ESPE Abstracts

Background: CCCTC-binding factor (coded by CTCF gene, OMIM *604167), as a transcription insulation protein, plays a key role for regulating the temporal and spatial transcription of genes related to growth in mammals and topologically associated chromatin loop formation. Pathogenic variants in CTCF gene are associated with mental retardation, autosomal dominant 21 (MRD21, MIM #615502) with short stature, mild facial deformities, and mental retardation. Patients with MRD21 consistently presented with short stature but how CTCF variants lead to short stature has not been studied.

Methods: We knocked down the CTCF gene in chondrosarcoma cell line (SW1353) by shRNA, and RNAseq detected differences in transcript levels. Further, we constructed a mouse model of chondrocyte-specific Ctcf knockout (Col2a1 -Cre; Ctcf ^fl/+; Col2a1 -Cre; Ctcf ^fl/fl) by microinjection of fertilized eggs combined with CRISPR/Cas9 technology. Pathological changes in the tibial metaphysis of the mouse models were analyzed to investigate the role of the CTCF gene in growth plate cartilage development.

Results: In cellular models, CTCF knockdown SW1353 cells showed significant differences in extracellular matrix, cell differentiation, and bone morphogenetic protein (BMP) signaling pathways compared to wild type. Among them, the expression of cartilage extracellular matrix-related genes was decreased, and the expression levels of matrix metalloproteinase genes and BMPs antagonists were increased. In the animal model, Col2a1 -Cre; Ctcf ^fl/fl mice were born with severe short limb deformities and died one hour after birth due to respiratory distress. Pathology of the tibial metaphysis of Col2a1 -Cre; Ctcf ^fl/+ showed that haploinsufficiency of the Ctcf gene resulted in impaired chondrogenesis and mainly affected the proliferative and hypertrophic regions of cartilage, and that the impaired chondrogenesis of cartilage tissues in the metaphyses of the long bones was more and more pronounced as the weekly age of Col2a1 -Cre; Ctcf ^fl/+ mice increased.

Conclusion: We constructed the chondrocyte-specific Ctcf gene knockout mouse model. We found that haploinsufficiency of the Ctcf gene affected the expression of key genes for chondrogenesis, leading to impaired chondrogenesis and short limb deformities in mice.