Biochemical and Biophysical Research Communications
Foxn3 is essential for craniofacial development in mice and a putative candidate involved in human congenital craniofacial defects
Research highlights
► A novel gene-trap mutation unravels the role of Foxn3 in mammalian development. ► Foxn3 affects craniofacial development in mice. ► Foxn3 regulates the expression of osteogenic genes during craniofacial development. ► Mutant mice phenotypes and clinical features of human congenital patients overlap.
Introduction
The fork-head family is a large family of transcription factors that share a structurally related DNA-binding domain: the fork-head box [1], [2]. More than 40 FOX genes have been discovered in the human genome. The human fork-head family of proteins is divided into distinct subfamilies (FOXA through FOXQ) and various members have been shown to play diverse roles in development, immune system, cell cycle, and cancer [3]. The FOXO family of transcription factors has been shown to play functional roles in tumor suppression, energy metabolism, and aging [4], [5] while the FOXP family members have been implicated in B-cell development, tumorigenesis, and neuronal development [6], [7], [8]. At the molecular level, fork-head box proteins bind DNA as monomers and regulate transcription independently or by interacting with transcriptional regulators that can lead to activation or repression of transcription [2], [9], [10]. In addition, FOX proteins can affect nucleosome positioning and promote gene activation [11], [12].
FOXN3 (also known as Checkpoint suppressor 1 and here forth referred to as FOXN3) belongs to the FOXN subfamily [2], and this subfamily consists of five additional members that have diverse physiological functions in mammals. Studies have shown that FOXN1 is a regulator of keratinocyte growth and differentiation of thymic epithelium [13]. The human FOXN2 protein (also known as human T-cell leukemia virus enhancer factor) binds to the human T-cell virus long terminal repeat and is implicated to play a role in transcriptional regulation [14]. The FOXN4 gene has been shown to be necessary for cardiac and nervous system development whereas FOXN5 and FOXN6 have been recently identified as FOXN family members [15], [16], [17], [18]. The human FOXN3 gene has been mapped to 14q32.11, and its C-terminal portion was first isolated as a high-copy suppressor of Saccharomyces cerevisiae G1-S and G2-M checkpoint mutants that conferred increased survival of the mutants to DNA damaging agents [19]. The C-terminal portion of the human FOXN3 protein was also found to interact with and inhibit Sin3, a component of the Sin3/Rpd3 histone deacetylase complex (HDAC) in budding yeast [20]. The Sin3/Rpd3 HDAC complex is targeted to specific promoter regions via Sin3 interactions with site-specific DNA-binding proteins [21]. While these heterologous genetic screens suggest a role for FOXN3 in the regulation of gene expression and possibly DNA damage responses, a direct role for the protein in mammalian development is not known due to the dearth of data relating to mammalian models that are defective in FOXN3 expression. In this study we describe a mutant mouse model that implicates a role for the murine Foxn3 protein in embryonic and craniofacial development.
Section snippets
Generation of Foxn3 mutant mice
The Baygenomics insertional mutagenesis strategy involves the use of an ATG-less splice-acceptor-beta-galactosidase-neomycin cassette. ES cell clones are then characterized by 5′RACE to identify upstream exons abutting the β-galactosidase-neomycin (βgeo) sequence. One of the ES cell clones that had been characterized to have a genetrap insertion within the Foxn3 gene was represented in the Baygenomics ES cell library. The Foxn3 gene trapped ES cells were obtained and further characterized to
Foxn3 mutation leads to embryonic and post natal lethality
To understand the role of Foxn3 in mammalian development, we generated a mutant mouse model using the Baygenomics gene-trap approach [22] (Supplementary Fig. 1). Survival analysis of homozygous mutant animals obtained from multiple heterozygous intercrosses indicated that the homozygous mutants exhibited partial embryonic and postnatal lethality that was lower than the expected Mendelian ratio (Table 1). Chi-squared analysis of the offspring numbers indicated that the differences were
Discussion
Genetic manipulation of the mouse germline has yielded opportunities to dissect the roles(s) of various gene products in mammalian development and the complexity of gene functions at the organismal level. The recent development of gene-trap based mutagenesis protocols have facilitated and expedited the generation of mutant mouse strains for a large number of genes and allowed the rapid analysis of gene functions in a variety of developmental and physiological pathways [29], [30]. In this study
Conclusion
Our results show that the Foxn3 protein is in involved in the regulation of craniofacial development in mammals and affects the expression of distinct osteogenic genes. The substantial overlap of the phenotypes of the Foxn3 mutant mouse model with the clinical features of human patients with deletion of the FOXN3 locus highlight its importance in the etiology of human craniofacial defects and provide a potential diagnostic tool in their assessment. Our studies could possibly pave way for
Acknowledgments
This work was supported by the Physicians Medical Education and Research Foundation and start-up funds from the UTK BCMB department.
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