaldehyde-assisted isolation of regulatory elements to extract protein-free DNA (FAIRE) and the MNase-mediated purification of mononucleosomes to extract histonebound DNA (MAINE), both techniques being coupled to high-throughput sequencing. We show that chromatin architecture undergoes drastic upheavals throughout the parasite's cycle, contrasting with targeted chromatin reorganization usually observed in eukaryotes. Chromatin loosens after the invasion of the red blood cell and then repacks prior to the next cycle. Changes in nucleosome occupancy within promoter regions follow this genome-wide pattern, with a few exceptions such as the var genes involved in virulence and genes expressed at early stages of the cycle. We postulate that chromatin structure and nucleosome turnover control massive transcription during the erythrocytic cycle. Our results demonstrate that the processes driving gene expression in Plasmodium challenge the classical eukaryotic model of transcriptional regulation occurring mostly at the transcription initiation level.

SUMMARY Cytosine DNA methylation is an epigenetic mark in most eukaryotic cells that regulates numerous processes, including gene expression and stress responses. We performed a genome-wide analysis of DNA methylation in the human malaria parasite Plasmodium falciparum. We mapped the positions of methylated cytosines and identified a single functional DNA methyltransferase, PfDNMT, that may mediate these genomic modifications. These analyses revealed that the malaria genome is asymmetrically methylated, in which only one DNA strand is methylated, and shares common features with undifferentiated plant and mammalian cells. Notably, core promoters are hypomethylated and transcript levels correlate with intra-exonic methylation. Additionally, there are sharp methylation transitions at nucleosome and exon-intron boundaries. These data suggest that DNA methylation could regulate virulence gene expression and transcription elongation. Furthermore, the broad range of action of DNA methylation and uniqueness of PfDNMT suggest that the methylation pathway is a potential target for anti-malarial strategies.

The partial purification of mouse mammary gland stem cells (MaSCs) using combinatorial cell surface markers (Lin − CD24 + CD29 h CD49f h ) has improved our understanding of their role in normal development and breast tumorigenesis. Despite the significant improvement in MaSC enrichment, there is presently no methodology that adequately isolates pure MaSCs. Seeking new markers of MaSCs, we characterized the stem-like properties and expression signature of label-retaining cells from the mammary gland of mice expressing a controllable H2b-GFP transgene. In this system, the transgene expression can be repressed in a doxycycline-dependent fashion, allowing isolation of slowly dividing cells with retained nuclear GFP signal. Here, we show that H2b-GFP h cells reside within the predicted MaSC compartment and display greater mammary reconstitution unit frequency compared with H2b-GFP neg MaSCs. According to their transcriptome profile, H2b-GFP h MaSCs are enriched for pathways thought to play important roles in adult stem cells. We found Cd1d, a glycoprotein expressed on the surface of antigen-presenting cells, to be highly expressed by H2b-GFP h MaSCs, and isolation of Cd1d + MaSCs further improved the mammary reconstitution unit enrichment frequency to nearly a single-cell level. Additionally, we functionally characterized a set of MaSC-enriched genes, discovering factors controlling MaSC survival. Collectively, our data provide tools for isolating a more precisely defined population of MaSCs and point to potentially critical factors for MaSC maintenance.

The software is available in the public domain at http://compbio.cs.ucr.edu/brat/.

Almost a decade after the publication of the complete sequence of the genome of the human malaria parasite Plasmodium falciparum, the mechanisms involved in gene regulation remain poorly understood. Like other eukaryotic organisms, P. falciparum’s genomic DNA organizes into nucleosomes. Nucleosomes are the basic structural units of eukaryotic chromatin and their regulation is known to play a key role in regulation of gene expression. Despite its importance, the relationship between nucleosome positioning and gene regulation in the malaria parasite has only been investigated recently. Using two independent and complementary techniques followed by next-generation high-throughput sequencing, our laboratory recently generated a dynamic atlas of nucleosome-bound and nucleosome-free regions (NFRs) at single-nucleotide resolution throughout the parasite erythrocytic cycle. We have found evidences that genome-wide changes in nucleosome occupancy play a critical role in controlling the rigorous parasite replication in infected red blood cells. However, the role of nucleosome positioning at remarkable locations such as transcriptional start sites (TSS) was not investigated. Here we show that a study of NFR in experimentally determined TSS and in silico-predicted promoters can provide deeper insights of how a transcriptionally permissive organization of chromatin can control the parasite’s progression through its life cycle. We find that NFRs found at TSS and core promoters are strongly associated with high levels of gene expression in asexual erythrocytic stages, whereas nucleosome-bound TSSs and promoters are associated with silent genes preferentially expressed in sexual stages. The implications in terms of regulatory evolution, adaptation of gene expression and their impact in the design of antimalarial strategies are discussed.

We present a new, accurate and efficient tool for mapping short reads obtained from the Illumina Genome Analyzer following sodium bisulfite conversion. Our tool, BRAT, supports single and paired-end reads and handles input files containing reads and mates of different lengths. BRAT is faster, maps more unique paired-end reads and has higher accuracy than existing programs. The software package includes tools to end-trim low-quality bases of the reads and to report nucleotide counts for mapped reads on the reference genome.

Forward genetic screens in zebrafish have been utilized to identify genes essential for the generation of primitive blood and the emergence of hematopoietic stem cells (HSCs), but have not elucidated genes essential for hematopoietic stem and progenitor cell (HSPC) proliferation and differentiation due to a lack of methodologies to functionally assess these processes. We previously described techniques to test the developmental potential of HSPCs by culturing them on zebrafish kidney stromal (ZKS) cells, derived from the main site of hematopoiesis in the adult teleost. Here we describe an additional primary stromal cell line we refer to as zebrafish embryonic stromal trunk (ZEST) cells, derived from tissue surrounding the embryonic dorsal aorta, the site of HSC emergence in developing fish. ZEST cells encouraged HSPC differentiation towards the myeloid, lymphoid, and erythroid pathways when assessed by morphological and qRT-PCR analyses. Additionally, ZEST cells significantly expanded the number of cultured HSPCs in vitro, indicating that these stromal cells are supportive of both HSPC proliferation and multilineage differentiation. Examination of ZEST cells indicates that they express numerous cytokines and Notch ligands, and possess endothelial characteristics. Further characterization of ZEST cells should prove to be invaluable in understanding the complex signaling cascades instigated by the embryonic hematopoietic niche required to expand and differentiate HSPCs. Elucidating these processes and identifying possibilities for the modulation of these molecular pathways should allow the in vitro expansion of HSPCs for a multitude of therapeutic uses.

Hematopoiesis is an essential and highly regulated biological process that begins with hematopoietic stem cells (HSCs). In healthy organisms, HSCs are responsible for generating a multitude of mature blood cells every day, yet the molecular pathways that instruct HSCs to self-renew and differentiate into post-mitotic blood cells are not fully known. To understand these molecular pathways, we investigated novel genes expressed in hematopoietic-supportive cell lines from the zebrafish (Danio rerio), a model system increasingly utilized to uncover molecular pathways important in the development of other vertebrate species. We performed RNA sequencing of the transcriptome of three stromal cell lines derived from different stages of embryonic and adult zebrafish and identified hundreds of highly expressed transcripts. For our studies, we focused on isthmin 1 (ism1) due to its shared synteny with its human gene ortholog and because it is a secreted protein. To characterize ism1, we performed loss-of-function experiments to identify if mature blood cell production was disrupted. Myeloid and erythroid lineages were visualized and scored with transgenic zebrafish expressing lineage-specific markers. ism1 knockdown led to reduced numbers of neutrophils, macrophages, and erythrocytes. Analysis of clonal methylcellulose assays from ism1 morphants also showed a reduction in total hematopoietic stem and progenitor cells (HSPCs). Overall, we demonstrate that ism1 is required for normal generation of HSPCs and their downstream progeny during zebrafish hematopoiesis. Further investigation into ism1 and its importance in hematopoiesis may elucidate evolutionarily conserved processes in blood formation that can be further investigated for potential clinical utility.