Fig 3

(A) Genetic map of the baldrian/Hdac1 gene locus. The bal mutation interval is flanked by the microsatellite markers z11872, z65805 and z3770. (B, C) Sequencing revealed the bal mutation to be a thymidine-to-guanine nucleotide transition in codon 125 within the zebrafish hdac1 gene. (D, D’) Homology model for Danio rerio Hdac1 (drHdac1), "type":"entrez-protein","attrs":{"text":"AAH85375","term_id":"55716026","term_text":"AAH85375"}}AAH85375, built from human HDAC1 structure 4BKX. wt and bal mutant structure of drHdac1, highlighting the active site zinc ion (cyan sphere) and L125. (D) Zoomed view of L125, showing the nonpolar interactions it makes with neighboring side chains within the hydrophobic core of drHDAC1. (D’) Modelled L125R mutant Hdac1. Steric clashes are represented as red disks. Modeled Arg rotamer was chosen based on the least number of steric clashes. (E-F”) IF staining on dissected hearts at 72 hpf, Hdac1 (turquoise) of Tg(myl7:mCherry) hearts (E-E”) or bal;Tg(myl7:mCherry) mutant hearts (F-F”) white arrows show Hdac1 costained cardiomyocytes of wt ventricle or atrium. (G) Western blot analysis of Hdac1 levels on protein lysates from bal and wt, demonstrating a drastic reduction in Hdac1 protein level in the bal mutants. (H) Western blot analysis of acetylation levels of Histone 3 (H3ac) and 4 (H4ac) of bal and wt protein lysates show a pronounced hyperacetylation of H3 and H4 in bal mutant embryos. pan Histone 3 served as loading control.