The TET-BMP regulatory axis in pathogenesis of CFM [hMeDIPseq]

Craniofacial microsomia (CFM) is a congenital defect that usually results from aberrant development of embryonic pharyngeal arches. However, the molecular basis of CFM pathogenesis is largely unknown. Here we employ zebrafish model to investigate the mechanism of CFM pathogenesis. In early embryos, tet2 and tet3 are highly expressed and are essential for pharyngeal cartilage development. Single-cell RNA sequencing and genetic analyses reveal that loss of Tet2/3 impaired chondrocyte differentiation largely due to insufficient BMP signaling. Mechanistically, Tet2/3-mediated 5-hydroxymethylcytosine modification allows the 5-hydroxymethylcytosine “reader”, Sall4, to specifically bind the bmp4 promoter, thereby promoting bmp4 expression and enabling efficient BMP signaling. These findings indicate the TET-BMP regulatory axis via 5-hydroxymethylcytosine to be critical for pharyngeal cartilage development. Whole-exome sequencing of CFM patient samples show that single nucleotide polymorphisms in TET and BMP pathway genes increase the risk of CFM. Collectively, our study provides novel insights into understanding craniofacial development and CFM pathogenesis. DNA was purified from tissues according to the manufacturer’s instructions. 5hmC DNA immunoprecipitation (hMeDIP) was performed as previously described. Briefly, genomic DNA was digested with proteinase K at 55°C overnight and then with RNase A at 37°C for 1 h. Genomic DNA was purified by phenol/chloroform extraction and recovered by ethanol precipitation. Purified genomic DNA was sonicated to an average size of 200–400 bp and heat-denatured (95°C, 10 min). Then, 5 μg of fragmented genomic DNA was diluted in 500 μL DNA-IP buffer [10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100] and pre-cleared with 50 μL protein A/G beads at 4°C for 3 h. Five percent of the pre-cleared genomic DNA was saved as an input sample. The DNA-IP sample was incubated with 5 μg 5hmC antibody (Active Motif, 39769) at 4°C overnight and then with 50 μL protein A/G beads for 4 h at 4°C. Three washes for a total of 30 min were then performed to remove non-specifically bound antibodies. The beads were then treated with 200 μL TE buffer (pH 8.0) plus proteinase K for at least 3 h at 1000 rpm, 55°C in a thermomixer. The DNA was then purified by phenol/chloroform extraction followed by ethanol precipitation. Purified DNA was dissolved in ultrapure H2O, and libraries were constructed using a VAHTS Universal DNA Library Prep Kit for MGI (Vazyme) according to the manufacturer’s instructions. Libraries were then sequenced on the MGISEQ-2000 platform.