The Cryptosporidium parvum ApiAP 2 gene family : insights into the evolution of apicomplexan AP 2 regulatory systems

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. Proteinbinding 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. INTRODUCTION Apicomplexan parasites are the causative agents of some of the world’s most devastating infectious diseases, including malaria (caused by Plasmodium), toxoplasmosis (T. gondii) and cryptosporidiosis (Cryptosporidium). Cryptosporidium species, primarily C. parvum and C. hominis, have recently been revealed to be the second leading cause of infant diarrhea globally (1). Plasmodium and Cryptosporidium diverged between 824 and 350 mya (2) with more recent estimates of ∼420 mya (3,4), a distance comparable to that between humans and the ancestral chordate (5). While RNA polymerase-associated factors and basal transcription factors have been identified in the Apicomplexa (6), examination of apicomplexan proteomes yielded a surprising dearth of ‘typical’ eukaryotic enhancer proteins and their characteristic binding sites (7,8). These findings were highly unexpected, given the extensive evidence for transcriptional control (9–11). The lack of recognizable, specific transcription factors initially suggested that the specific transcription factors were likely so divergent from those found in other eukaryotes that they were unrecognizable. Balaji et al. (8) took a more sophisticated approach to tackle this issue by generating new Hidden Markov Models (HMMs) to perform sensitive sequence analyses of several apicomplexan genomes (Plasmodium, Cryptosporidium, Theileria) for all known DNA-binding domains (8). The team confirmed the dearth of ‘typical’ DNA-binding enhancer proteins, but they did identify Myb and zinc-finger proteins, as well as an unexpected family of proteins with multiple members present in all examined apicomplexan genomes. This Apicomplexan AP2 family of proteins (ApiAP2) bears resemblance to the AP2/ERF family of DNA-binding tran*To whom correspondence should be addressed. Tel: +1 706 542 6562; Fax: +1 706 542 3582; Email: jkissing@uga.edu