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Tatiana Eliseeva, Ko Park, Joseph A Brzezinski; Characterization of a Blimp1-specific enhancer in the developing retina. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1341.
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© ARVO (1962-2015); The Authors (2016-present)
Diseases associated with photoreceptor death result in irreversible vision loss. Current cell replacement therapies utilizing stem cells have been handicapped by poor understanding of the mechanisms that govern the earliest steps in photoreceptor development. The transcription factor Blimp1 (Prdm1) is required for photoreceptor development and expressed very early in photoreceptor genesis. To identify the gene regulatory network that controls photoreceptor genesis, we have identified a cis-regulatory element (enhancer) that controls the spatial and temporal aspects of Blimp1 expression within the retina.
Putative enhancer regions identified by retinal DNase hypersensitivity sequencing were cloned into reporter plasmids containing a minimal promoter driving nuclear-localized GFP. Explanted newborn mouse retinas were co-electroporated with the enhancer constructs and a plasmid that ubiquitously expressed nuclear-localized Cherry. After one day of culture, explants were screened by immunohistochemistry to determine what fraction of GFP+ and Cherry+ cells co-expressed Blimp1. To identify necessary sequences, enhancer elements were further subdivided and key putative transcription factor binding sites destroyed by site-directed mutagenesis.
Of ten putative DNase hypersensitive sites flanking Blimp1, we identified a single 1.9kb region that recapitulated Blimp1 expression in retinal explant cultures. This region was subdivided into three highly conserved fragments, only one of which could recapitulate the Blimp1 expression pattern. Within this fragment, we found two highly-conserved Otx2 transcription factor binding sites. Mutagenesis of one of these sites resulted in partial loss of enhancer activity, while mutagenesis of the other led to complete loss of activity. A construct containing these two Otx2 binding sites and the intervening sequence was unable to recapitulate Blimp1 expression.
We have identified an enhancer that recapitulates Blimp1 expression during retinal development. This enhancer element requires Otx2 binding sites for activity. However, these Otx2 sites alone are not sufficient for enhancer activation. These data suggest that combinatorial action of Otx2 and an as of yet unidentified factor are necessary for Blimp1 expression during retinal development. Further work will characterize this element in vivo and assess its regulation by other transcription factors.
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