Ancient Lamprey Gene Offers Clues to Evolution of AFF Transcription Factors

Lampreys are among the most ancient living vertebrates, and their genomes preserve a molecular record of the distant evolutionary past. A new study published in Development Genes and Evolution has turned to these jawless fish to trace the history of the AF4/FMR2 family member (AFF) gene family, a group of transcriptional regulators with important roles in development and cancer biology. The research team, led by Difan Sun, Xinyu Du and Peng Su, identified for the first time an AFF3 homolog in the lamprey Lethenteron reissneri and showed that its conserved protein domains can promote cell proliferation in laboratory experiments.

The AFF family consists of four members in vertebrates, AFF1 through AFF4, all of which act as components of the transcriptional super elongation complex that regulates gene expression. Every AFF protein carries three conserved regions: an N-terminal homology domain (NHD), an AF4/lymphoid nuclear protein domain, and a C-terminal homology domain (CHD). The NHD controls transcription by binding other subunits of the elongation complex, the AF4/lymphoid nuclear protein domain regulates proteasome-mediated protein degradation, and the CHD participates in intranuclear localization and binds G-quadruplex RNA structures.

The motivation for the study came from earlier transcriptome analyses of lamprey spinal cord injury. Unlike mammals, lampreys spontaneously regenerate and recover spinal cord function after injury, and complex transcriptional responses accompany this repair. From that dataset the researchers identified a hub gene they named Lr-AFF3. In the current work, they cloned its full-length open reading frame, a 5,235-base-pair sequence encoding a protein of 1,744 amino acids, and deposited it in GenBank under accession number PP350802.

To place Lr-AFF3 in evolutionary context, the team constructed a neighbor-joining phylogenetic tree from 61 complete AFF protein sequences across species. The tree resolved the family into four groups corresponding to AFF1, AFF2, AFF3 and AFF4. Strikingly, lamprey contained only one AFF homolog, and it fell within the AFF3 group. The authors suggest that AFF3 represents the most ancient member of the vertebrate AFF family and speculate that Lr-AFF3 may resemble the common ancestor of all four vertebrate AFF subfamilies, which likely diversified through the two rounds of whole-genome duplication early in the vertebrate lineage.

Analyses of conserved motifs and protein domains reinforced this picture. Lr-AFF3 possesses both the N-terminal and C-terminal homology domains, and specific motifs appear to underpin the formation of each domain. The gene structure of AFF3, however, was not conserved across species: the number of exons and introns in Lr-AFF3 differs markedly from that of other vertebrates, with exon numbers increasing notably at the mammalian stage, possibly reflecting intron insertion and exon reassembly during evolution. In contrast, the structures of AFF1, AFF2 and AFF4 remained relatively stable among higher vertebrates.

Synteny analysis, which compares the neighboring genes flanking AFF genes across genomes, added further evidence. Lr-AFF3 sits in a conserved linkage group with the REV1 and EIF5B genes, which are also found near AFF3 in higher vertebrates, confirming its identity as an AFF3 ortholog. Yet the neighboring genes of Lr-AFF3 showed higher diversity than those of jawed vertebrates, a pattern the authors attribute to chromosomal rearrangements or homologous recombination during vertebrate evolution. The analysis also revealed two AFF3 genes lying adjacent to each other on the crocodile chromosome, likely the result of local tandem duplication.

To probe the protein’s structure, the researchers used SWISS-MODEL to build a three-dimensional model of the Lr-AFF3 CHD, with the human AFF4 CHD as a template because no complete AFF3 crystal structure was available. The two models shared a highly homologous protein fold, with a loop region and eight alpha helices. The comparison also identified two clusters of conserved active sites: one potentially involved in forming a heterodimer with the CHD of AFF1, and another that could be phosphorylated by P-TEFb, suggesting Lr-AFF3 may have functional activity similar to that of its human counterpart.

The functional experiments centered on whether Lr-AFF3 influences cell proliferation. Working in HEK293T cells, the team expressed the full-length protein as well as its NHD and CHD separately, using FGF2 as a positive control and an empty pcDNA3.1 vector as a negative control. In MTT assays measuring cell viability over 0 to 72 hours, cells expressing the NHD or CHD showed significantly increased viability compared with the empty-vector group. EdU staining, which labels cells undergoing DNA synthesis, likewise showed significantly improved proliferative capacity in the NHD and CHD groups.

Taken together, these results indicate that the NHD and CHD of Lr-AFF3 promote cell proliferation, and that the full-length protein likely plays a promoting role as well. The authors note that previous studies have linked AFF4 to the proliferation of melanoma and pancreatic ductal carcinoma cells, and that AFF proteins participate in key signaling pathways including Wnt/beta-catenin, PPARG, Rap1, Hippo and PI3K-Akt. Their findings suggest that the proliferation-related function of AFF proteins may be deeply rooted in vertebrate evolution, predating the split between jawless and jawed vertebrates.

The study carries some important limitations. The proliferation assays were performed in a human embryonic kidney cell line rather than in lamprey cells or tissues, so the findings demonstrate what Lr-AFF3 domains can do in an experimental cell culture setting, not how the gene functions during lamprey spinal cord regeneration in living animals. The 3D structure was a homology model rather than an experimentally determined structure, and the evolutionary interpretations rest on computational analyses of available genome data. Even so, by identifying AFF3 in lamprey for the first time and demonstrating conserved domains that drive cell proliferation, the work offers new insight into the origin and evolution of the AFF gene family and opens avenues for exploring how these ancient transcriptional regulators contribute to regeneration in one of nature’s most remarkable healers.

Subject of Research: Molecular evolution of the AFF transcription factor gene family and the role of lamprey AFF3 in cell proliferation

Article Title: Molecular evolution of transcription factors AF4/FMR2 family member (AFF) gene family and the role of lamprey AFF3 in cell proliferation

Article References: Sun, D., Du, X., & Su, P. (2024). Molecular evolution of transcription factors AF4/FMR2 family member (AFF) gene family and the role of lamprey AFF3 in cell proliferation. Development Genes and Evolution, 234(1), 45-53. https://doi.org/10.1007/s00427-024-00717-1

Image Credits: AI Generated

DOI: 10.1007/s00427-024-00717-1

Keywords: lamprey, AFF gene family, transcription factors, phylogenetics, Lr-AFF3, cell proliferation, MTT assay, EdU assay, synteny analysis, evolution, spinal cord injury, Development Genes and Evolution