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Abstract Background Epigenetic modifications of histones and regulation of chromatin structure have been implicated in regulation of virulence gene families in P. falciparum. To better understand chromatin-mediated gene regulation, we used a high-density oligonucleotide microarray to map the position and enrichment of nucleosomes across the entire genome of P. falciparum at three time points of the intra-erythrocytic developmental cycle (IDC) in vitro. We used an unmodified histone H4 antibody for chromatin immunoprecipitation of nucleosome-bound DNA. Results We observed generally low nucleosomal occupancy of intergenic regions and higher occupancy of protein coding regions. In contract to the overall small fluctuation of nucleosomal occupancy in most coding regions throughout the IDC, subtelomeric genes encoding surface proteins such as var and rif, as well as some core chromosomal genes such as transcription factors, showed large changes in chromatin structure. Telomeres harbored a region with the highest nucleosomal occupancy of the genome and also exhibited large changes with higher nucleosomal occupancy at schizont stages. While many of these subtelomeric genes were previously shown to be modified by H3K9 trimethylation, we also identified some housekeeping genes in core chromosome regions that showed extensive changes in chromatin structure but do not contain this modification. tRNA and basal transcription factor genes showed low nucleosomal occupancy at all times, suggesting of an open chromatin structure that might be permissive for constitutively high levels of expression. Generally, nucleosomal occupancy was not correlated with the steady-state mRNA levels. Several var genes were exceptions: the var gene with the highest expression level showed the lowest nucleosomal occupancy, and selection of parasites for var2CSA expression resulted in lower nucleosomal occupancy at the var2CSA locus. We identified nucleosome-free regions in intergenic regions that may serve as transcription start sites or transcription factor binding sites. Using the nucleosomal occupancy data as the baseline, we further mapped the genome-wide enrichment of H3K9 acetylation and detected general enrichment of this mark in intergenic regions. Conclusions These data on nucleosome enrichment changes add to our understanding of the influence of chromatin structure on the regulation of gene expression. Histones are generally enriched in coding regions, and relatively poor in intergenic regions. Histone enrichment patterns allow for identification of new putative gene-coding regions. Most genes do not show correlation between chromatin structure and steady-state mRNA levels, indicating the dominant roles of other regulatory mechanisms. We present a genome-wide nucleosomal occupancy map, which can be used as a reference for future experiments of histone modification mapping.

Abstract Background The structurally complex network of minicircles and maxicircles comprising the mitochondrial DNA of kinetoplastids mirrors the complexity of the RNA editing process that is required for faithful expression of encrypted maxicircle genes. Although a few of the guide RNAs that direct this editing process have been discovered on maxicircles, guide RNAs are mostly found on the minicircles. The nuclear and maxicircle genomes have been sequenced and assembled for Trypanosoma cruzi, the causative agent of Chagas disease, however the complement of 1.4-kb minicircles, carrying four guide RNA genes per molecule in this parasite, has been less thoroughly characterised. Results Fifty-four CL Brener and 53 Esmeraldo strain minicircle sequence reads were extracted from T. cruzi whole genome shotgun sequencing data. With these sequences and all published T. cruzi minicircle sequences, 108 unique guide RNAs from all known T. cruzi minicircle sequences and two guide RNAs from the CL Brener maxicircle were predicted using a local alignment algorithm and mapped onto predicted or experimentally determined sequences of edited maxicircle open reading frames. For half of the sequences no statistically significant guide RNA could be assigned. Likely positions of these unidentified gRNAs in T. cruzi minicircle sequences are estimated using a simple Hidden Markov Model. With the local alignment predictions as a standard, the HMM had an ~85% chance of correctly identifying at least 20 nucleotides of guide RNA from a given minicircle sequence. Inter-minicircle recombination was documented. Variable regions contain species-specific areas of distinct nucleotide preference. Two maxicircle guide RNA genes were found. Conclusion The identification of new minicircle sequences and the further characterization of all published minicircles are presented, including the first observation of recombination between minicircles. Extrapolation suggests a level of 4% recombinants in the population, supporting a relatively high recombination rate that may serve to minimize the persistence of gRNA pseudogenes. Characteristic nucleotide preferences observed within variable regions provide potential clues regarding the transcription and maturation of T. cruzi guide RNAs. Based on these preferences, a method of predicting T. cruzi guide RNAs using only primary minicircle sequence data was created.

Abstract Background The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. Results We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. Conclusion The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network.