A Major Role for the Plasmodium falciparum ApiAP2 Protein PfSIP2 in Chromosome End Biology

Abstract

The heterochromatic environment and physical clustering of chromosome ends at the nuclear periphery provide a functional and structural framework for antigenic variation and evolution of subtelomeric virulence gene families in the malaria parasite Plasmodium falciparum. While recent studies assigned important roles for reversible histone modifications, silent information regulator 2 and heterochromatin protein 1 (PfHP1) in epigenetic control of variegated expression, factors involved in the recruitment and organization of subtelomeric heterochromatin remain unknown. Here, we describe the purification and characterization of PfSIP2, a member of the ApiAP2 family of putative transcription factors, as the unknown nuclear factor interacting specifically with cis-acting SPE2 motif arrays in subtelomeric domains. Interestingly, SPE2 is not bound by the full-length protein but rather by a 60kDa N-terminal domain, PfSIP2-N, which is released during schizogony. Our experimental re-definition of the SPE2/PfSIP2-N interaction highlights the strict requirement of both adjacent AP2 domains and a conserved bipartite SPE2 consensus motif for high-affinity binding. Genome-wide in silico mapping identified 777 putative binding sites, 94% of which cluster in heterochromatic domains upstream of subtelomeric var genes and in telomere-associated repeat elements. Immunofluorescence and chromatin immunoprecipitation (ChIP) assays revealed co-localization of PfSIP2-N with PfHP1 at chromosome ends. Genome-wide ChIP demonstrated the exclusive binding of PfSIP2-N to subtelomeric SPE2 landmarks in vivo but not to single chromosome-internal sites. Consistent with this specialized distribution pattern, PfSIP2-N over-expression has no effect on global gene transcription. Hence, contrary to the previously proposed role for this factor in gene activation, our results provide strong evidence for the first time for the involvement of an ApiAP2 factor in heterochromatin formation and genome integrity. These findings are highly relevant for our understanding of chromosome end biology and variegated expression in P. falciparum and other eukaryotes, and for the future analysis of the role of ApiAP2-DNA interactions in parasite biology.

Systematic CRISPR-Cas9-Mediated Modifications of Plasmodium yoelii ApiAP2 Genes Reveal Functional Insights into Parasite Development

“…Previous studies have individually investigated 11 Plasmodium ApiAP2 genes functioning in sexual, mosquito, and liver stages in other species (12)(13)(14)(15)(16)(17)(18)(19). In particular, a recent study attempted to knock out all of the ApiAP2 genes and succeeded in disrupting 10 genes in P. berghei (9).…”

ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst

“…AP2IX-9 binds DNA in a sequence-specific manner, and the AP2IX-9 binding motif is repeated in many bradyzoite promoters where direct binding of this factor to the BAG1 promoter in chromatin was established. ApiAP2 factors have roles in activating developmental gene expression (13)(14)(15)(16), and there is evidence an ApiAP2 factor may assist the silencing of var gene expression in P. falciparum (17). The AP2IX-9 factor is the first ApiAP2 transcriptional repressor to be characterized in the Apicomplexa, and this mechanism begins to explain how the early steps in tachyzoite to bradyzoite development are regulated in Toxoplasma.…”

“…In Toxoplasma, 24 ApiAP2 factors are cell cycle regulated in tachyzoites (8), whereas 18 are expressed in a cascading fashion during the P. falciparum intraerythrocytic cycle (12). Relatively few ApiAP2 proteins are characterized; however, stage-specific gene activation (13)(14)(15)(16) as well as chromatin biology and genome maintenance (17) are functions identified so far. Like their plant counterparts, ApiAP2 factors can bind promoter elements of distinct coregulated gene clusters (12) and, therefore, it is likely these proteins will be major regulators of apicomplexan transcription.…”

Insulator-like pairing elements regulate silencing and mutually exclusive expression in the malaria parasite Plasmodium falciparum

“…Within these upstream regulatory regions DNA elements have been identified by deletion analysis of the 5′UTR and EMSA (16,17). It was demonstrated that one of these motifs, SPE2, specifically binds a member of a newly discovered DNA-binding protein family, ApiAP2, which has a role in chromosome end biology (18). Recently, an additional protein-binding DNA element implicated in mutually exclusive locus recognition was identified in 44 of the var 5′ UTRs (17).…”