Fig 2: p53 downregulation rescues neurodevelopmental defects in XLID JMS patient hiPSCs(A) Relative number of rosettes upon neural differentiation of WT-DYS0100, JMS-cl.1, and JMS-cl.1-expressing shRNA control (shCtrl) or shRNA targeting p53 (shP53a and shP53b) hiPSCs (n = 3, biological replicates). JMS cells harbor HUWE1 p.G4310R.(B–D) mRNA expression levels of NES/NESTIN (B), TUBB3/TUJ1 (C), and DCX (D) analyzed by qRT-PCR in WT and JMS neural cells (collected at day 13) addressed by qRT-PCR.(E) Immunofluorescence analysis of the TUJ1 signal in WT-DYS0100, JMS-cl.1, JMS-cl.1 shCtrl, and JMS-cl.1 shP53a and shP53b at day 13 of neural differentiation.(F–H) qRT-PCR analysis of CDKN1A/p21 (F), GADD45a (G), and BAX (H) expression in WT and JMS neural cells (collected at day 13).All error bars indicate mean ± SEM (n = 3, biological replicates); one-way ANOVA followed by Dunnett’s post-test (A); one-tailed t test (B–D and F–H); *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001, n.s. = 0.05. See also Figures S4 and S5.

Fig 3: A model of HUWE1-mediated cytosolic PQC. On translation, proteins are assembled into multi-subunit protein complexes. The assembly process usually shields hydrophobic surfaces that would otherwise be exposed on unassembled subunit. Although assembled protein complexes can be transported to their final destination (for example, nucleus), unassembled protein subunits bearing an exposed hydrophobic segment (marked by the star) is subject to HUWE1-mediated ubiquitination, which targets them for degradation by the proteasome in the cytosol.

Fig 4: UV-induced H1 ubiquitylation is mediated by HUWE1 and stimulates the RNF8-RNF168 pathway. Model of HUWE1 functioning in the DNA damage-induced ubiquitin pathway. HUWE1 ubiquitylates histone H1, with a mono-ubiquitin or short poly-ubiquitin chain, in response to DNA damage, thereby providing additional substrates for RNF8-mediated K63-linked poly-ubiquitylation. These K63-chains are recognized and bound by RNF168 which ubiquitylates histone H2A at lysine K13/15, stimulating the recruitment of downstream factors like 53BP1. P = phosphorylation of H2AX, Ub = ubiquitylation.

Fig 5: Oxidative stress response is impaired in HUWE1 p.R4187C XLID individual cells. (a) Efficiency and accuracy of dATP versus dCTP incorporation opposite frequent oxidative DNA lesion 7,8-dihydro-8-oxo-guanine (Goxo), using increasing amounts of healthy and XLID individual (HUWE1-R4187C) whole cell extracts (WCEs) in an in vitro DNA polymerase assay. (b) Quantification of (a) (n = 5). Error bars indicate mean ± SEM. The significance of changes between healthy and XLID individual WCEs was determined by unpaired t-test, one-tailed p-value; *p < 0.05–1 µM dATP t(7) = 2.29 p = 0.0277; 5 µM dATP t(8) = 2.11 p = 0.034; 1 µM dCTP t(7) = 2.4 p = 0.0238. (c) Cell viability of healthy and XLID individual (HUWE1-R4187C) cells after 24 hours of continuous treatment with increasing concentrations of potassium bromate (KBrO3) (n = 3). The significance of changes in the survival upon the treatment was determined by unpaired t-test, two-tailed p-value; n.s ≥ 0.05, *p < 0.05 and **p < 0.01. (d) Single-cell gel electrophoresis (Comet) analysis of DNA repair kinetics upon H2O2-treatment in healthy and XLID individual cells, represented as the relative percentage of DNA in the tail. Mean of 3 independent experiments presented. In each experiment at least 100 cells were analyzed per each time point and each cell type. Error bars indicate mean ± SEM.