Missense mutations in POU4F3 cause autosomal dominant hearing impairment DFNA15 and affect subcellular localization and DNA binding.

In a Dutch pedigree suffering from autosomal dominant nonsyndromic hearing impairment (ADNSHI), linkage was found to the locus for DFNA15, with a two-point logarithm of the odds (LOD) score of 5.1. Sequence analysis of the POU4F3 gene that is involved in DFNA15 revealed the presence of a missense mutation (c.865C>T), segregating with the deafness in this family. The mutation is predicted to result in the substitution of a phenylalanine residue for a leucine residue (p.L289F) in the POU homeodomain of the transcription factor POU4F3. Mutation analysis of the POU4F3 gene in 30 patients suffering from dominantly inherited hearing impairment revealed a second novel missense mutation (c.668T>C), resulting in the substitution of a proline for a leucine residue (p.L223P) within the POU-specific DNA-binding domain of the protein. In a computer model describing the structure of the two DNA-binding domains, the alterations are predicted to affect the tertiary structure of these domains. Transient transfection studies showed that whereas the wild-type POU4F3 is located almost exclusively in the nucleus, part of the mutant proteins was also present in the cytoplasm. In addition, both mutant proteins showed greatly reduced capability for binding to DNA as well as transcriptionally activating reporter gene expression. Together, our results describe the identification of the first missense mutations in POU4F3 causing DFNA15. Furthermore, mutations in this gene do not seem to be a rare cause of hearing impairment in the Dutch population, and the POU4F3 gene may thus be suitable for implementation in diagnostic testing.

Barhl1 regulatory sequences required for cell-specific gene expression and autoregulation in the inner ear and central nervous system.

The development of the nervous system requires the concerted actions of multiple transcription factors, yet the molecular events leading to their expression remain poorly understood. Barhl1, a mammalian homeodomain transcription factor of the BarH class, is expressed by developing inner ear hair cells, cerebellar granule cells, precerebellar neurons, and collicular neurons. Targeted gene inactivation has demonstrated a crucial role for Barhl1 in the survival and/or migration of these sensory cells and neurons. Here we report the regulatory sequences of Barhl1 necessary for directing its proper spatiotemporal expression pattern in the inner ear and central nervous system (CNS). Using a transgenic approach, we have found that high-level and cell-specific expression of Barhl1 within the inner ear and CNS depends on both its 5' promoter and 3' enhancer sequences. Further transcriptional, binding, and mutational analyses of the 5' promoter have identified two homeoprotein binding motifs that can be occupied and activated by Barhl1. Moreover, proper Barhl1 expression in inner ear hair cells and cerebellar and precerebellar neurons requires the presence of Atoh1. Together, these data delineate useful Barhl1 regulatory sequences that direct strong and specific gene expression to inner ear hair cells and CNS sensory neurons, establish a role for autoregulation in the maintenance of Barhl1 expression, and identify Atoh1 as a key upstream regulator.

Regulation of unsaturated fatty acid biosynthesis in Saccharomyces: the endoplasmic reticulum membrane protein, Mga2p, a transcription activator of the OLE1 gene, regulates the stability of the OLE1 mRNA through exosome-mediated mechanisms.

The Saccharomyces cerevisiae OLE1 gene encodes a membrane-bound Delta9 fatty-acid desaturase, whose expression is regulated through transcriptional and mRNA stability controls. In wild type cells grown on fatty acid-free medium, OLE1 mRNA has a half-life of 10 +/- 1.5 min (basal stability) that becomes highly unstable when cells are exposed to unsaturated fatty acids (regulated stability). Activation of OLE1 transcription is dependent on N-terminal fragments of two membrane proteins, Mga2p and Spt23p, that are proteolytically released from the membrane by a ubiquitin-mediated mechanism. Surprisingly, disruption of the MGA2 gene also reduces the half-life of the OLE1 transcript and abolishes fatty acid regulated instability. Disruption of its cognate, SPT23, has no effect on the half-life of the mRNA. Mga2p appears to have two distinct functions with respect to the OLE1 mRNA stability: a stabilizing effect in cells grown in fatty acid-free medium and a destabilizing function in cells that are exposed to unsaturated fatty acids. These functions are independent of OLE1 transcription and can confer basal and regulated stability on OLE1 mRNAs that are produced under the control of the unrelated GAL1 promoter. Expression of soluble, N-terminal fragments of Mga2p stabilize the transcript but do not confer fatty acid-regulated instability on the mRNA suggesting that the stabilizing functions of Mga2p do not require membrane processing and that modifications to the protein introduced during proteolysis may play a role in the destabilizing effect. An analysis of mutants that are defective in mRNA degradation indicate that the Mga2p-requiring control mechanism that regulates the fatty acid-mediated instability of the OLE1 transcript acts by activating exosomal 3' --> 5'-exonuclease degradation activity.

Maintenance and regulation of mRNA stability of the Saccharomyces cerevisiae OLE1 gene requires multiple elements within the transcript that act through translation-independent mechanisms.

The Saccharomyces cerevisiae OLE1 gene encodes a membrane-bound Delta-9 fatty acid desaturase, whose expression is regulated by unsaturated fatty acids through both transcriptional and mRNA stability controls. In fatty acid-free medium, the mRNA has a half-life of 10 +/- 1.5 min (basal stability) that drops to 2 +/- 1.5 min when cells are exposed to unsaturated fatty acids (regulated stability). A deletion analysis of elements within the transcript revealed that the sequences within the protein-coding region that encode transmembrane sequences and a part of the cytochrome b5 domain are essential for the basal stability of the transcript. Deletion of any of the three essential elements produced unstable transcripts and loss of regulated instability. By contrast, substitution of the 3'-untranslated region with that of the stable PGK1 gene did not affect the basal stability of the transcript and did not block regulated decay. Given that Ole1p is a membrane-bound protein whose activities are a major determinant of membrane fluidity, we asked whether membrane-associated translation of the protein was essential for basal and regulated stability. Insertion of stop codons within the transcript that blocked either translation of the entire protein or parts of the protein required for co-translation insertion of Ole1p had no effect. We conclude that the basal and regulated stability of the OLE1 transcript is resistant to the nonsense-mediated decay pathway and that the essential protein-encoding elements for basal stability act cooperatively as stabilizing sequences through RNA-protein interactions via a translation-independent mechanism.