PfGCN5-mediated histone H3 acetylation plays a key role in gene expression in Plasmodium falciparum

Abstract

Histone acetylation, regulated by the opposing actions of histone acetyltransferases (HATs) and deacetylases, is an important epigenetic mechanism in eukaryotic transcription. Although an acetyltransferase (PfGCN5) has been shown to preferentially acetylate histone H3 at K9 and K14 in Plasmodium falciparum, the scale of histone acetylation in the parasite genome and its role in transcriptional activation are essentially unknown. Using chromatin immunoprecipitation (ChIP) and DNA microarray, we mapped the global distribution of PfGCN5, histone H3K9 acetylation (H3K9ac) and trimethylation (H3K9m3) in the P. falciparum genome. While the chromosomal distributions of H3K9ac and PfGCN5 were similar, they are radically different from that of H3K9m3. In addition, there was a positive, though weak correlation between relative occupancy of H3K9ac on individual genes and the levels of gene expression, which was inversely proportional to the distance of array elements from the putative translational start codons. In contrast, H3K9m3 was negatively correlated with gene expression. Furthermore, detailed mapping of H3K9ac for selected genes using ChIP and real-time PCR in three erythrocytic stages detected stage-specific peak H3K9ac enrichment at the putative transcriptional initiation sites, corresponding to stage-specific expression of these genes. These data are consistent with H3K9ac and H3K9m3 as epigenetic markers of active and silent genes, respectively. We also showed that treatment with a PfGCN5 inhibitor led to reduced promoter H3K9ac and gene expression. Collectively, these results suggest that PfGCN5 is recruited to the promoter regions of genes to mediate histone acetylation and activate gene expression in P. falciparum.

FIG. 1.

Histograms showing the distribution of enrichment of array elements from ChIP-chip experiments. Hybridizations were performed using synchronized P. falciparum late trophozoites (∼28 h) with a preimmune serum (A) and antibodies to H3K9ac (B). For each antibody, ChIP was performed in triplicate, and the averages of three experiments were used to construct the histograms. Each bar indicates the number of array elements with similar log₂(IP/input) values binned together.

FIG. 2.

Genome-wide H3K9ac and PfGC5 occupancy profiles are highly correlated but different from that of H3K9m3 occupancy. (A) Correlation between the ChIP-chip datasets. R indicates Pearson correlation values. (B) Venn diagram comparing genes highly enriched (≥4-fold) for H3K9ac, PfGCN5, and H3K9m3.

FIG. 3.

Correlation of H3K9ac and H3K9m3 enrichment with gene expression divided on the positions of the array elements (bp from the putative ATG). For the four groups of elements, the correlations (R) between H3K9ac and gene expression were 0.285 (P < 0.102), 0.453 (P < 0.002), 0.135 (P < 0.236), and 0.011 (P < 0.468), whereas the correlations (R) between H3K9m3 and gene expression were −0.035 (P < 0.019), −0.29 (P < 0.001), −0.061 (P < 0.135), and 0.009 (P < 0.553).

FIG. 4.

Mapping of H3K9ac and H3K9m3 at the MAL7P1.37-38 locus in trophozoites (28 h). The array element for MAL7P1.37 was highly enriched in the H3K9ac and PfGCN5 ChIP-chip data sets. (Top) RT-PCR analysis of MAL7P1.37 and MAL7P1.38 expression in asexual erythrocytic stages. R, rings; ET, early trophozoites; LT, late trophozoites; S, schizonts. Cycles indicate RT-PCR cycles for the respective genes, and numbers above the gels indicate average 2^−ΔΔCT values by real-time RT-PCR with ring stage as the reference stage (42). (Middle) Schematic drawing to show the ORF of the active (filled arrow) and inactive (open arrow) genes in the trophozoite stage and their intergenic region (line). Primers were designed to amplify nine regions of this locus. Primer pairs 2 and 8 were used in RT-PCR analysis. (Bottom) ChIP enrichment (IP/input) at different regions of the locus to show that H3K9ac was higher in the 5′ region of MAL7P1.37, whereas H3K9m3 was highly enriched near the inactive MAL7P1.38 gene.

FIG. 5.

Mapping of H3K9ac to the promoters of active genes using ChIP and real-time PCR analysis. Selected genes represent stage-specific and constitutively expressed genes (marked on the right). (A and B) Expressed in trophozoite stage (DNA pol δ and GBP130); (C and D) expressed in ring stage (MAL7P1.170 and HSP101); (E and F) expressed in schizont stage (EBA175 and MSP1); (G and H) expressed in all stages (actin-related and calmodulin). ChIP was performed with synchronized parasites at the ring, trophozoite, and schizont stages. Real-time PCR was done using input DNA and DNA enriched by ChIP with anti-H3K9ac. For the gene schemes, filled and open block arrows indicate the orientation of expressed and silent genes, respectively. Intergenic regions are shown as thin lines. The positions of previously determined transcription initiation sites for DNA pol δ, GBP130, and calmodulin are indicated as arrows. The relative positions of PCR fragments (A to F) are indicated under the gene schemes. In each graph, the x axis indicates the position of the PCR fragments, and the y axis shows fold enrichment (IP/input) determined by real-time PCR. The inset in panel G shows the expression of the PF14_0218 gene in rings (R), early trophozoites (ET), late trophozoites (LT), and schizonts (S) by real-time RT-PCR compared to constitutive expression of seryl-tRNA synthetase in Fig. 4. Numbers below the gel indicate the average 2^−ΔΔCT values with the ring stage as the reference stage (42).

FIG. 6.

Curcumin treatment is associated with reduced gene expression and promoter acetylation. (A) Curcumin treatment (5 and 20 μM) of early trophozoites (18 h) for 12 h resulted in the reduced expression of DNA pol δ, GBP130, actin-related, and calmodulin genes. Shown here are gel pictures of semiquantitative RT-PCR for the PF07_0047 gene (20 cycles) and four other genes (25 cycles). Numbers above each panel indicate average 2^−ΔΔCT values of the genes compared to that of PF07_0047 gene by real-time PCR with untreated parasites as the reference (42). (B) Reduced acetylation in the upstream regions of genes. Fragments used for real-time PCR analysis are DNA pol δ (c), GBP130 (d), actin-related (c), and calmodulin (b) as shown in Fig. 4. The region of the PF07_0047 gene was selected from a mapping experiment and the primers are shown in Table S1 in the supplemental material.