Genome-wide analysis of the human malaria parasite Plasmodium falciparum transcription factor PfNF-YB shows interaction with a CCAAT motif

Abstract

Little is known about transcription factor regulation during the Plasmodium falciparum intraerythrocytic cycle. In order to elucidate the role of the P. falciparum (Pf)NF-YB transcription factor we searched for target genes in the entire genome. PfNF-YB mRNA is highly expressed in late trophozoite and schizont stages relative to the ring stage. In order to determine the candidate genes bound by PfNF-YB a ChIP-on-chip assay was carried out and 297 genes were identified. Ninety nine percent of PfNF-YB binding was to putative promoter regions of protein coding genes of which only 16% comprise proteins of known function. Interestingly, our data reveal that PfNF-YB binding is not exclusively to a canonical CCAAT box motif. PfNF-YB binds to genes coding for proteins implicated in a range of different biological functions, such as replication protein A large subunit (DNA replication), hypoxanthine phosphoribosyltransferase (nucleic acid metabolism) and multidrug resistance protein 2 (intracellular transport).

Keywords: CCAAT-box; Plasmodium falciparum; malaria; signaling; transcription factor.

Figure 1. PfNF-YB expression throughout the intraerythrocytic cycle of P. falciparum

Samples of a synchronized parasite population were collected at various time points (10-48 h) after invasion. RT-PCR analysis of PfNF-YB mRNA level reveals differential expression during the intraerythrocytic cycle. Statistical analysis was performed by t test (p<0.05). The amount of mRNA in each sample was normalized by the level of 18S RNA.

Figure 2. Genome-wide PfNF-YB occupancy by high-density ChIP-on-chip assay

The display (Deva) shows the chromosomal map position and location of DNA enrichment due to PfNF-YB binding. (A) The blue panel peaks shows the ChiP/Input ratio for all 14 chromosome in schizont stage 3D7 parasites. PfNF-YB occupancy for each gene was calculated as the average of log₂ ratios of hybridization values for immunoprecipitated and input chromatin. (B). PfNF-YB occupancy on chromosome 7. Schematic display (Deva) of the blue peaks on chromosome 7 shows PfNF-YB occupancy in the gene region (red bars) and the exon region (green bars).The candidate promoter regions were estimated from false discovery rate (FDR) values (0.095) and by Log2 value ratio ≥ 0.5 (dotted red line). The ratio data were randomized 20 times to evaluate the probability of “false positives”. Each peak was assigned an FDR score based on the randomization. Chromosome numbers are indicated on the right, chromosomal position (Kb) on top.

Figure 3. ChIP-on-chip validation

Target genes were selected randomly to perform ChIP/qPCR from schizont stage parasites. Twenty-one different genes showed a PfNF-YB enrichment of at least 2-fold compare to control beads. The last three genes were selected as negative binding targets for PfNF-YB. Input samples were used to normalize the relative values. The control beads were used as a negative control for the ChIP assay. These analyses were derived from three independent assays. The primers used in the analyses can be found in Supplementary Table 3. Gene accession numbers (www.PlasmoDB.org) are indicated at the bottom of the graphic.

Figure 4. PfNF-YB binds to different gene functions in P. falciparum

(A) PfNF-YB target genes classified by RNA type and protein function. Genes coding for proteins with unknown function (42%) represent the biggest group, followed by the groups represented by the genes with putative (40%) and known (16%) function. (B) Identification of biological processes associated to the target genes bound by PfNF-YB. A group containing 167 coding and putative genes that have a biological process attribute according to the Gene Ontology section from PlasmoDB database were plotted. Fifty-two per cent of these genes are involved in RNA translation, intracellular transport, metabolism, protein folding and cell redox homeostasis.

Figure 5. Identification of CCAAT and AATAT motifs bound by PfNF-YB transcription factor

(A) The CCAAT motif sequence recognized by PfNF-YB was found in the promoter regions of 140 genes. Forty-five out of these 140 regions containing 49 CCAAT motifs were investigated for putative consensus sequences. (B) AATAT motif sequence was found in the promoter regions of 297 genes recognized by PfNF-YB. One hundred and twenty-three of these 297 regions containing 313 AATAT motifs were investigated for putative consensus sequences. The probe sequences covering the target genes were selected and assembled, and the putative promoter sequences were analysed by motif finder WebLogo [67]. The graphic shows the relative entropy-based logo of the detected motif. Values in parenthesis represent the number of gene promoters belonging to the consensus motifs. (C) The PfNF-YB target genes present a diversity of binding site regions. The scheme shows the matches to the CCAAT motif and to the putative TATA-box motif. The location of these motifs is both upstream and downstream of the ATG start codon. (*) putative gene.

Figure 6. Motif matching in the putative promoter region of PfNF-YB target genes

(A) The Monte Carlo test was performed taking into account the gene target lengths to obtain new random subsequences. This procedure was executed 100,000 times to estimate the p-value (percentage of executions in which the number of subsequences that matched the considered motifs equalled or exceeded the actual number of target matches). The frequency of the CCAAT motif is significantly higher in predicted genes (p-value: 0.00043) than in the rest of the genome. The three motifs CCAAT, AAATG and ATTTG are also significantly more frequent in the PfNF-YB target genes group compared to the rest of the genome (p-value: 0.00074), which suggests that these motifs are strongly associated. (B) A Venn diagram to better illustrate the distribution of the three motifs among the 297 predicted genes. There are 140 matches for CCAAT, 263 matches for AAATG and 242 matches for AATTG. 120 predicted genes (40.4%) match with the three CCAAT, AAATG and ATTTG motifs. Note that only twelve genes (4%) contain none of the three motifs.