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Nora Blanca Caberoy, Peipei Pan, Arnold Salazar, Adrienne Bugayong, Lorena Samentar; Tubby regulates photoreceptor survival via Estrogen-related receptor beta (Esrrβ). Invest. Ophthalmol. Vis. Sci. 2017;58(8):4517.
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Mutation in tubby gene causes adult-onset obesity, blindness, and hearing loss with unknown mechanisms. We have previously identified tubby as a MerTK ligand for phagocytosis in the retinal pigment epithelium and microglial cells. Tubby was also reported to be involved in GPCR signaling, transport in primary cilium, and predicted as a transcription factor. In order to delineate tubby’s pathological mechanisms, we set out to define its interaction network. Using ORF phage display for protein-protein interactions, we identified Esrrβ as a putative tubby-binding protein. Esrrβ is an orphan nuclear hormone receptor homologous to the classical estrogen receptor but can activate transcription in the absence of estrogen. Esrrβ is expressed in differentiating and mature mouse rod photoreceptors and in human retina. This study aims to characterize interaction of tubby and Esrrβ to elucidate their roles in retinal homeostasis and disease pathogenesis.
The binding specificity of tubby to Esrrβ was analyzed by protein pull-down assay. The interaction of tubby and Esrrβ was demonstrated in vivo using co-immunoprecipitation and FRET analyses. Co-localization of tubby and Esrrβ in the retina was examined through immunohistochemistry. Since tubby was predicted as a transcription factor, we examined whether it transcriptionally regulates Esrrβ. Promoter binding and transcriptional activation assays were done to demonstrate tubby binding and activation of Esrrβ expression.
Tubby and Esrrβ interact in vitro and in vivo and the interaction is mediated through tubby N-terminal domain. Promoter binding and transcriptional activation studies showed that tubby directly binds to Esrrβ response elements and activates its transcription.
The results revealed that tubby interacts with Esrrβ protein and also regulates Esrrβ expression. Previous studies have shown that Esrrβ is a critical regulator of rod photoreceptor function and survival. Tubby’s direct regulation of Esrrβ further sheds light as to why mutation in tubby results to death of photoreceptors that leads to retinal degeneration. Tubby was previously implicated as a membrane-bound, G protein-activated transcription factor with no reported target gene(s). To our knowledge, this is the first to describe a gene targeted for tubby regulation.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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