Fig. 4: Mutational analysis of HIM-8 residues responsible for base-specific binding in vitro and in vivo.

a Single-point mutations of base-specific residues on ZF1 are shown to reduce the binding affinities to the 12-bp X chromosome PC DNA sequence, as quantified by ITC. b Single-point mutations of base-specific residues on ZF2 lead to reduced binding affinities to the 12-bp X chromosome PC DNA sequence, as assessed by ITC. c A schematic diagram of him-8 gene and HIM-8 protein. There are two variants of the him-8 gene denoted by numbers T07G12.12a and T07G12.12b. Exons are represented by black boxes on the line. Single mutations were labeled on the HIM-8 protein. d Observation within the pachytene nucleus reveals typically one HIM-8 focus in HIM-8_WT::GFP::3 × FLAG worms. Conversely, an increase in unpaired foci is observed in HIM-8_R289A::GFP::3 × FLAG mutant worms, while the foci in HIM-8_Y293A::GFP::3 × FLAG mutant nematodes appear diffused. Three times were repeated independently with similar results. e Quantification of the ratio of worm pachytene germline cells exhibiting either one or two HIM-8 foci. The variable “n” denotes the number of cells analyzed. f The brood size of the indicated animals at 20 °C is depicted. Data are presented as Mean ± SD. A two-sided t-test was performed. Statistical significance was denoted by ***p < 0.001, **p < 0.01, *p < 0.05. WT vs N2, p = 0.0770; him-8(−/−) vs N2, p = 0.0010; R289A vs N2, p = 0.7189; Y293A vs N2, p = 0.7741. The “n” represents the number of P0 generation animals, whose self-progeny were tested. g The male ratio of self-progeny produced by wild-type N2, him-8(−/−), HIM-8_WT::GFP::3 × FLAG, HIM-8_R289A::GFP::3 × FLAG and HIM-8_Y293A::GFP::3 × FLAG hermaphrodites serves as a measure of X chromosome nondisjunction, with “n” representing the number of animals counted. Source data are provided as a Source Data file.