Wang Jianbo, Galvao Joana, Beach Krista M, Luo Weijia, Urrutia Raul A, Goldberg Jeffrey L, Otteson Deborah C
From the Departments of Physiological Optics and Vision Science and.
the Byers Eye Institute, School of Medicine, Stanford University, Palo Alto, California 94303, the Shiley Eye Institute, University of California San Diego, La Jolla, California 92093, and.
J Biol Chem. 2016 Aug 26;291(35):18084-95. doi: 10.1074/jbc.M116.732339. Epub 2016 Jul 11.
Regenerative medicine holds great promise for the treatment of degenerative retinal disorders. Krüppel-like factors (KLFs) are transcription factors that have recently emerged as key tools in regenerative medicine because some of them can function as epigenetic reprogrammers in stem cell biology. Here, we show that KLF16, one of the least understood members of this family, is a POU4F2 independent transcription factor in retinal ganglion cells (RGCs) as early as embryonic day 15. When overexpressed, KLF16 inhibits RGC neurite outgrowth and enhances RGC growth cone collapse in response to exogenous ephrinA5 ligands. Ephrin/EPH signaling regulates RGC connectivity. The EphA5 promoter contains multiple GC- and GT-rich KLF-binding sites, which, as shown by ChIP-assays, bind KLF16 in vivo In electrophoretic mobility shift assays, KLF16 binds specifically to a single KLF site near the EphA5 transcription start site that is required for KLF16 transactivation. Interestingly, methylation of only six of 98 CpG dinucleotides within the EphA5 promoter blocks its transactivation by KLF16 but enables transactivation by KLF2 and KLF15. These data demonstrate a role for KLF16 in regulation of RGC neurite outgrowth and as a methylation-sensitive transcriptional regulator of EphA5 expression. Together, these data identify differential low level methylation as a novel mechanism for regulating KLF16-mediated EphA5 expression across the retina. Because of the critical role of ephrin/EPH signaling in patterning RGC connectivity, understanding the role of KLFs in regulating neurite outgrowth and Eph receptor expression will be vital for successful restoration of functional vision through optic nerve regenerative therapies.
再生医学在退行性视网膜疾病的治疗方面具有巨大潜力。Krüppel样因子（KLFs）是一类转录因子，最近已成为再生医学中的关键工具，因为其中一些因子在干细胞生物学中可作为表观遗传重编程因子发挥作用。在此，我们表明KLF16是该家族中了解最少的成员之一，早在胚胎第15天，它就是视网膜神经节细胞（RGCs）中一种独立于POU4F2的转录因子。过表达时，KLF16会抑制RGC轴突生长，并增强RGC生长锥对外源性ephrinA5配体的反应性塌陷。ephrin/EPH信号传导调节RGC的连接性。EphA5启动子包含多个富含GC和GT的KLF结合位点，染色质免疫沉淀分析表明，这些位点在体内与KLF16结合。在电泳迁移率变动分析中，KLF16特异性结合EphA5转录起始位点附近的单个KLF位点，该位点是KLF16反式激活所必需的。有趣的是，EphA5启动子内98个CpG二核苷酸中只有6个发生甲基化，会阻断KLF16对其的反式激活，但能使KLF2和KLF15进行反式激活。这些数据证明了KLF16在调节RGC轴突生长中的作用，以及作为EphA5表达的甲基化敏感转录调节因子的作用。总之，这些数据确定了差异低水平甲基化是调节整个视网膜中KLF16介导的EphA5表达的一种新机制。由于ephrin/EPH信号传导在RGC连接模式形成中起关键作用，了解KLFs在调节轴突生长和Eph受体表达中的作用，对于通过视神经再生疗法成功恢复功能性视力至关重要。
