Hundreds of thousands of cis-regulatory DNA sequences are predicted in vertebrate genomes, but unlike genes themselves, few have been characterized at the functional level or even unambiguously paired with a target gene. Here we serendipitously identified and started investigating the first reported long-range regulatory region for the Nr2f1 (Coup-TFI) transcription factor gene. NR2F1 is temporally and spatially regulated during development and required for patterning and regionalization in the nervous system, including sensory hair cell organization in the auditory epithelium of the cochlea. Analyzing the deaf wanderer (dwnd) spontaneous mouse mutation, we traced back the cause of its associated circling behavior to a 53 kb deletion removing five exons and adjacent intronic regions of the poorly characterized Mctp1 gene. Interestingly, loss of Mctp1 function cannot account for the hearing loss, inner ear dysmorphology and sensory hair cell disorganization observed in dwnd mutants. Instead, we found that the Mctp1dwnd deletion affects the Nr2f1 gene located 1.4 Mb away, downregulating transcription and protein expression in the embryonic cochlea. Remarkably, the Mctp1dwnd allele failed to complement a targeted inactivation allele of Nr2f1, and transheterozygotes or Mctp1dwnd homozygotes exhibit the same morphological defects observed in inner ears of Nr2f1 mutants without sharing their early life lethality. Defects include improper separation of the utricle and saccule in the vestibule not described previously, which can explain the circling behavior that first brought the spontaneous mutation to attention. By contrast, mice homozygous for a targeted inactivation of Mctp1 have normal hearing and inner ear structures. We conclude that the 53 kb Mctp1dwnd deletion encompasses a long-range cis-regulatory region essential for proper Nr2f1 expression in the embryonic inner ear, providing a first opportunity to investigate Nr2f1 function in postnatal inner ears. This work adds to the short list of long-range regulatory regions characterized as essential to drive expression of key developmental control genes.
Keywords: Cis-regulation; Deafness; Inner ear development; Long-range enhancer; Mctp1; Nr2f1.
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