Additional remarks phenotype | Mutant/mutation
The mutant expresses a mutated form of Zfp (lacking the C-terminal C2H2 ZnFs) and expresses mCherry under control of the constitutive eef1a promoter.
Protein (function)
Studies on identification and genome-wide mapping of recombination have revealed a correlation between hotspot activity and enrichment of trimethylated histone 3 at lysine 4 (H3K4me3). Further studies in mammals have identified PRDM9, a meiosis-specific chromatin-modifying protein, as the key determinant of recombination hotspots and a master transcription regulator of entry into meiosis. The PRDM9 protein contains a C-terminal module comprised of multiple C2H2-type zinc fingers (ZnF) which binds the degenerate 13-bp motif that was previously found to be associated with ~40% of human hotspots, formation of doublestrand breaks (DSB), and recruitment of the recombination machinery leading to exchange of chromosomal material and DNA repair. Additionally, the ZnF domain (Zfd) in PRDM9 is fused to a SET domain which catalyzes the trimethylation of histone 3 lysine 4 (H3K4) and histone 3 lysine 36 (H3K36) associated with the hotspots.
The SET domain initially characterized in Drosophila is an evolutionarily well-conserved domain associated with many chromosomal proteins and with proteins necessary for histone lysine trimethylation in mammalian cells.
The C2H2-type Zfd, represented by two cysteine and two histidine residues separated by ~12 amino acids acting as ligands to a zinc atom, are widespread in most DNA-binding proteins. In animals, proteins with multiple C2H2 ZnFs are transcription factors regulating gene expression and acting during cell differentiation and development. Binding of C2H2 ZnF proteins to DNA often helps recruit other proteins which mediate epigenetic changes, including posttranslational modifications of histones. These include histone methylases as well as enzymes catalyzing demethylation of H3K4me3 and other histone methyl transferases transferring methyl groups to H3K9 and H3K27.
While proteins with runs of multiple (>3) instances of C2H2 ZnF are common in animals, the P. berghei Zfp (PbZfp) protein is the only such example conserved across Plasmodium species. The protein is comprised of a poorly conserved N-terminal low-complexity region and a C-terminal region with 11 C2H2 ZnFs, which is where the sequence similarity is most pronounced. The C2H2 ZnFs are arranged in an initial run of 3 ZnFs followed by a disordered poorly conserved linker connecting them to the remaining 8 ZnFs. Unlike the animal PRDM9 protein, PbZfp lacks the methyltransferase SET domain and does not have KRI domains typical of the members of the KRAB family of animal C2H2 ZnF proteins. Thus, while not directly related to these animal proteins, PbZfp shows an unusual resemblance to animal C2H2 ZnF proteins in having a multiplicity of C2H2 Zfd. The presence of a run of multiple C2H2 ZnFs indicates that the protein might contact an extended binding site as is typical of recombination hotspots. These observations pointed to potential roles in addition to or beyond that of a conventional transcription factor and prompted the authors to investigate if it might play a role in recombination.
Phenotype
See also mutant RMgm-4316 which lacks expression of Zfp (Zfp-KO) which has a comparable phenotype as Zfp-Trunc, which expresses a truncated form of Zfp (lacking the C-terminal C2H2 ZnFs).
Normal growth/multiplication characteristics of blood stages. Mice infected with Zfp-Trunc parasites survived longer than mice infected with wild type parasites.
No differences in gametocyte production and fertilsation.
Oocyst numbers in mosquitoes infected with Zfp-Trunc parasites were 75% to 80% lower than those seen with mosquitoes infected with wild type parasites.
Additional information
The authors present evidence for: 'KO parasites revealed a total lack of trimethylation of histone 3 at several lysine residues (K4, K27, and K36) without any effect on acetylation patterns (H3K9, H3K14, and H4K16). Reduced DNA damage and reduced expression of topoisomerase-like Spo11 in the KO parasites with normal Rad51 expression further suggest a functional role for PbZfp during genetic recombination that involves DNA double-strand break (DSB) formation followed by DNA repair. These finding raise the possibility of some convergent similarities of PbZfp functions to functions of mammalian PRDM9, also a C2H2 ZnF protein with histone 3 lysine 4 (H3K4) methyltransferase activity. These functions include the major role played by the latter in binding recombination hotspots in the genome during meiosis and trimethylation of the associated histones and subsequent chromatin recruitment of topoisomerase-like Spo11 to catalyze DNA DSB formation and DMC1/Rad51-mediated DNA repair and homologous recombination' .
Other mutants
RMgm-4316- A mutant lacking expression of Zfp |