Elsevier

Gene

Volume 432, Issues 1–2, 1 March 2009, Pages 82-90
Gene

Characterization of the murine Dfna5 promoter and regulatory regions

https://doi.org/10.1016/j.gene.2008.11.017Get rights and content

Abstract

Mutations in DFNA5 cause a non-syndromic autosomal dominant type of hearing loss. Although not much is known regarding the physiological function of DFNA5, it is not only related to hearing loss. A clear link with cancer exists. For example, methylation of the 5′ flanking region of DFNA5 was detected in breast, colorectal and gastric cancer. So far, this 5′ flanking region has not been studied in detail. Here, we describe the identification of the cochlear transcription initiation site (TIS), the identification of the core promoter region between − 120 and + 70 relative to the TIS and the identification of an enhancer (between − 121 and − 356 bp) and a silencer element (between − 356 and − 670 bp). Results were similar in HEK293 cells and in the organ of Corti cell line OC-k3. Transfection with a reversely-oriented construct resulted in high transcriptional activity. We subsequently confirmed this antisense activity and identified a novel antisense transcript partly overlapping Dfna5.

Introduction

Mutations in DFNA5 cause a non-syndromic, progressive, autosomal dominant, sensorineural type of hearing loss. Patients from the four families segregating DFNA5 mutations all have a highly similar phenotype. At the genomic DNA level, four different mutations were detected in DFNA5 in different families. All these mutations led to skipping of exon 8 at the mRNA level (Van Laer et al., 1998, Yu et al., 2003, Bischoff et al., 2004, Cheng, 2007). Moreover, a mutation in exon 5 that did not affect exon 8 splicing did not cause hearing loss in an Iranian family (Van Laer et al., 2007). The discovery of four mutations all leading to exon 8 skipping, led to the formulation of the hypothesis that DFNA5-related hearing loss was associated with a very specific gain of function event, in which only skipping of exon 8 causes disease while other mutations in the gene do not (Van Laer et al., 2004). This hypothesis was supported by experimental evidence demonstrating that transfections with mutant DFNA5 lead to cell death, both in yeast (Gregan et al., 2003) and in mammalian cells (Van Laer et al., 2004).

DFNA5 is ubiquitously expressed. The highest expression levels were detected in placenta, while lower expression was observed in brain, heart and kidney (Thompson and Weigel, 1998, Van Laer et al., 1998). Using RT-PCR, DFNA5 expression, albeit at low levels, was found in every tissue investigated so far (nine in total, unpublished results).

The physiological function of DFNA5 remains largely unknown. For a long time, DFNA5 was considered an orphan gene. Recently, DFNB59, a paralogue of DFNA5, has been described. Mutations in DFNB59, encoding Pejvakin, cause auditory neuropathy and non-syndromic hearing impairment in mice and men (Delmaghani et al., 2006, Hashemzadeh Chaleshtori et al., 2007, Schwander, 2007). As DFNB59 mutations may cause hearing loss through diverse pathogenic mechanisms, unfortunately the identification of DFNB59 did not shed light on the mechanism by which DFNA5 may cause hearing loss.

Although not much is known regarding the physiological function of DFNA5, we can certainly state that it is not only related to hearing loss. A clear link with cancer exists. In a recent paper DFNA5 was even catalogued as tumour suppressor gene (Kim et al., 2008b). DFNA5 has also been designated ICERE-1 (inversely correlated with estrogen receptor expression) due to its lower expression in estrogen receptor (ER)-positive breast cancers compared to ER-negative tumours (Thompson and Weigel, 1998). In melanoma cells, etoposide resistance was associated with decreased expression of DFNA5 (Lage et al., 2001). Etoposide is used as an anti-neoplastic agent and causes DNA strand breakage via inhibition of DNA topoisomerase II (Glisson and Ross, 1987). These results may indicate that DFNA5 plays a role in melanoma and breast cancer progression and in the resistance to chemotherapy. P53 is a well-known tumour suppressor gene. Upon DNA damage or other cellular stress situations, various pathways will lead to the activation of p53, inducing either cell cycle arrest to allow repair and survival or apoptosis, depending on the damage. Expression of DFNA5 was strongly induced both by endogenous and exogenous p53 in human cancer cell lines and a functional p53-binding site was identified in intron 1 of DFNA5. These observations suggested that DFNA5 may be involved in the p53-mediated cellular response to genotoxic stress (Masuda et al., 2006). Further evidence supporting a role for DFNA5 in cancer came from methylation studies. DFNA5 was silenced in breast cancer cell lines by methylation of the 5′ flanking region of the gene (Kim et al., 2008b). Previously, methylation of the DFNA5 promoter region was associated with gastric (Akino et al., 2007) and colorectal cancer (Kim et al., 2008a).

So far, the promoter region and the regulatory motifs for DFNA5 expression have not been characterized. However, knowledge of the regulatory regions of DFNA5 is particularly interesting as the above-mentioned studies implicate that pathophysiological downregulation of DFNA5 plays a role in breast, gastric and colorectal cancer. This implies that therapies based on the activation of DFNA5 expression might provide an effective treatment for these types of cancers. Here, we describe the identification of the Dfna5 transcription initiation site (TIS), the characterization of the regions responsible for basal transcriptional activity, and the presence of enhancing and silencing elements in the Dfna5 5′-flanking region. Furthermore, we provide evidence for the presence of a Dfna5 antisense transcript.

Section snippets

In silico analysis

Promoter regions were predicted in silico using different computer modelling programs, listed in Supplementary Table 1. CpG island prediction was performed using both the CpG island searcher (www.uscnorris.com/cpgislands2/cpg.aspx) (Takai and Jones, 2003) and the EMBOSS CpG prediction tool (www.ebi.ac.uk/cpgplot/). Transcription factor (TF) binding sites were scored using three programs, MatInspector (www.genomatix.de), NSITE (http://softberry.com/) and ProScan (//www-bimas.cit.nih.gov/molbio/proscan/

In silico investigation of the Dfna5 upstream region

In order to localize the promoter region, the 5′ flanking region of Dfna5 was first investigated in silico. The Eldorado, Proscan, TSSG, NNPP and Promoter 2,0 modelling programs respectively predicted 2, 3, 3, 21 and 3 potential promoter regions in an 11 kb fragment flanking Dfna5 (Refseq Genbank accession no. NT_039353.7, nt2523000 to nt2534000). Promoser and McPromoter did not predict any promoter elements in the investigated region. A detailed view of the predicted promoter regions contained

Discussion

Here we report the localization of the TIS of cochlear dfna5 mRNA, the identification of the core promoter and additional regulatory regions required for the expression of dfna5. Furthermore we report the identification of an antisense Dfna5 transcript.

The computer programs that we have used to identify the putative core promoter in silico resulted in diverging predictions. As recent studies that compared eight different promoter prediction programs indicated that true prediction rates vary

Acknowledgments

The authors thank Dr F. Kalinec from the House Ear Institute, LA, USA for providing the OC-k3 cell line. This work is supported by FWO grant 1.5.048.05N and by the European Commission FP6 Integrated Project: Eurohear (LSHG-CT-20054-512063).

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