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 5

Biallelic JMJD5 pathogenic variants are associated with impaired DNA replication fidelity and reduced colony survival.

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Biallelic JMJD5 pathogenic variants are associated with impaired DNA rep...
(A) DNA fiber assays were performed by incubation of cells with thymidine analog CldU followed by IdU, with detection by immunofluorescence (top). Examples of different labeling outcomes are shown (bottom). (B) Stalled replication forks were significantly increased in Sib4In/CY immortalized fibroblasts. The prevalence of stalled forks was calculated as a percentage of total fork structures counted. A minimum of 200 forks were counted per condition in each experiment. (C) Asymmetric replication forks were significantly increased in Sib4In/CY immortalized fibroblasts. Fork symmetry was determined by measurement of the ratio between the two IdU tracts found in first-label origin structures. Symmetric forks should approach a ratio of about 1.0. An increase indicates fork asymmetry. At least 50 structures were measured per sample in each experiment. (D) The total length of ongoing DNA fiber fork structures was measured and converted to replication fork speed. No significant difference in replication fork speed was observed. A minimum of 200 forks were analyzed per sample. (E) Fibroblasts were plated at limited density and monitored for colony formation. Sib4In/CY fibroblasts had reduced colony formation. (B–D) Data represent mean ± SEM from 4 independent experiments. (E) Data represent mean ± SEM from 3 independent experiments. Statistical analyses used 1-way ANOVA with Tukey’s post hoc test (B and E) or Kruskal-Wallis with Dunn’s correction (C and D); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.