A non-syndromic orofacial cleft risk locus links tRNA splicing defects to neural crest cell pathologies [4C-seq]
Orofacial clefts are the most common form of congenital craniofacial malformations worldwide. The etiology of these birth defects is multifactorial, involving genetic and environmental factors. In most cases, however, the underlying causes remain unexplained, precluding molecular understanding of disease mechanisms. Here, we integrated genome-wide association data, targeted re-sequencing of case and control cohorts, cell type-specific epigenomic profiling, and genome architecture analyses, to functionally and molecularly dissect a genomic locus associated with an increased risk of non-syndromic orofacial cleft. We found that common and rare risk variants associated with orofacial cleft intersect with a conserved enhancer (e2p24.2) that becomes activated in cranial neural crest cells—the embryonic cell type responsible for sculpting the craniofacial complex. We mapped e2p24.2 long-range interactions to a topologically associated domain harboring MYCN and DDX1 and demonstrated that both MYCN and DDX1 are required for craniofacial development in chicken embryos. Molecularly, we found that e2p24.2 regulates the expression of MYCN, but not DDX1, in cranial neural crest cells. In turn, DDX1 is a target of the MYC family of transcription factors and a component of the tRNA splicing complex. The loss of DDX1 in cranial neural crest cells resulted in the accumulation of unspliced tRNA fragments, and impaired both global protein synthesis and cranial neural crest cell migration. We further showed that the induction of tRNA fragments is sufficient to disrupt craniofacial development. Together, these results uncovered a molecular mechanism in which impaired tRNA splicing, and the concomitant accumulation of tRNA fragments, affect neural crest and craniofacial development and positioned MYCN, DDX1, and tRNA processing defects as risk factors in the pathogenesis of orofacial clefts. Overall design: Circular Chromatin Conformation Capture (4C) was performed as previously described (Laugsch et al., 2019), based on a published protocol (Stadhouders et al., 2013) with minor modifications. 2-3x107 neural crest cells or 25 frontonasal prominences of stage HH24 chicken embryos were crosslinked with 1% formaldehyde. NlaIII (NEB, R0125) was used as the first restriction enzyme, DpnII (NEB, R0543) as the second restriction enzyme. The resulting 4C DNA was amplified by inverse PCR with the expand long template PCR system (11681842001, Roche) using 32 amplification cycles and primers designed as previously described (Stadhouders et al., 2013) (see Supplemental Table 1) Illumina adaptors P5 and P7 were added to the primers close to the NlaIII and the DpnII restriction site, respectively, with barcodes for multiplex sequencing. 4C-seq libraries were sequenced on the HiSeq2500 platform, generating reads of either 74 or 100 bp in length. As described in Laugsch et al. (2019), 4C-seq reads were assigned to samples based on the first 10 bases of the read. Then, the primer sequences were removed from the reads and the remaining sequence was trimmed to 36 bp. These 36 bases were then aligned to the human (hg19) or the chicken (galGal4) reference genome using Bowtie (Langmead et al., 2009). Finally, the resulting reads were analyzed with R3C-seq  to generate RPM-normalized bedgraph files for downstream visualization and analysis (Thongjuea et al., 2013).