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Hum Genet
2010 Oct 01;1284:411-9. doi: 10.1007/s00439-010-0864-x.
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Multiple enhancers located in a 1-Mb region upstream of POU3F4 promote expression during inner ear development and may be required for hearing.
Naranjo S
,
Voesenek K
,
de la Calle-Mustienes E
,
Robert-Moreno A
,
Kokotas H
,
Grigoriadou M
,
Economides J
,
Van Camp G
,
Hilgert N
,
Moreno F
,
Alsina B
,
Petersen MB
,
Kremer H
,
Gómez-Skarmeta JL
.
???displayArticle.abstract??? POU3F4 encodes a POU-domain transcription factor required for inner ear development. Defects in POU3F4 function are associated with X-linked deafness type 3 (DFN3). Multiple deletions affecting up to ~900-kb upstream of POU3F4 are found in DFN3 patients, suggesting the presence of essential POU3F4 enhancers in this region. Recently, an inner ear enhancer was reported that is absent in most DFN3 patients with upstream deletions. However, two indications suggest that additional enhancers in the POU3F4 upstream region are required for POU3F4 function during inner ear development. First, there is at least one DFN3 deletion that does not eliminate the reported enhancer. Second, the expression pattern driven by this enhancer does not fully recapitulate Pou3f4 expression in the inner ear. Here, we screened a 1-Mb region upstream of the POU3F4 gene for additional cis-regulatory elements and searched for novel DFN3 mutations in the identified POU3F4 enhancers. We found several novel enhancers for otic vesicle expression. Some of these also drive expression in kidney, pancreas and brain, tissues that are known to express Pou3f4. In addition, we report a new and smallest deletion identified so far in a DFN3 family which eliminates 3.9 kb, comprising almost exclusively the previous reported inner ear enhancer. We suggest that multiple enhancers control the expression of Pou3f4 in the inner ear and these may contribute to the phenotype observed in DFN3 patients. In addition, the novel deletion demonstrates that the previous reported enhancer, although not sufficient, is essential for POU3F4 function during inner ear development.
Fig. 1. Localization of DFN3 deletions and HCNRs in the upstream genomic region of POU3F4. Green arrows show the position of the identified enhancers. a Deletion map within 1.5 Mb around POU3F4 as reported previously (de Kok et al. 1996; Cremers et al. 2008). Dashed lines represent the deleted regions in each patient. The red box marks the 13-kb EcoRI fragment in which the 2540 8-kb deletion is located. b Vista view showing the distribution of HCNRs within 1-Mb upstream of POU3F4. Shown from top to bottom are mouse versus human, chicken versus human and Xenopus tropicalis versus human global alignments. Coloured peaks (purple coding, pink non-coding) indicate regions of at least 100 bp and 75% similarity. Red dots are HCNRs with no enhancer activity in Xenopus assays. Green arrows point at the HCNRs that showed enhancer activity (also shown with green dots). Their genomic positions are depicted in green. The red box marks the 13-kb EcoRI fragment in which the 2540 8-kb deletion is located. c Close-up of this 13-kb EcoRI fragment. Below is shown the new deletion identified in this work (dotted line). The blue line shows the region that was reported to have inner ear enhancer activity in mouse transgenic assays (Ahn et al. 2009)
Fig. 2. Expression patterns promoted by different Pou3f4 HCNRs in Xenopus and zebrafish transgenic assays. Xenopus embryos show in situ hybridization to detect EGFP mRNA in fixed transgenic animals while zebrafish embryos show EGFP in living transgenic specimens. All panels, except (l) that show a dorsal view, are lateral views of transgenic Xenopus (a, c, e, g, i) at stage 35 or transgenic zebrafish (b, d, f, h, j, l) at 36–72-h post fertilization (hpf). The position of the HCNRs in the human genome is shown in the upper right corner of each panel. Different tissues are pointed at with coloured arrowheads. Blue otic vesicle, red midbrain, orange hindbrain, yellow kidney and purple pancreas. Note the equivalent patterns promoted by each HCNR in Xenopus and zebrafish transgenic embryos. k Expression pattern of XenopusPou3f4
Fig. 3. Transverse sections at the level of the otic vesicle of 65–75 hpf embryos showing the expression patterns promoted by the different Pou3f4 HCNRs in stable zebrafish transgenic lines. All panels show in situs hybridization to detect EGFP mRNA in fixed embryos from the different transgenic lines. Note that enhancers at positions 81675 (a), 81728 (b) and 82637 (d) promoted expression, although in somehow different areas, in the periotic mesenchyme (red arrows). In addition, all enhancers are active in the otic epithelium (white arrows) and enhancer 82478 (c) is, in addition, active in the developing kidneys (yellow arrows)
Fig. 4. Genetic analysis of family W06-205. a Pedigree of family W06-205 and segregation of the deletion in the family. Filled squares represent DFN3 males. δ deletion, + wildtype. The genotype of females who are not obligate carriers has not been indicated for privacy reasons. b Sequence chromatogram showing the deletion breakpoints in patient II.6. Above this chromatogram, the deletion is schematically indicated
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