Fig. 9

CHD1L function is mediated through its macrodomain and is implicated in neurodevelopmental disorders. (A) Sashimi plot of CHD1L mRNA transcripts expressed in hNPC showing two main isoforms corresponding to CHD1L-202 (ENST00000369258.8) and CHD1L-203 (ENST00000369259.4). (B) Schematic representation of the human protein isoforms of CHD1L including CHD1L-FL (CHD1L-202), CHD1L-ΔLobe1 (CHD1L-203), and two truncated forms of CHD1L (CHD1L-FL-ΔMacro and CHD1L-ΔLobe1-ΔMacro). The homozygous variant p.Arg392His is shown on the protein. (C) Dot plot showing the distance between the eyes (head size) of 5 dpf control larvae and larvae injected with mRNA for each of the two isoforms and the two truncated forms of CHD1L. Data shown as mean ± SEM of triplicate batches; Kruskal–Wallis test performed between chd1l−/- alone versus all the other conditions. (D) Dot plot showing the body length of 5 dpf control larvae and larvae injected with mRNA for each of the two isoforms and the two truncated forms of CHD1L. Data shown as mean ± SEM of triplicate batches; ordinary one-way ANOVA performed between chd1l−/- alone versus all the other conditions to validate phenotypic rescue. (E) Dot plot showing the distance between the eyes (head size) of 5 dpf control larvae or larvae injected with human CHD1L-FL mRNA either WT or carrying the mutation p.Arg392His. Data shown as mean ± SEM of triplicate batches; ordinary one-way ANOVA. (F) Dot plot showing the distance between the eyes (head size) of 5 dpf control, noninjected chd1l mutant larvae and chd1l mutant larvae injected with the human CHD1L-FL mRNA either WT or carrying the mutation p.Arg392His. Data shown as mean ± SEM of triplicate batches; ordinary one-way ANOVA. (G) Ribbon representation of the structure of the self-inhibited form of CHD1L (PDB entry 7epu) in full (left panel) and with a closeup on the region harboring Arg392 residue (boxed right panel). Arg392 structures a specific region of CHD1L ATPase lobe 2 (orange) by interacting with main chain carbonyls and aspartate side chains. This region is key in interacting with the CHD1L macrodomain (blue) in the inhibited form. (H) Total protein expression levels in E. coli of the three constructs used in this study. No significant changes are observed between the WT and p.Arg392His constructs. (I) Levels of the soluble CHD1L constructs from panel (H). The p.Arg392His mutation causes a major decrease of the solubility of CHD1L constructs, irrespective of the construct considered.