Departments of Surgery/Otolaryngology, La Jolla, CA 92093-0666, USA.
Dev Biol. 2012 Dec 1;372(1):68-80. doi: 10.1016/j.ydbio.2012.09.002. Epub 2012 Sep 15.
Transcription factors (TFs) can regulate different sets of genes to determine specific cell types by means of combinatorial codes. We previously identified closely-spaced TF binding motifs located 8.2-8.5 kb 5' to the ATG of the murine Pou4f3 gene, a gene required for late hair cell (HC) differentiation and survival. These motifs, 100% conserved among four mammalian species, include a cluster of E-boxes preferred by TCF3/ATOH1 heterodimers as well as motifs for GATA factors and SP1. We hypothesized that these factors might interact to regulate the Pou4f3 gene and possibly induce a HC phenotype in non-sensory cells of the cochlea. Cochlear sensory epithelium explants were prepared from postnatal day 1.5 transgenic mice in which expression of GFP is driven by 8.5 kb of Pou4f3 5' genomic DNA (Pou4f3/GFP). Electroporation was used to transfect cells of the greater epithelial ridge with multiple plasmids encoding human ATOH1 (hATOH1), hTCF3 (also known as E2A or TEF2), hGATA3, and hSP1. hATOH1 or hTCF3 alone induced Pou4f3/GFP cells but hGATA3 and hSP1 did not. hATOH1 but not hTCF3 induced conversion of greater epithelial ridge cells into Pou4f3/GFP and myosin VIIa double-positive cells. Transfection of hATOH1 in combination with hTCF3 or hGATA3 induced 2-3X more Pou4f3/GFP cells, and similarly enhanced Pou4f3/GFP and myosin VIIa double-positive cells, when compared to hATOH1 alone. Triple or quadruple TF combinations were generally not more effective than double TF combinations except in the middle turn, where co-transfection of hATOH1, hE2A, and hGATA3 was more effective than hATOH1 plus either hTCF3 or hGATA3. The results demonstrate that TFs can cooperate in regulation of the Pou4f3 gene and in the induction of at least one other element of a HC phenotype. Our data further indicate that combinations of TFs can be more effective than individual TFs in the inner ear.
转录因子 (TFs) 可以通过组合密码来调节不同的基因，从而确定特定的细胞类型。我们之前在小鼠 Pou4f3 基因的 ATG 前 8.2-8.5kb 处发现了紧密间隔的 TF 结合基序，该基序是晚期毛细胞 (HC) 分化和存活所必需的。这些基序在四种哺乳动物中 100%保守，包括 TCF3/ATOH1 异二聚体偏好的 E 盒簇，以及 GATA 因子和 SP1 的基序。我们假设这些因子可能相互作用以调节 Pou4f3 基因，并可能在耳蜗的非感觉细胞中诱导 HC 表型。从出生后 1.5 天的转基因小鼠中制备耳蜗感觉上皮外植体，其中 GFP 的表达由 8.5kb 的 Pou4f3 5'基因组 DNA (Pou4f3/GFP) 驱动。电穿孔用于转染高尔基上皮嵴细胞多个质粒，编码人 ATOH1(hATOH1)、hTCF3(也称为 E2A 或 TEF2)、hGATA3 和 hSP1。hATOH1 或 hTCF3 单独诱导 Pou4f3/GFP 细胞，但 hGATA3 和 hSP1 没有。hATOH1 但不是 hTCF3 诱导高尔基上皮嵴细胞转化为 Pou4f3/GFP 和肌球蛋白 VIIa 双阳性细胞。与单独转染 hATOH1 相比，hATOH1 与 hTCF3 或 hGATA3 联合转染可诱导 2-3 倍的 Pou4f3/GFP 细胞，并同样增强 Pou4f3/GFP 和肌球蛋白 VIIa 双阳性细胞。除了中圈，三重或四重 TF 组合通常不如双重 TF 组合有效，在中圈，hATOH1、hE2A 和 hGATA3 的共转染比 hATOH1 加 hTCF3 或 hGATA3 更有效。结果表明，TFs 可以在调节 Pou4f3 基因和诱导至少一个 HC 表型的另一个元件方面进行合作。我们的数据进一步表明，在内耳中，TFs 的组合比单个 TFs 更有效。
