The Cryptosporidium parvum ApiAP2 gene family: insights into the evolution of apicomplexan AP2 regulatory systems

Abstract

We provide the first comprehensive analysis of any transcription factor family in Cryptosporidium, a basal-branching apicomplexan that is the second leading cause of infant diarrhea globally. AP2 domain-containing proteins have evolved to be the major regulatory family in the phylum to the exclusion of canonical regulators. We show that apicomplexan and perkinsid AP2 domains cluster distinctly from other chromalveolate AP2s. Protein-binding specificity assays of C. parvum AP2 domains combined with motif conservation upstream of co-regulated gene clusters allowed the construction of putative AP2 regulons across the in vitro life cycle. Orthologous Apicomplexan AP2 (ApiAP2) expression has been rearranged relative to the malaria parasite P. falciparum, suggesting ApiAP2 network rewiring during evolution. C. hominis orthologs of putative C. parvum ApiAP2 proteins and target genes show greater than average variation. C. parvum AP2 domains display reduced binding diversity relative to P. falciparum, with multiple domains binding the 5'-TGCAT-3', 5'-CACACA-3' and G-box motifs (5'-G[T/C]GGGG-3'). Many overrepresented motifs in C. parvum upstream regions are not AP2 binding motifs. We propose that C. parvum is less reliant on ApiAP2 regulators in part because it utilizes E2F/DP1 transcription factors. C. parvum may provide clues to the ancestral state of apicomplexan transcriptional regulation, pre-AP2 domination.

Figure 1.

Distribution and quantification of AP2 proteins and domains across chromalveolates and algae. Counts of AP2 domain-containing proteins and the number of AP2 domains per species as determined by sensitive sequence profile analysis using either the AP2 HMM available from PFAM or our custom ApiAP2 HMM. Analyses on most species were run on fully annotated protein sets. **Dinoflagellate analyses were run on clustered EST data. *P. marinus analyses were run on clustered genome ORFs. These counts represent profile matches at or below a permissive e-value of 10. Approximately 85% of the hits were at or below 1e-3.

Figure 2.

Evolutionary relationships of AP2 domains across chromalveolates and algae. Maximum likelihood tree constructed of top-scoring AP2 domains (hmmsearch domain e-value of 1e-3 or better using the AP2 HMM) from selected taxa. Bootstrap support obtained from 100 replicates are indicated on nodes where support = 50% or greater. Tree constructed with RAxML using a gamma rate estimation and a Dayhoff model of codon evolution, and visualized using FigTree (v. 1.4.0; http://tree.bio.ed.ac.uk/software/figtree/). Species abbreviation prefixes have been added to non-apicomplexan gene IDs for ease of understanding: At_, A. tamarense; Cr_, C. reinhardtii; K_brevis_, K. brevis; Micro_, Micromonas; Pm_, P. marinus; Ptric_, P. tricornutum; Tt_, T. thermophila; Tp_, T. pseudonana. Perkinsid and ApiAP2 domains group together outside of other chromalveolate and green algae AP2 domains with high bootstrap support, indicating possible independent origins for these domains.

Figure 3.

C. parvum and P. falciparum AP2 domain ortholog binding motifs as determined by PBM. C. parvum domains are color-coded according to evolutionary groups based on OrthoMCL clustering at 1e-6 as discussed in Materials and Methods. Data for core DNA motifs determined for P. falciparum AP2 domains obtained from (37).

Figure 4.

Maximum likelihood tree of P. falciparum and C. parvum AP2 domains and their DNA-binding motifs. Domain sequences were extracted from full-length proteins using HMM-defined coordinates, aligned and edited as described in Materials and Methods. A maximum likelihood tree was constructed from the edited alignment using RAxML with a Dayhoff protein evolution model, then visualized using FigTree. Domains in red were not identified in (37); as all domains were numbered from N-terminus to C-terminus, the numbering scheme therefore shifts slightly from (37). The previous domains PF11_0404_D3, PF13_025_D3, PF13_0267 and PF13_0026 correspond in this figure to domains PF11_0404_D4, PF13_0235_D4, PF13_0267_D2 and PF13_0026_D2, respectively. *Denotes domains that when tested alone, have no detectable binding specificity, but when tested with an adjacent domain, have the indicated binding specificity. **Denotes tested domains with no binding motifs over threshold.

Figure 5.

Overrepresented C. parvum motifs bound by AP2 domains. Overrepresented motifs as determined by (49). Overrepresented motifs that are (i) recognized by known non-AP2 proteins (E2F; CAAT-box) or (ii) are not AP2 binding motifs (GAGA-like; Unknown Set 1, Unknown Set2 and Unknown Motifs 14, 21, 22 and 25) are not included.

Figure 6.

Expression patterns of genes containing overrepresented C. parvum G-box motifs and potential G-box-binding ApiAP2 regulators. (A) The PBM-determined binding motif as well as expression data for each G-box-binding AP2 domain is displayed for seven time points from 2 to 72 h across the in vitro life cycle (expression data from (52)). (B) Two G-box motifs were identified as overrepresented in upstream regions of C. parvum genes clustered by expression profile (49). The number of clusters, and the number of genes having the motif out of those clusters is shown. (C) Expression data for all genes in clusters identified as having overrepresented G-box motifs. Genes with overrepresented G-box motifs are expressed across the life cycle; there is a G-box-binding ApiAP2 protein expressed at each of those time points, suggesting ApiAP2s could be driving expression of these genes.