The Promoter and Multiple Enhancers of the pou4f3 Gene Regulate Expression in Inner Ear Hair Cells.
The Promoter and Multiple Enhancers of the pou4f3 Gene Regulate Expression in Inner Ear Hair Cells.
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DOI：
10.1007/s12035-016-0060-7
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发表时间：
2017-09
影响因子：
5.1
通讯作者：
Ryan AF
中科院分区：
文献类型：
--
作者：
Masuda M;Li Y;Pak K;Chavez E;Mullen L;Ryan AF
很少有增强子能够将基因表达靶向内耳毛细胞 (HC)。利用 eGFP 报告基因构建体的转基因分析和生物信息学，我们评估了 pou4f3 基因表达的控制，因为它仅在内耳内的 HC 中表达，并且在整个生命过程中持续表达。起始密码子 5' 处的 8.5 kb 基因组 DNA 片段包含三个高度跨物种同源性区域，驱动所有胚胎和新生儿 HC、成人前庭和内部 HC 中的表达，但不影响成人外部 HC。具有 0.4、0.8、2.5 或 6.5 kb 5' DNA 的转基因不会产生 HC 表达。然而，添加 6.5 至 7.2 kb 区域会在前庭 HC 和新生儿基底转外 HC 中产生表达，这也暗示 7.2 kb 至 8.5 kb 区域在内和顶端外 HC 表达中。从 8.5 kb 构建体中删除 0.4 至 5.5 kb 5' 区域并不影响 HC 表达，进一步表明缺乏 HC 调控元件。当 1 至 0.4 kb 的区域被 Ela1 基因的最小启动子取代时，HC 表达得以维持，但水平大幅降低。生物信息学鉴定了 8.5 kb 之外的高度保守序列区域，其中包含 POU4F3、GFI1 和 LHX3 结合位点。这些区域可能参与维持成人外部 HC 中 POU4F3 的表达。我们的结果确定了不同位置的单独增强子，这些增强子在不同年龄指导表达不同的 HC 类型，并确定 0.4 kb 的上游序列决定表达水平。这些数据将有助于识别该耳聋基因的非编码调节区域的突变。
Few enhancers that target gene expression to inner ear hair cells (HCs) have been identified. Using transgenic analysis of eGFP reporter constructs and bioinformatics, we evaluated the control of pou4f3 gene expression, since it is expressed only in HCs within the inner ear, and continues to be expressed throughout life. An 8.5 kb genomic DNA fragment 5’ to the start codon, containing three regions of high cross-species homology drove expression in all embryonic and neonatal HCs, and adult vestibular and inner HCs, but not adult outer HCs. Transgenes with 0.4, 0.8, 2.5 or 6.5 kb of 5’ DNA did not produce HC expression. However, addition of the region from 6.5 to 7.2 kb produced expression in vestibular HCs and neonatal basal turn outer HCs, which also implicated the region from 7.2 kb to 8.5 kb in inner and apical outer HC expression. Deletion of the region from 0.4 to 5.5 kb 5’ from the 8.5 kb construct did not affect HC expression, further indicating lack of HC regulatory elements. When the region from 1 to 0.4 kb was replaced with the minimal promoter of the Ela1 gene, HC expression was maintained, but at a drastically reduced level. Bioinformatics identified regions of highly conserved sequence outside of the 8.5 kb, which contained POU4F3, GFI1, and LHX3 binding sites. These regions may be involved in maintaining POU4F3 expression in adult outer HCs. Our results identify separate enhancers at various locations that direct expression to different HC types at different ages, and determine that 0.4 kb of upstream sequence determines expression level. These data will assist in the identification of mutations in noncoding, regulatory regions of this deafness gene.