Impaired protein hydroxylase activity causes replication stress and developmental abnormalities in humans
Sally C. Fletcher, Charlotte Hall, Tristan J. Kennedy, Sander Pajusalu, Monica H. Wojcik, Uncaar Boora, Chan Li, Kaisa Teele Oja, Eline Hendrix, Christian A.E. Westrip, Regina Andrijes, Sonia K. Piasecka, Mansi Singh, Mohammed E. El-Asrag, Anetta Ptasinska, Vallo Tillmann, Martin R. Higgs, Deanna A. Carere, Andrew D. Beggs, John Pappas, Rachel Rabin, Stephen J. Smerdon, Grant S. Stewart, Katrin Õunap, Mathew L. Coleman
Sally C. Fletcher, Charlotte Hall, Tristan J. Kennedy, Sander Pajusalu, Monica H. Wojcik, Uncaar Boora, Chan Li, Kaisa Teele Oja, Eline Hendrix, Christian A.E. Westrip, Regina Andrijes, Sonia K. Piasecka, Mansi Singh, Mohammed E. El-Asrag, Anetta Ptasinska, Vallo Tillmann, Martin R. Higgs, Deanna A. Carere, Andrew D. Beggs, John Pappas, Rachel Rabin, Stephen J. Smerdon, Grant S. Stewart, Katrin Õunap, Mathew L. Coleman
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Research Article Cell biology Genetics

Impaired protein hydroxylase activity causes replication stress and developmental abnormalities in humans

Abstract

Although protein hydroxylation is a relatively poorly characterized posttranslational modification, it has received significant recent attention following seminal work uncovering its role in oxygen sensing and hypoxia biology. Although the fundamental importance of protein hydroxylases in biology is becoming clear, the biochemical targets and cellular functions often remain enigmatic. JMJD5 is a “JmjC-only” protein hydroxylase that is essential for murine embryonic development and viability. However, no germline variants in JmjC-only hydroxylases, including JMJD5, have yet been described that are associated with any human pathology. Here we demonstrate that biallelic germline JMJD5 pathogenic variants are deleterious to JMJD5 mRNA splicing, protein stability, and hydroxylase activity, resulting in a human developmental disorder characterized by severe failure to thrive, intellectual disability, and facial dysmorphism. We show that the underlying cellular phenotype is associated with increased DNA replication stress and that this is critically dependent on the protein hydroxylase activity of JMJD5. This work contributes to our growing understanding of the role and importance of protein hydroxylases in human development and disease.

Authors

Sally C. Fletcher, Charlotte Hall, Tristan J. Kennedy, Sander Pajusalu, Monica H. Wojcik, Uncaar Boora, Chan Li, Kaisa Teele Oja, Eline Hendrix, Christian A.E. Westrip, Regina Andrijes, Sonia K. Piasecka, Mansi Singh, Mohammed E. El-Asrag, Anetta Ptasinska, Vallo Tillmann, Martin R. Higgs, Deanna A. Carere, Andrew D. Beggs, John Pappas, Rachel Rabin, Stephen J. Smerdon, Grant S. Stewart, Katrin Õunap, Mathew L. Coleman

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Figure 6

Decreased replication fidelity and viability of affected patient cells are due to loss of JMJD5 hydroxylase activity.

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Decreased replication fidelity and viability of affected patient cells a...
Reconstitution of Sib4In/CY fibroblasts with JMJD5. Sib4In/CY cells were transduced with lentivirus encoding doxycycline-inducible WT or inactive (H321A) JMJD5 cDNA or a control empty vector (EV). Sib1WT/WT cells were transduced with EV as a control. (A) Cells were treated with doxycycline for 14 days before Western blotting and immunofluorescence (Supplemental Figure 22A). “Low” and “High” refer to the exposure time. Note the leaky physiological re-expression of exogenous JMJD5 in the absence of doxycycline. (B–E) Because of modest heterogeneity in re-expression levels between cells (Supplemental Figure 22A), and slightly reduced expression of H321A compared with WT JMJD5, all subsequent experiments were conducted at 0 and 10 ng/mL doxycycline. Increased 53BP1 bodies (B), stalled replication forks (C), and replication fork asymmetry (D) in Sib4In/CY cells are suppressed by JMJD5 hydroxylase activity. (E) Decreased colony survival of Sib4In/CY cells is rescued by JMJD5 hydroxylase activity. Reconstituted Sib4In/CY cells were plated at limited density and monitored for colony formation. Left: Example of stained colonies. Right: Quantification of colonies expressed as percentage survival relative to Sib4In/CY EV control cells. In B, a minimum of 300 cells were counted per condition. In C, at least 200 forks were counted per sample. In D, at least 50 structures per condition were measured in each experiment. (B) Data represent mean ± SEM from 4 independent experiments. (C–E) Data represent mean ± SEM from 3 independent experiments. Statistical analyses used 1-way ANOVA with Tukey’s post hoc test (B, C, and E) or Kruskal-Wallis with Dunn’s correction (D); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.