The Drosophila toothrin Gene Related to the d4 Family Genes: An Evolutionary View on Origin and Function

<i>D. melanogaster</i> has two paralogs, <i>tth</i> and <i>dd4</i>, related to the evolutionarily conserved <i>d4</i> family genes. In mammals, the family consists of <i>Dpf1-3</i>, encoding transcription co-factors involved in the regulation of development and cell fate determination. The function of <i>tth</i> and <i>dd4</i> in <i>Drosophila</i> remains unclear. The typical domain structure of the proteins encoded by the <i>d4</i> family consists of an N-terminal 2/3 domain (Requiem_N), a central Kruppel-type zinc finger, and a C-terminal D4 domain of paired PHD zinc fingers (DPFs). In <i>Drosophila</i>, both paralogs lack the Kruppel-type ZF, and <i>tth</i> encodes a protein that contains only Requiem_N. In contrast, vertebrate <i>Dpf1-3</i> paralogs encode all the domains, but some paralogs have specific splice isoforms. For example, the DPF3a isoform lacks the D4 domain necessary for histone reading. The occurrence of proteins without the D4 domain in mammals and flies implies functional significance and analogous roles across animal taxa. In this study, we reconstructed the evolutionary events that led to the emergence of <i>Drosophila tth</i> by analyzing the divergence of <i>d4</i> paralogs across different evolutionary lineages. Our genomic and transcriptomic data analysis revealed duplications and gene copy loss events. Among insects, gene duplication was only observed in Diptera. In other lineages, we found the specialization of paralogs for producing isoforms and further specialization for coding proteins with specific domain organizations. We hypothesize that this pathway is a common mechanism for the emergence of paralogues lacking the D4 domain across different evolutionary lineages. We, thus, postulate that TTH may function as a splice isoform of the ancestral single-copy gene, possibly a DPF3a-like isoform characteristic of related insect species. Our analysis provides insights into the possible impact of paralogue divergence, emphasizing the functional significance of the 2/3 domain and the potential roles of isoforms lacking the D4 domain.