The association of nucleosomes with coding regions and their relative absence from intergenic regions is distinctly different from the findings in model eukaryotes, where nucleosomes are usually absent only at promoters and intergenic regions are condensed. In P. falciparum
genome, this may be the result of the extreme AT-richness in the intergenic regions. Since poly-A tracts induce bending of the DNA helix [39
] and this bending tends to exclude nucleosomes from these regions [40
], the high AT content (95%) of intergenic regions of P. falciparum
may selectively exclude histones from these regions. While model organisms generally have small nucleosome-free regions observed at TSSs [11
], our analysis using the P. falciparum
full-length cDNA data was inconclusive. Instead, a more extensively nucleosome-free region may be present in the intergenic regions of the parasite genome allowing easy access to RNA polymerase machinery and transcription factors. This may be partially related to the diverse transcription initiation sites found in many P. falciparum
]. Yet, in spite of the lower nucleosomal occupancy at the intergenic regions, there were spikes of modified histones in the intergenic regions, which may play additional roles in gene expression regulation. The higher nucleosomal occupancy in coding regions of most genes may provide a means to safeguard against spurious transcription initiation in the coding region of the gene.
In model eukaryotes, gene expression is positively correlated with the nucleosome occupancy at the promoter regions. In contrast, the intergenic putative promoter regions of P. falciparum showed little or no change in nucleosomal occupancy, and generally remained nucleosome-free, compared to the large changes observed in coding regions. Comparing the nucleosomal occupancy of the coding regions, we found no general correlation with steady-state mRNA levels determined by microarrays. This is not surprising, since chromatin structure only represents one mechanism of transcription regulation. In addition, transcription initiation is regulated by sequence-specific transcriptional activators and repressors. Steady-state mRNA levels are determined by the level of transcription, and post-transcriptional regulation of mRNA stability and degradation. Therefore, we would not expect chromatin structure to be the dominant determinant of steady-state mRNA levels.
The highly condensed heterochromatin that comprises most non-coding regions of higher eukaryotes represses transcription in those regions. However, in P. falciparum
, a recent study found the repressive histone modification of H3K9me3 only at subtelomeric loci and not in other intergenic regions [43
]. It may be that the generally relaxed, non-condensed structure of P. falciparum
intergenic chromatin in core chromosomal regions is sufficiently permissive of transcription such that further relaxation is not required to recruit transcription factors and initiate high levels of transcription. Our Q-PCR validation at promoters of selected individual genes supported such a generalization (e.g., var
genes, msp2). Another study found that the transcription pre-initiation complex was pre-assembled on promoters of all erythrocytic-expressed genes, independent of any histone acetylation [44
], supporting this hypothesis, and challenging the model that chromatin acetylation precedes transcription initiation in Plasmodium
In human cells, regulation of transcription by RNA Pol III requires histone acetylation and changes in chromatin condensation [45
]. However, the consistently low nucleosomal occupancy of tRNA and 5S rRNA genes in P. falciparum
may indicate a different mechanism for regulating Pol III-derived transcripts that may not involve interaction with modified histones but may instead depend on other Pol III associated transcription factors. Therefore, it may be advantageous to keep these genes in a perpetually permissive state to allow for rapid transcription of tRNAs when protein synthesis is rapidly accelerated in growing trophozoites. This same situation may apply to the RNA polymerase II transcribed genes that constitute the basal transcription machinery, which may need to be rapidly and highly expressed to initiate a general transcriptional increase in ring stage.
The expression of var
genes is inversely correlated with nucleosomal occupancy. This has been confirmed from the dramatic changes in nucleosomal occupancy between active and silent var2CSA gene. The changes in nucleosomal occupancy observed at the intron of var
genes supports the hypothesis that var
gene regulation is based on cooperative interactions between the two promoters of each var
]. The higher nucleosome occupancy in exon 2 of most var
genes may inhibit full-length gene transcription compared with the lower nucleosomal occupancy in exon 1. This may provide an explanation for the low-level production of many var
transcripts that are detectable by RT-PCR, but not detectable by Northern blots. The generally permissive chromatin structure of all var
genes exon 1 may allow for abortive transcripts to be formed and rapidly degraded and for the generation of antisense transcript directed by the var
intron. Transcription factor genes with patterns of nucleosome enrichment similar to VSAs may be involved in regulating the expression of these genes. The subnuclear localization of the singly expressed var
gene may be shared with some of these similarly regulated core chromosomal genes.
The lack of histones in intergenic regions may prevent the condensation of chromosomes during late stages of mitotic replication, which does not occur in Plasmodium
parasites as it does in other eukaryotic organisms. The telomeric regions display the highest chromatin condensation of any region of the genome. In other eukaryotes where chromosomes condense upon mitotic cell division, the centromere region is responsible for binding to the kinetochore and microtubules for chromosomal segregation in daughter cells. In Plasmodium
, not all chromosomes have annotated centromeres, and the chromosomes do not condense at schizogony. The telomeric regions with TAREs and Rep20 that have higher nucleosomal enrichment may play a role in chromosome segregation into daughter cells [35
]. This may also be an adaptation to the schizogony process where multiple rounds of nuclear replication precede cytokinesis in the development of merozoites. The highly repetitive genes that show telomere-like chromatin changes may be targeted by chromatin remodeling machinery that recognizes repetitive regions throughout the genome.