Abstract
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.
Highlights
Progressive loss of retinal ganglion cells (RGCs)2 leads to blindness in diseases such as glaucoma, the leading cause of irreversible bilateral blindness worldwide [1]
We show that overexpression of KLF16 inhibits neurite outgrowth of RGCs
KLF16 is expressed in RGCs at the appropriate developmental time to be an in vivo transcriptional regulator of EphA5, and using chromatin immunoprecipitation (ChIP), KLF16 occupies the proximal promoter region of EphA5 in the retina
Summary
KLF16 Expression Is Enriched in the Developing Retina—Previous RT-PCR analysis showed KLF16 expression in the developing retina [7]. A–C, retinal section from an E15.5 mouse showing immunoreactivity for KLF16 (A) and POU4F2 (B) in nascent retinal ganglion cells in the central retina. Bars show mean pixel intensity/mm Ϯ S.E. K–M, retinal section double-stained with antibodies against KLF16 (K) and PAX6 (L), with merged image (M) showing co-localization in P7 the mouse retina. PAX6 is expressed in RGCs, amacrine cells, and horizontal cells in the mature retina, in a pattern similar to that observed for KLF16 (Fig. 1, K–M) [30]. In the inner nuclear layer, KLF16 immunoreactivity was closely associated with PAX6-positive nuclei of amacrine and horizontal cells. Consistent with the PAX6 double-staining, horizontal and amacrine cells in the inner nuclear layer were double labeled for KLF16 and calbindin (Fig. 1, N–P). Effects of KLF16 overexpression on total neurite length in rat retinal ganglion cells
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