Figure 1
From: Loss of PHF6 leads to aberrant development of human neuron-like cells
Overexpression analysis of variants in PHF6 from females vs males. (a) Protein scheme of PHF6 (Q8IWS0) with all missense variants previously published in male and female individuals2,4,6,21,23,24 as well as three selected truncating variants2,4 analysed via immunofluorescence coloured in red. Analysed variants (immunofluorescence) are printed in bold. Coding domains (according to uniprot91) are displayed in colour. NLS nuclear localization ___domain, NoLS nucleolar localization ___domain. (b) Table including all published missense variants occurring in male and female individuals, their VIPUR score, their localization in regard to ePHD and their origin (inherited/de novo). Variants p.(Met1Tyr) and p.(Ser138Cys) do not reside in a ___domain and cannot be modelled. Analysed variants (immunofluorescence) are printed in bold. ePHD extended atypical PHD-like zinc finger ___domain, 1 = N-terminal, 2 = C-terminal. (c) Immunofluorescence of HEK-293 cells transiently transfected with HA-tagged wildtype or mutant PHF6 co-stained with nucleolin (NCL). While wildtype PHF6 was expressed in the nucleoplasm as well as the nucleolus co-localizing with NCL, all three variants occurring in females led to an accumulation of PHF6 into aggregates, partly co-localizing with NCL. The truncating variant c.677delG (p.(Gly226Glufs*53)) resulted in small and more aggregates in the nucleus, and the two variants in the C-terminal ePHD resulted in large PHF6 aggregates while the expression of aberrant PHF6 in the nucleoplasm is reduced. In contrast, the three variants occurring in males resembled the wildtype with PHF6 expression in both the nucleoplasm and the nucleolus, co-localizing with NCL. Per variant, at least 40 cells were analyzed. Scale bar depicts 20 µm. Cells were stained with rabbit anti-HA (H6908, Sigma-Aldrich, 1:75) and mouse anti-nucleolin (39–6400, Thermo Fisher Scientific, 1:200). (d) Quantification of nuclear aggregates. The number of cells with or without aberrant large or small PHF6 aggregates in the nucleus was quantified for each variant and the wildtype. At least 40 cells were evaluated for each variant. ♀, variant identified in a female individual; ♂, variant identified in a male individual.
