Inter-chromosomal enhancer-promoter interaction critical for the appropriate level of Tead4 gene expression at the blastocyst stage [FAIRE-Seq]

Although Tead4 is well-known to be critical for blastocyst development and trophoblast lineage differentiation, the higher-order chromatin organization around this gene remains poorly understood. We assayed long-range chromosomal interactions on the Tead4 promoter in mouse embryonic stem (ES) cells and trophoblast stem (TS) cells using the circular chromosome conformation capture (4C)-seq technique. We observed frequent overlaps of genomic regions interacting with the Tead4 promoter and open chromatin regions identified by FAIRE-seq in TS cells. By systematic reporter assays using ES and TS cells, we identified five genomic fragments possessing a TS-specific enhancer activity to the Tead4 promoter, consisting of two intra- and three inter-chromosomal loci relative to the Tead4 gene on chromosome 6. We established five mouse lines deleted with one of the five enhancer elements using CRISPR/Cas9 genome editing, and evaluated the effect of each of the enhancer deletions on the Tead4 gene expression at the blastocyst stage. We observed 35-50% decrease of the Tead4 expression in the blastocysts with heterozygous or homozygous deletion of an inter-chromosomally located enhancer on chromosome 19 compared to that in wild-type blastocysts, whereas the other four deletions did not affect the Tead4 expression level. Our results demonstrate that inter-chromosomal enhancer-promoter interaction bears a critical role in assuring the appropriate level of Tead4 gene expression at the trophoblast lineage specification.