Precocious chondrocyte differentiation disrupts skeletal growth in Kabuki syndrome mice
Jill A. Fahrner, Wan-Ying Lin, Ryan C. Riddle, Leandros Boukas, Valerie B. DeLeon, Sheetal Chopra, Susan E. Lad, Teresa Romeo Luperchio, Kasper D. Hansen, Hans T. Bjornsson
Jill A. Fahrner, Wan-Ying Lin, Ryan C. Riddle, Leandros Boukas, Valerie B. DeLeon, Sheetal Chopra, Susan E. Lad, Teresa Romeo Luperchio, Kasper D. Hansen, Hans T. Bjornsson
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Research Article Genetics

Precocious chondrocyte differentiation disrupts skeletal growth in Kabuki syndrome mice

Abstract

Kabuki syndrome 1 (KS1) is a Mendelian disorder of the epigenetic machinery caused by mutations in the gene encoding KMT2D, which methylates lysine 4 on histone H3 (H3K4). KS1 is characterized by intellectual disability, postnatal growth retardation, and distinct craniofacial dysmorphisms. A mouse model (Kmt2d+/βGeo) exhibits features of the human disorder and has provided insight into other phenotypes; however, the mechanistic basis of skeletal abnormalities and growth retardation remains elusive. Using high-resolution micro-CT, we show that Kmt2d+/βGeo mice have shortened long bones and ventral bowing of skulls. In vivo expansion of growth plates within skulls and long bones suggests disrupted endochondral ossification as a common disease mechanism. Stable chondrocyte cell lines harboring inactivating mutations in Kmt2d exhibit precocious differentiation, further supporting this mechanism. A known inducer of chondrogenesis, SOX9, and its targets show markedly increased expression in Kmt2d–/– chondrocytes. By transcriptome profiling, we identify Shox2 as a putative KMT2D target. We propose that decreased KMT2D-mediated H3K4me3 at Shox2 releases Sox9 inhibition and thereby leads to enhanced chondrogenesis, providing a potentially novel and plausible explanation for precocious chondrocyte differentiation. Our findings provide insight into the pathogenesis of growth retardation in KS1 and suggest therapeutic approaches for this and related disorders.

Authors

Jill A. Fahrner, Wan-Ying Lin, Ryan C. Riddle, Leandros Boukas, Valerie B. DeLeon, Sheetal Chopra, Susan E. Lad, Teresa Romeo Luperchio, Kasper D. Hansen, Hans T. Bjornsson

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Figure 5

Genome-wide transcriptome profiling identifies Shox2 as a target of KMT2D in vitro and in vivo.

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Genome-wide transcriptome profiling identifies Shox2 as a target of KMT2...
Volcano plots showing genome-wide differential gene expression in Kmt2d–/– and Kmt2d+/+ (A) chondrocytes differentiated for 7 days and (B) undifferentiated cells. (C) PCA reveals tight clustering within and distinct separation between genotypes and differentiation states, separating the cells into 4 distinct groups. Kmt2d+/+ cells cluster toward the upper end of PC2 and Kmt2d–/– cells cluster toward the lower end of PC2. Undifferentiated cells cluster toward the lower end of PC1 and chondrocytes cluster toward the upper end of PC1. Each color represents a biological replicate; each biological replicate is a distinct clonal cell line. Three (Kmt2d+/+) or 2 (Kmt2d–/–) biological replicates were used for each differentiation state (chondrocytes vs. undifferentiated cells). Each point represents a technical replicate; 2 technical replicates were performed for each cell line. Sox9 and Col2a1 are upregulated and Shox2 is downregulated in (A) Kmt2d–/– chondrocytes and (B) Kmt2d–/– undifferentiated cells. (D) Venn diagrams summarizing differential gene expression from RNA-seq analyses in Kmt2d+/+ and Kmt2d–/– chondrocytes and undifferentiated cells. (E) Validation of downregulation of Shox2 in 3 independent experiments in vitro; mean ± SEM; n = 9 (Kmt2d+/+) or n = 6 (Kmt2d–/–) per group (undifferentiated cells or chondrocytes); 1-sided unpaired Student’s t test; ***P < 0.001; ****P < 0.0001. (F) Immunofluorescence and (G) quantification of SHOX2 protein levels in vivo in Kmt2d+/βGeo and Kmt2d+/+ proximal tibia growth plates (outlined in yellow dashed lines). SHOX2 intensity was normalized to number of DAPI-stained cells within the growth plate. Data represent mean ± SD; n = 5 male Kmt2d+/+, n = 5 male Kmt2d+/βGeo, n = 3 female Kmt2d+/+, n = 3 female Kmt2d+/βGeo; 1-sided unpaired Student’s t test; **P < 0.01. Scale bar: 400 μM. Chondro, chondrocytes differentiated for 7 days; Undiff, undifferentiated cells.