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The heterochronic LIN-14 protein is a BEN domain transcription factor

Sharrell Greene, Ji Huang, Keith Hamilton, Liang Tong, Oliver Hobert, and HaoSheng Sun

Heterochrony is a foundational concept in animal development and evolution, first introduced by Ernst Haeckel in 1875 and later popularized by Stephen J. Gould1. A molecular understanding of heterochrony was first established by genetic mutant analysis in the nematode C. elegans, revealing a genetic pathway that controls the proper timing of cellular patterning events executed during distinct postembryonic juvenile and adult stages2. This genetic pathway is composed of a complex temporal cascade of multiple regulatory factors, including the first-ever discovered miRNA, lin-4, and its target gene, lin-14, which encodes a nuclear, DNA-binding protein2,3,4. While all core members of the pathway have homologs based on primary sequences in other organisms, homologs for LIN-14 have never been identified by sequence homology. We report that the AlphaFold-predicted structure of the LIN-14 DNA binding domain is homologous to the BEN domain, found in a family of DNA binding proteins previously thought to have no nematode homologs5. We confirmed this prediction through targeted mutations of predicted DNA-contacting residues, which disrupt in vitro DNA binding and in vivo function. Our findings shed new light on potential mechanisms of LIN-14 function and suggest that BEN domain-containing proteins may have a conserved role in developmental timing.

Structural homology of LIN-14 DNA-binding domain to BEN domain.
(A) The motif logo of the LIN-14 DNA-binding domain is minimally similar to that of the BEN domain. The motif logos are generated using WebLogo 3. The alignment of 4,441 sequences from Pfam (PF10524) was used to generate the logo for BEN domains, while the alignment of 139 LIN-14 homologs across many nematode species (obtained from the WormBase ParaSite and aligned using ClustalOmega) was used to generate the logo for the LIN-14 domain. The dotted black boxes denote the five alpha-helices in the predicted LIN-14 structure (B,C), and the red dots denote the four arginine residues predicted to be important in DNA binding (C–E). (B) The predicted structure of the LIN-14 DNA-binding domain by AlphaFold (green, left, Uniprot Q21446) is homologous to the structures of BEN domains (Drosophila melanogaster insv, gray, middle, PDB 4IX7; Homo sapiens BEND3 BEN4, pink, right, PDB 7W27)5,8. See Supplemental Experimental Procedures in Supplemental information for how homologous structures were identified. (C) Predicted structure of the LIN-14 BEN domain (green) overlaid with the structure of Drosophila insv BEN domain (gray) bound to DNA (orange), showing the LIN-14 arginine residues (purple) that contact DNA that we mutate in panels D and E. (D) Wild-type LIN-14 proteins bind to the nlp-45 promoter sequence while two separate arginine (R) to alanine (A) mutant LIN-14 proteins do not, as demonstrated in the electrophoretic mobility shift assay. The black arrow indicates unbound 6-FAM labeled nlp-45 promoter sequence dsDNA probes. (E) Animals with mutation of arginine residues in LIN-14’s BEN domain, lin-14(syb5772), mimic lin-14(ma135) null mutant animals in adult morphology and the de-repression of nlp-45 expression in first larval stage animals. Both lin-14(ma135) and lin-14(syb5772) adult animals are dumpy, egg-laying defective, sterile, and display a protruding vulva (indicated by white arrows, upper panel). The bottom panels show de-repressed nlp-45 expression in the head of L1 lin-14(ma135) and lin-14(syb5772) animals compared to control animals. See Figure S1F for cell identification details. The red bars in the bottom right represent 10 μm.

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