Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development
Fbx15 is a novel target of Oct3/4 but is dispensable for embryonic stem cell self-renewal and mouse development
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DOI：
10.1128/mcb.23.8.2699-2708.2003
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发表时间：
2003-04-01
影响因子：
5.3
通讯作者：
Yamanaka, S
中科院分区：
文献类型：
--
作者：
Tokuzawa, Y;Kaiho, E;Yamanaka, S
胚胎干（ES）细胞是源自早期哺乳动物胚胎的永生多能细胞。转录因子 Oct3/4 对于 ES 细胞的自我更新和小鼠早期发育至关重要。然而，仅鉴定了少数 Oct3/4 靶基因。在这项研究中，我们发现含有F-box的蛋白Fbx15主要在小鼠未分化ES细胞中表达。 ES 细胞中 Oct314 的失活导致 Fbx15 表达迅速消失。报告基因分析表明，这种 ES 细胞特异性表达需要位于转录起始位点上游约 500 个核苷酸处的 18 bp 增强子元件。该增强子包含一个八聚体样基序和一个相邻的 Sox 结合基序。任一基序的删除或点突变都会消除增强子活性。当 Oct3/4 和 Sox2 共表达时，18 bp 片段在 NIH 3T3 细胞中变得活跃。凝胶迁移率变化测定证明了 Oct3/4 和 Sox2 与增强子序列的协同结合。在 Fbx15 基因座敲入 β-半乳糖苷酶基因的小鼠中，在 ES 细胞、早期胚胎（两细胞至囊胚阶段）和睾丸组织中检测到 5-溴-4-氯-3-吲哚基-β-D-吡喃半乳糖苷染色。尽管 Fbx15 具有如此特异性的表达，但纯合突变小鼠没有表现出明显的发育缺陷并且具有生育能力。 Fbx15 缺失的 ES 细胞形态、增殖和分化均正常。这些数据表明 Fbx15 是 Oct3/4 的新靶标，但对于 ES 细胞的自我更新、发育和生育力来说是可有可无的。
Embryonic stem (ES) cells are immortal and pluripotent cells derived from early mammalian embryos. Transcription factor Oct3/4 is essential for self-renewal of ES cells and early mouse development. However, only a few Oct3/4 target genes have been identified. In this study, we found that F-box-containing protein Fbx15 was expressed predominantly in mouse undifferentiated ES cells. Inactivation of Oct314 in ES cells led to rapid extinction of Fbx15 expression. Reporter gene analyses demonstrated that this ES cell-specific expression required an 18-bp enhancer element located approximately 500 nucleotides upstream from the transcription initiation site. The enhancer contained an octamer-like motif and an adjacent Sox-binding motif. Deletion or point mutation of either motif abolished the enhancer activity. The 18-bp fragment became active in NIH 3T3 cells when Oct3/4 and Sox2 were coexpressed. A gel mobility shift assay demonstrated cooperative binding of Oct3/4 and Sox2 to the enhancer-sequence. In mice having a beta-galactosidase gene knocked into the Fbx15 locus, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining was detected in ES cells, early embryos (two-cell to blastocyst stages), and testis tissue. Despite such specific expression of Fbx15, homozygous mutant mice showed no gross developmental defects and were fertile. Fbx15-null ES cells were normal in morphology, proliferation, and differentiation. These data demonstrate that Fbx15 is a novel target of Oct3/4 but is dispensable for ES cell self-renewal, development, and fertility.