POU Transcription Factors

The POU-domain family of transcription factors was identified on the basis of amino acid sequence homology in the DNA binding domain of the transcription factors PIT1/GHF1, OCT1 and OCT2, and UNC86 (Herr et al. 1988). The POU domain consists of 147 to 156 amino acids and is comprised of two distinct DNA binding domains: a 69- to 78-amino-acid POU-specific domain located amino terminal to a 60-amino-acid POU homeodomain (Rosenfeld 1991). The two POU domains are separated by a variable linker, a flexible stretch of amino acids that increases the repertoire of the specific sequences to which these proteins can bind to and improves the kinetics of the binding (reviewed in Phillips and Luisi 2000).

The class III of POU domain genes were identified using PCR and degenerate oligonucleotides representing codons of the nine conserved amino acids in the original POU genes (He et al. 1989). All mammalian class III POU domain transcription factors are broadly expressed within the developing nervous system and assume more restricted expression patterns in the adult nervous system. Mouse knockout models for genes in this group reveal both the redundant and unique functions of these genes.

3.1.1 POU3F4/DFN3

The POU3F4 (BRN4) gene has a clear role in development, manifested clinically, as children born with mutations in this gene suffer from congenital pro found deafness. As described below, not only is the expression strong during early development of the embryo, but the Pou3f4 mouse mutants have structural abnormalities in their auditory system during development. Mutations in the POU3F4 transcription factor are associated with the DFN3 locus on chromosome X (de Kok et al. 1995). The DFN3 phenotype is variable, and at the very least it is characterized by profound sensorineural hearing loss but is also often associated with conductive hearing loss and with stapes fixation. Furthermore, as DFN3 maps to chromosome Xq21 in the region containing mental retardation and choroideremia, some patients with POU3F4 mutations have additional symptoms due to deletions of larger portions of the chromosome. Both point mutations in the POU3F4 gene, as well as larger deletions, duplications, and inversions of the Xq21 region are associated with the DFN3 phenotype. The deletions account for a little over half of DFN3 mutations and many of them do not encompass the POU3F4 coding region. A detailed molecular analysis of the region proximal to the POU3F4 gene revealed small deletions 900 kb proximal to the gene that is associated with DFN3 deafness (de Kok et al. 1996). It was suggested that the DNF3 phenotype, without coding region mutations, could be caused by the loss of the POU3F4 enhancer, repressor, or promoter.

Most of our knowledge regarding the role of POU3F4 during development comes from expression studies in the mouse, as well as from spontaneous and gene targeted mutagenesis models. Indeed, the POU3F4 gene has a unique role in the development of the inner ear. During mouse development, Pou3f4 is first expressed at E9.5 in the neural tube, and then in the otic capsule, the hindbrain, and the branchial arch mesenchyme (Phippard et al. 1998) (Fig. 7.4). Notably, the subcellular localization of this transcription factor shifts to the cytoplasm in areas of mesenchymal remodeling that will further develop to acellular structures, demonstrating a potential mechanism for crucial silencing of the gene during normal otic development.

Several Pou3f4 mouse mutants are available and two of these, the sex-linked fidget mutation and a targeted null mutation, have both cochlear and temporal bone abnormalities (Phippard et al. 1999, 2000) (Fig. 7.4). These include a constricted superior semicircular canal, widening of the internal auditory mea-tus, thinning of various structures in the temporal bone; a misshaped stapes footplate; and a shortening of the cochlea, demonstrated as a reduction in the number of cochlear coils in most of the mutants. In addition, the mice have a generally hypoplastic cochlea, with widening of the scala tympani, and dysplasia of fibrocytes in the spiral limbus, that may lead to the hydrops observed in these animals. Notably, no structural abnormalities are observed in the organ of Corti. Since the Pou3f4 protein is expressed in mesenchymal tissue (Phippard et al. 1998), the widened structures may result from disruption of mesenchymal remodeling, the shortening of the cochlea from disruption of epithelial-mesenchymal interactions, and the mishaping of the stapes from disruption of mesenchymal-mesenchymal interactions. This phenotype resembles the temporal bone phenotype of the people suffering from POU3F4 mutations.

Another mouse model created by a targeted deletion of the Pou3f4 gene on

Figure 7.4. Gene-targeted mutagenesis, with the lacZ gene replacing Pou3f4, reveals its expression during development. (A) Pou3f4 is expressed in most of the neuraxis and in some mesodermally derived tissues in the head, including the otic capsule. (B) Pou3f4 is expressed in the hindbrain of a 9.5dpc embryo, but is not seen in the mesenchyme surrounding the otic vesicle. (C) In an E10.5 embryo, Pou3f4 is expressed in the condensing mesenchyme of the otic vesicle. (D) Expression patterns of lacZ in a parasagittal section of a E14.5, Pou3f4 is detected throughout the otic capsule (Phippard et al. 1999). BA, branchial arches; HB, hindbrain; OV, otic vesicle.

Figure 7.4. Gene-targeted mutagenesis, with the lacZ gene replacing Pou3f4, reveals its expression during development. (A) Pou3f4 is expressed in most of the neuraxis and in some mesodermally derived tissues in the head, including the otic capsule. (B) Pou3f4 is expressed in the hindbrain of a 9.5dpc embryo, but is not seen in the mesenchyme surrounding the otic vesicle. (C) In an E10.5 embryo, Pou3f4 is expressed in the condensing mesenchyme of the otic vesicle. (D) Expression patterns of lacZ in a parasagittal section of a E14.5, Pou3f4 is detected throughout the otic capsule (Phippard et al. 1999). BA, branchial arches; HB, hindbrain; OV, otic vesicle.

a different genetic background showed no gross temporal or inner ear defect, nor in the neuroepithelium or cochlear length, while the mice displayed profound deafness and a reduced endocochlear potential (Minowa 1999). The pathological effects appeared in the fibrocytes that line the stria vascularis that are hypothesized to be involved in potassium recycling back to the endolymph (Spicer and Schulte 1996).

3.1.2 POU4F3/DFNA15

A mutation in the POU4F3 (BRN3.1/BRN3C) gene leads to progressive autosomal dominant hearing loss in an Israeli kindred (Vahava et al. 1998). While Pou4f3 has a clear role in development based on the recessive mouse mutant described below, in humans, there is only a late-onset effect. The DFNA15 locus was discovered in a linkage project performed to determine the underlying cause of late-onset hearing loss in a family of Libyan descent, now living in Israel. Although close to the first autosomal dominant locus defined, DFNA1, on human chromosome 5q31, DFNA15 defined a new locus. Examination of the mouse syntenic region revealed the presence of the Pou4f3 gene, known to cause deaf ness when removed by gene-targeted mutagenesis in the mouse (Erkman et al. 1996; Xiang et al. 1996). Subsequent sequencing of the POU4F3 gene in affected individuals revealed an eight-base-pair deletion in the second coding exon of the gene, which leads to a frameshift and premature stop codon. As a result, a bipartite nuclear localization signal is lost, and in a cell culture model of the mutation, leads to loss of expression in the nucleus (Weiss et al. 2003). The mutation does not appear to cause a dominant-negative effect, but rather may be unavailable to bind to its targets at a sufficient and necessary threshold over time.

In the mouse inner ear, Pou4f3 is a hair cell-specific protein (Fig. 7.5). In the adult mouse, all hair cell nuclei both in the auditory and vestibular systems express Pou4f3 (Erkman et al. 1996; Xiang et al. 1997). Pou4f3 protein can be detected as early as E12.5 in scattered cells of the developing otocyst (Xiang et al. 1998). Double staining of inner ear sections from bromodeoxy uridine (BrdU)-labeled embryos, with anti-BrdU and anti-Pou4f3 antibodies, revealed that Pou4f3 expression is confined to postmitotic cells (Xiang et al. 1998). Realtime RT-PCR of auditory sensory epithelia indicated that very low levels of Pou4f3 mRNA can be detected as early as E12, and constantly increase thereafter, up to P3, the latest time point measured (Hertzano et al. 2004).

Mice with a targeted deletion of Pou4f3 suffer from deafness and vestibular dysfunction, due to a loss of all hair cells by early postnatal days (Erkman et al. 1996; Xiang et al. 1997). Innervated hair cell-like cells with some sterociliary-like structures do form in the absence of Pou4f3 and express hair cell markers such as myosin VI, myosin VIIa, parvalbumin, and calretinin (Xiang et al. 1998, 2003; Hertzano et al. 2004). However, the patterning of these cells is markedly disrupted and inner hair cell loss from the base of the cochleae can

Figure 7.5. Pou4f3 expression in E13.5 and E16.5 inner ears, demonstrated by immu-nostaining with a Pou4f3 antibody (provided by Ronna Hertzano).

e16.5

Figure 7.5. Pou4f3 expression in E13.5 and E16.5 inner ears, demonstrated by immu-nostaining with a Pou4f3 antibody (provided by Ronna Hertzano).

be detected as early as E16.5, accompanied by an increase in apoptotic cell death, as detected by a terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay, as early as E17.5 (Xiang et al. 1998; Hertzano et al. 2004). Reactive loss of sensory ganglion neurons and sensory epithelia supporting cells is also observed (Erkman et al. 1996; Xiang et al. 1997, 1998, 2003; Hertzano et al. 2004).

The gene encoding growth factor independence 1 (Gfi1), a known deafness gene in the mouse (Wallis et al. 2003), was recently identified as a target gene of Pou4f3 by using a transcription profiling approach of inner ears from wild type and Pou4f3 mutant mice (Hertzano et al. 2004). Gfi1 expression is markedly reduced in the ears of the Pou4f3 mutant mice. A comparison of cochleae from Pou4f3 and Gfi1 mutants suggested that outer hair cell loss in the Pou4f3-deficient cochleae may result from the loss of Gfi1 expression.

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