Investigation Of The Function Of Fox12 In Mouse Facial And Ovarian Development And Identification Of Its Long-range Regulatory Elements

Blepharophimosis, ptosis, epicanthus inversus syndrome (BPES) is an autosomal genetic disorder in human. The BPES patients are characterized by their eyelid malformations associated with (type I) or without (type II) premature ovarian failure (POF). The winged-helix/forkhead transcription factor FOXL2 is mutated in BPES. The mutations of FOXL2 include intragenic mutations and intergenic genomic rearrangements such as chromosomal translocations and genomic deletions, which indicate the existence of long-range regulatory elements.We identified a mouse line B61aR14 from the piggyBac (PB) insertional mutagenesis studies in our lab. The homozygous mice display characteristic facial appearance including eyelid malformations and maxillary hypoplasia. The PB transposon is inserted in a TTAA-site located in the intron of AK087804 on chromosome 9. AK087804 is expressed in the ovary but absent in the facial region, and its expression is disrupted by the PB insertion. A BAC transgene expresssing AK087804 can not resuce the facial defects, suggesting that AK087804 is not involved in these defects. Foxl2, a gene located 160kb away from the PB insertion, is partially disrupted by the PB insertion in the facial region and its downstream target Osr2 is down-regulated accordingly. The Fox12 expression is also decreased in the mutant ovaries. The homozygous female mice are subfertile and produce less progenies. The defective phenotypes and the disturbed expression of Foxl2 in the mutant mice mimic the symptoms of the BPES patients, and this mutant may serve as a new BPES mouse model.We hypothesized that the PB insertion causes Fox12 reduction by interfering with its potential long-range regulatory elements. Based on human-mouse genomic sequence alignment, we identified four evolutionarily conserved regions (ECRs) near PB insertion site. One of the regions ECR1 is able to enhance the transcription of reporter genes. In the maxillary and eyelid tissues where Fox12 is expressed, ECR1 possesses accessible chromatin conformation and could form a looping structure with the Foxl2 promoter region, which are typical behaviors of long-range regulatory elements. These observations indicate that ECR1 may function as a long-range regulatory element to control the expression of Foxl2.