Loss of DDRGK1 modulates SOX9 ubiquitination in spondyloepimetaphyseal dysplasia
Adetutu T. Egunsola, … , Mordechai Shohat, Brendan H. Lee
Adetutu T. Egunsola, … , Mordechai Shohat, Brendan H. Lee
Published March 6, 2017
Citation Information: J Clin Invest. 2017;127(4):1475-1484. https://doi.org/10.1172/JCI90193.
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Research Article Bone biology Genetics

Loss of DDRGK1 modulates SOX9 ubiquitination in spondyloepimetaphyseal dysplasia

Abstract

Shohat-type spondyloepimetaphyseal dysplasia (SEMD) is a skeletal dysplasia that affects cartilage development. Similar skeletal disorders, such as spondyloepiphyseal dysplasias, are linked to mutations in type II collagen (COL2A1), but the causative gene in SEMD is not known. Here, we have performed whole-exome sequencing to identify a recurrent homozygous c.408+1G>A donor splice site loss-of-function mutation in DDRGK domain containing 1 (DDRGK1) in 4 families affected by SEMD. In zebrafish, ddrgk1 deficiency disrupted craniofacial cartilage development and led to decreased levels of the chondrogenic master transcription factor sox9 and its downstream target, col2a1. Overexpression of sox9 rescued the zebrafish chondrogenic and craniofacial phenotype generated by ddrgk1 knockdown, thus identifying DDRGK1 as a regulator of SOX9. Consistent with these results, Ddrgk1–/– mice displayed delayed limb bud chondrogenic condensation, decreased SOX9 protein expression and Col2a1 transcript levels, and increased apoptosis. Furthermore, we determined that DDRGK1 can directly bind to SOX9 to inhibit its ubiquitination and proteasomal degradation. Taken together, these data indicate that loss of DDRGK1 decreases SOX9 expression and causes a human skeletal dysplasia, identifying a mechanism that regulates chondrogenesis via modulation of SOX9 ubiquitination.

Authors

Adetutu T. Egunsola, Yangjin Bae, Ming-Ming Jiang, David S. Liu, Yuqing Chen-Evenson, Terry Bertin, Shan Chen, James T. Lu, Lisette Nevarez, Nurit Magal, Annick Raas-Rothschild, Eric C. Swindell, Daniel H. Cohn, Richard A. Gibbs, Philippe M. Campeau, Mordechai Shohat, Brendan H. Lee

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Figure 4

Ddrgk1 deficiency decreases SOX9 protein and Col2a1 mRNA expression.

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Ddrgk1 deficiency decreases SOX9 protein and Col2a1 mRNA expression. (A...
(A and B) ATDC5 cells were transiently transfected with control or Ddrgk1 siRNA and treated with either DMSO or ITS to induce differentiation 24 hours later. The cells were harvested 7 days after treatment. (A) RT-PCR of total RNA from ATDC5 cells treated with control siRNA + DMSO (n = 3), Ddrgk1 siRNA + DMSO (n = 3), control siRNA + ITS (n = 3), and Ddrgk1 siRNA + ITS (n = 3). Values are represented as mean ± SEM. **P < 0.01, 2-way ANOVA followed by Tukey’s post-hoc test. (B) Immunoblots of total cell lysates from ATDC5 cells. The immunoblots are representative of 3 independent experiments. (C) ddrgk1 morphants have less mRNA expression of col2a1, but not of sox9, than control morphants. Zebrafish embryos were injected with 5 pg control MO (n = 3) or 5 pg ddrgk1 MO (n = 3), and total RNA was collected for RT-PCR 72 h.p.f. later. Values are represented as mean ± SEM. **P < 0.01; ***P < 0.001, 2-tailed t test. (D and E) Deletion of Ddrgk1 decreases transcript levels of Col2a1 via SOX9 protein reduction in E11.5 limb buds. (D) RT-PCR of total RNA from E11.5 WT (n = 3) and Ddrgk1–/– (n = 3) limb buds. Values are represented as mean ± SEM. *P < 0.05, 2-tailed t test. (E) Immunoblots of total cell lysates from E11.5 WT (n = 3) and Ddrgk1–/– (n = 3) limb buds.