P. falciparum-specific Genomic Context Protein-Binding Microarray

Development of the human malaria parasite, Plasmodium falciparum is regulated by a limited number of sequence-specific transcription factors (TFs). However, the mechanisms by which these TFs recognize genome-wide binding sites to regulate target genes is still largely unknown. To address TF target specificity, we investigated the binding of two TF subsets that either bind CACACA or GTGCAC and further characterized PfAP2-G and PfAP2-EXP which bind unique DNA motifs (GTAC and TGCATGCA). We interrogated the impact of DNA sequence context and the chromatin landscape on P. falciparum TF binding using high-throughput in vitro and in vivo binding assays, DNA shape predictions, epigenetic post-translational modifications, and chromatin accessibility. We determined that DNA sequence context does not greatly impact binding site selection for CACACA-binding TFs, while chromatin accessibility, epigenetic patterns, co-factor recruitment, and dimerization contribute to differential binding. In contrast, GTGCAC-binding TFs prefer different sequence contexts, DNA shape profiles, and chromatin dynamics. Finally, we find that TFs that preferentially bind divergent DNA motifs may bind overlapping genomic regions in vivo due to low-affinity binding to other sequence motifs. Our results demonstrate that TF binding site selection relies on a combination of DNA sequence and chromatin features, thereby contributing to the complexity of P. falciparum gene regulatory mechanisms. 4x180k high-density Genomic Context Protein-Binding Microarray (gcPBM) representing over 24,000 unique genomic regions from the P. falciparum genome. This array design was used to test eight specific DNA-binding domains (DBDs) against motif-containing sites from the P. falciparum genome. Using position weight matrix (PWM) data from published work (De Silva et al. 2008 & Campbell et al. 2010), all instances of each motif of interest (i.e., CACACA, GTGCAC, GTAC, and TGCATGCA) were identified in the P. falciparum genome (Plasmodium falciparum 3D7 strain genome release v38), using a motif E-score cutoff of >0.45. Only intergenic regions (excluding telomeric regions) were used for this gcPBM design. The numbers of probes found containing each motif recognized by the eight DBDs are as follows: 2848 probes with putative sites for PF3D7_0420300_D1 (500 negative controls), 4251 probes with putative sites for AP2-LT_D1 (500 negative controls), 3864 probes with putative sites for PF3D7_1305200_D1 (500 negative controls), 4321 probes with putative sites for AP2-HC_D1 (500 negative controls), 1459 probes with putative sites for SIP2_D1 (500 negative controls), 3742 probes with putative sites for AP2-I_D3 (500 negative controls), 8998 probes with putative sites for AP2-G_D1 (1000 negative controls), and 1059 probes with putative sites for AP2-EXP_D1 (1000 negative controls). Any sequence containing another instance of the centered motif in the left or right flanks was mutated to prevent multiple binding sites per 36-bp window. The number of 36-bp genomic regions designed for each TF. HDP1 motif-specific genomic sequences were not initially included in the gcPBM design due to its identification and characterization after the initial design of the gcPBM experiment, and was included after the fact. Due to the similarities between the CACACA and GTGCAC PWMs, there were genomic DNA sequences that led to redundant probe designs, which were discarded, leaving only one instance of the sequence. After discarding redundant probe designs with motif types, the total number of probes per motif group is as follows: 9388 probes with putative CACACA sites (1834 CACACA negative controls), 1394 probes with putative GTGCAC sites (736 GTGCAC negative controls), 8998 probes with putative GTAC sites (620 GTAC negative controls), and 1059 probes with putative TGCATGCA sites (612 TGCATGCA negative controls). Overall, the P. falciparum gcPBM design reached a total of 24,641 unique genomic regions. Each DNA probe was replicated in random areas of the microarray surface (CACACA/GTGCAC with 8 replicates per sequence and GTAC/TGCATGCA with 6 replicates per sequence), which brought the total number of DNA probes to 174,550 spots for a 4x180k microarray (Agilent Technologies). Additional spots on the array were set aside for control grid alignment, microarray scanning, and downstream analysis.