Abstract
Purpose :
During retinal development, the Nuclear Factor I (NFI) transcription factors help drive differentiation of late stage retinal progenitor cells into Müller glia and bipolar cells. How NFI factors contribute to terminal differentiation of these cell types has not been defined. This work seeks to investigate how NFI factors independently and in concert drive changes in genomic organization resulting in cell type specification and loss of pluripotency.
Methods :
A fusion protein of an NFI factor and a small molecule stabilized degron allows for temporal control of gene expression in the liver cancer line, HepG2. The degron, a mutated form of FKBP, causes protein degradation in the absence of aquashield-1 in the culture media. Relying on a fluorescence cassette encoded within pCAGIG, we flow sorted transfected cells with robust fusion protein expression. Bulk ATAC- and RNA-sequencing libraries were then prepared from sorted transfected cells to determine how these fusion proteins drive genomic reorganization and gene expression associated with developmental changes in the retinal progenitor cell population.
Results :
Exploring the impacts associated with transcription factors outside of their developmental context allowed us to observe changes without the redundancy of the factors associated with retinal development. The ATAC-seq profiles of NFI fusion protein transfected HepG2 varied from control transfected cells. However, RNA-sequencing did not show large differences between control and fusion protein transfected HepG2 cells, likely due to the short duration of exposure to the degron stabilizing small molecule.
Conclusions :
Determining if an individual transcription factor drives genomic organization changes is a difficult task during development due to overlap in both expression and target sites in gene families. By investigating a gene’s effect outside of the developmental context, we can observe changes linked directly to a gene of interest. In addition, this approach reveals potential targets directly associated with a transcription for future in vivo testing. This work shows how the NFI family dictates organizational changes during retinal development in their expression timeline.
This is a 2020 ARVO Annual Meeting abstract.