Abstract
Purpose :
North Carolina Macular Dystrophy is an inherited retinal disease that has been linked to alterations in PRDM13, but how PRDM13 functions as a transcription factor during retinal development is not well understood. We have employed a stem cell-based platform to uncover how PRDM13 controls retinal gene expression. We hypothesize that the overexpression (OE) of PRDM13 represses retinal differentiation in mouse retinal organoids by localizing to and regulating genes required for retinal differentiation.
Methods :
PRDM13 and a series of PRDM13 zinc-finger inactivating mutants were generated and cloned into dox-inducible plasmids, transfected into mouse embryonic stem cells carrying an Rx-GFP/Sox1-mCherry+ dual reporter, and differentiated into mouse retinal organoids (mROs). These mutants were analyzed with flow-cytometry to determine their effect on Rx-GFP+ retinal and Sox1-mCherry+ brain differentiation, respectively. To identify genes directly regulated by PRDM13, RNA-seq and ChIP-seq were performed in mouse embryonic stem cells overexpressing full-length PRDM13 and an inactive PRDM13 mutant. Additionally, co-immunoprecipitation followed by mass spectrometry was performed with FLAG-tagged PRDM13 in HEK293T cells to identify PRDM13 protein-protein interactions.
Results :
OE of full-length PRDM13 (+dox 20ng/mL) completely represses Rx-GFP+ mRO differentiation. OE of PRDM13 zinc-finger inactivating mutants demonstrates zinc-finger 1 and 2 domains are required for repression of mRO differentiation. Integration of genes downregulated by greater than 2-fold from PRDM13 OE with ChIP-seq data have identified repressed candidate target genes of PRDM13, such as Trim2. Mass spectrometry has identified the novel PRDM13 protein interactor, HCFC1, a transcription factor implicated in epigenetic regulation of cell cycle control.
Conclusions :
Our results provide mechanistic insights into the function of PRDM13 during development of the retina. We have identified candidate target genes of PRDM13, such as Trim2, that are repressed during retinal organoid differentiation. In addition, we have confirmed a novel protein interaction between PRDM13 and HCFC1. Further studies are required to determine the biochemical function of PRDM13 as a transcriptional repressor and regulator of pre-mRNA processing during retinal differentiation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.