Lineage-specific expansion of the Zinc Finger Associated Domain ZAD

Lineage-specific expansion of the Zinc Finger Associated Domain ZAD Ho-Ryun Chung,1 Ulrike Löhr, and Herbert Jäckle Max-Planck-Institut für biophysikalische Chemie, Abteilung Molekulare Entwicklungsbiologie, Am Fassberg 11, 37077 Göttingen, Germany The zinc finger associated domain (ZAD), present in almost 100 distinct proteins, characterizes the largest subgroup of C2H2 zinc finger proteins in Drosophila melanogaster and was initially found to be encoded by arthropod genomes only. Here, we report that the ZAD was also present in the last common ancestor of arthropods and vertebrates, and that vertebrate genomes contain a single conserved gene that codes for a ZAD-like peptide. Comparison of the ZAD proteomes of several arthropod species revealed an extensive and species-specific expansion of ZAD-coding genes in higher holometabolous insects, and shows that only few ZAD-coding genes with essential functions in Drosophila melanogaster are conserved. Furthermore, at least 50% of the ZAD-coding genes of Drosophila melanogaster are expressed in the female germline, suggesting a function in oocyte development and/or a requirement during early embryogenesis. Since the majority of the essential ZAD coding genes of Drosophila melanogaster were not conserved during arthropod or at least during insect evolution, we propose that the LSE of ZAD-coding genes shown here may provide the raw material for the evolution of new functions that allow organisms to pursue novel evolutionary paths. Introduction 1 Present address: Max-Planck-Institut für molekulare Genetik, Department of Computational Molecular Biology, Ihnestr. 63-73, 14195 Berlin, Germany. Key words: Drosophila melanogaster, lineage-specific expansion, Zinc Finger Associated Domain. E-mail: . Mol. Biol. Evol. 24(9):1934–1943. 2007 doi:10.1093/molbev/msm121 Advance Access publication June 14, 2007 Results and Discussion Based on initial searches in expressed sequence tag databases the ZAD was proposed to be restricted to arthropods (Chung et al. 2002). Meanwhile, however, we found a ZAD-like domain among the zinc finger proteins reported by the database of protein families Pfam (Finn et al. 2006). This zinc finger protein, termed ZFP276, initially described in mouse and subsequently in human, was previously reported to contain C2H2 zinc fingers only (Wong et al. Ó 2007 The Authors This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/ uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. C2H2 zinc finger proteins (ZFPs) represent the most abundant nucleic acid binding proteins in the eukaryotic kingdom (e.g. Böhm, Frishman, and Mewes 1997; Lander et al. 2001; Chung et al. 2002; Englbrecht, Schoof, and Böhm 2004). Many vertebrate ZFPs are characterized by associated domains, like the KRAB and SCAN domains (Collins, Stone, and Williams 2001). Recently, a novel zinc finger associated domain (ZAD) was found in the N-terminal portion of many ZFPs of Drosophila melanogaster (Chung et al. 2002). The structure of this domain, as revealed by X-ray crystallography, resembles that of a treble-clef fold. The ZAD structure is stabilized by zinc coordination via four cysteine residues found to be invariantly present in all identified ZAD peptides (Jauch et al. 2003). Similar to the KRAB and SCAN domains, the ZAD can function as protein-protein interaction module as shown for the Grauzone protein (Jauch et al. 2003) and a ZAD protein called Serendipity-d (Payre et al. 1997; Ruez, Payre, and Vincent 1998). The zinc finger associated domains (ZAD, SCAN and KRAB) characterize protein families whose members independently proliferated in distinct lineages, a phenomenon referred to as lineage-specific expansion (LSE; Jordan et al. 2001; Lespinet et al. 2002). For example the genomes of mouse and humans encode a comparable number of KRAB domains (Huntley et al. 2006), but many of these KRAB domain-coding genes were independently generated by gene duplication in the lineages leading to either mouse or humans (e.g. Urrutia 2003; Huntley et al. 2006). It appears that KRAB-coding genes are prone to duplicate, suggesting that there is positive selection for these duplication events. However, despite their large numbers in mammals, only few KRAB-coding sequences have been found in other deuterostomes (Birtle and Ponting 2006 and referen- ces therein). Thus, the tendency to duplicate is not a unique feature of the genes per se but has to be viewed in the context of the lineage-specific organismal constraints and demands. Many aspects of the ZAD are very similar to the KRAB domain. Like the KRAB domain in mammals, it characterizes the largest subgroup of ZFPs in Drosophila melanogaster and has previously only been identified in arthropods (Chung et al. 2002). Here, we show that the ZAD was present in the last common ancestor of arthropods and vertebrates. Although present in vertebrates we were able to identify only a single gene encoding a ZAD-like peptide, suggesting that the ZAD-coding genes underwent LSE in the lineage leading to Drosophila melanogaster. Moreover, by comparing the ZAD sequences of six arthropod species, we show that ZAD-coding genes were subject to LSE especially in the higher holometabolous insects. Only few Drosophila melanogaster ZAD-coding genes with known and essential functions are conserved in evolution. This observation indicates that some or even the majority of the ZAD-coding genes exert lineagespecific or even species-dependent functions. We speculate that at least some of the lineage-specific ZAD-coding genes may be involved in processes that lead to developmental differences. This hypothesis is supported by our observation that many ZAD-coding genes of Drosophila melanogaster are expressed in the female germline, implying that they are involved in some aspects of oogenesis and/ or contribute to the maternal effect on early embryonic development. Consistent with this view, we find that the maternal effect gene pita, which plays a critical role during Drosophila melanogaster oogenesis (Laundrie et al. 2003), is conserved in all holometabolous insects examined. The Zinc Finger Associated Domain 1935 FIG. 1.—Multiple sequence alignment of the ZAD peptide sequence of vertebrate orthologs of ZFP276 and Drosophila melanogaster Grauzone. The ZAD peptide sequences were extracted from protein sequences deposited in GenBank with the accession numbers Q8N554 (Homo sapiens), Q8CE64 (Mus musculus), XP_414213 (Gallus gallus), AAI26057 (Xenopus laevis) and AAI33089 (Danio rerio). Asterisks mark residues present in all sequences; the four invariable cysteine residues are in red; conserved hydrophobic residues are marked with yellow boxes; black arrows indicate the bstrands found in ZADgrau; black cylinders mark the a-heli (...truncated)