Histone PTMs in P. falciparum

The combination of mass-spectrometry (MS) and ChIP-Seq-based studies, at different parasite life-cycle stages, is allowing us to unravel the spatial and temporal distribution of histone PTMs and is starting to shed light on the roles different histone PTMs play in transcriptional regulation

falciparum histones 228,232,233,253–262, but only acetylation and methylation, the most abundant marks, have been identified along multiple life cycle stages and thoroughly characterized. It has become increasingly clear that not only individual PTMs but also combinations of these PTMs carry specific functional consequences ²⁶³, adding a layer of complexity to the histone PTMs landscape. A recent study also demonstrated that histone PTMs presence changes dynamically with life-cycle progression, and that there is a marked difference in the palette of histone PTMs used during asexual proliferation and sexual differentiation ²⁵⁴.

Although some histone PTMs might just be a mere consequence of previous transcriptional events, the ubiquity of histone PTMs, their combinatorial and dynamic nature and the fact different life cycle stages have unique and specific histone PTM signatures ^253,254, seem to be clear indicators of the pivotal role of histone PTMs in the regulation of gene expression in P. falciparum (Figure 9).

Figure 9. Histone PTMs identified in P. falciparum by quantitative methods. Reproduced from Connacher et al. 2022 ⁴²¹.

3.5.5.1 Histone Acetylation

Acetylation is the most abundant histone PTM during the IDC and is mainly associated with a transcription-permissive state ^253,254. Acetylation of histones H3K9, H3K14, H3K56 and H4K5, H4K8, H4K12, H4K16 are widespread across the euchromatic regions of the genome 254,255,263–266

and their disruption by HAT/HDAC inhibitors resulted in global transcriptional disruption ^267,268, highlighting their implication in active transcription.

Although all these marks are found in euchromatin, only H3K9ac and H3K14ac were found to be enriched in highly transcribed genes when paired with RNA-Seq data ²⁶⁶. Both marks are more abundant at the 5’ end of genes, near the TSS, but they spread along the whole coding region ²⁶⁶. H3K9ac colocalizes with H3K4me3 and histone variant H2A.Z and its enrichment levels not only correlate with highly expressed genes but also with temporal patterns of gene expression ²³⁴.

Some of these marks display specific distributions that vary along the parasite’s life cycle. During the IDC, H3K18ac and H3K27ac, together with the histone variant PfH2A.Z, dynamically mark the TSS of active genes and colocalize with PfAP2-I and bromodomain protein 1 (PfBDP1) ²⁶⁵. H4K8ac is higher in schizont stages, while H3K9ac and H3K14ac are higher in trophozoites ^254,255. H3K4ac is abundant in active promoter regions, and its abundance increases greatly during the schizont stage ²⁵⁴ (a very transcriptionally active stage ¹⁸⁷). H4K16ac has a very similar distribution profile to H3K4ac and correlated with active transcription in a mass spectrometry -based study, but its role is less clear ²⁵⁴.

The fact that histone acetylation is so widely distributed, marking not only actively transcribed genes but also silent genes found in euchromatin, suggests that many of these marks somehow

3.5.5.2 Histone Methylation

Methylation is also widely present along the genome, and unlike acetylation, it might be associated with both transcriptionally active and repressed chromatin.

H3K4me2-3 is a widespread active mark with increased abundance in schizonts. H3K4 methylation also correlated with levels of transcription when paired with RNA-Seq data, although not in a temporal manner ²⁶⁶. H3K4me3 can recruit the transcriptional activator PfGCN5/ADA2 and its associated reader complexes containing PfBDP1 and PfBDP2 ^269,270. The PfGCN5/ADA2 complex acetylates H3K9 generating H3K9ac, which is a clear example of histone PTMs crosstalk and positive feedback. H3K4me2 has also been implicated in var gene regulation, where it decorates the active member of the family, found in a poised state between cell divisions, thus contributing to the active state memory ^271,272.

Moving onto repressive marks, H3K9me3, the landmark of constitutive heterochromatin in most eukaryotes, is found in both constitutive and facultative heterochromatin in P. falciparum. It spreads from TAREs into the subtelomeric genes, and it is also found in some internal regions in a few chromosomes 231,266,273. Given its importance as the main marker of heterochromatin in P.

falciparum, H3K9me3 will be further discussed on a latter chapter.

Other repressive methylation marks have been found in P. falciparum, although unlike H3K9me3 they are not restricted to heterochromatin, nor to silenced genes ²⁶⁶. H3K36me3, which is associated with transcription elongation in other eukaryotes, has a broad distribution along the genome on both active and silent genes in P. falciparum, but it’s ratio to activating marks has been found to correlate negatively with transcription ²⁶⁶ . In var genes, it has been found to be enriched in the TSS of all silent var genes as well as the coding sequence of both the silenced and the active members of the family but absent from the TSS of the active var gene. Its disruption led to the expression of virtually all members of the family ²⁷⁴ .

The highly conserved heterochromatin mark H3K27me3, which for a long time was thought to be absent in P. falciparum, has been found to be present only during gametocytogenesis ²⁵⁴. In fact, repressive histone PTMs seem to increase considerably during the initial steps of gametocyte maturation ²⁵⁴, when many genes related to the IDC need to be silenced. Also during gametocytogenesis, both H3K27me3 and H3K36me3 were found to have a very high pairwise correlation with the occupancy of the repressive TF PfAP2-G2 ¹⁹⁵, suggesting an interesting functional link in gene silencing between these marks and the TF.

H4K20me3, which is associated with heterochromatin in most eukaryotes, has a very unspecific and widespread distribution in P. falciparum, without a clear relation with transcriptional status

231,275.

3.5.6 Histone Modifying enzymes in P. falciparum