Understanding the chromatin architectural landscape of retinal development at the single-cell level

Project Abstract: The 3D organization of the genome is important in controlling neuronal cell lineage commitment. As an extension of the CNS, the retina is an excellent model for studying neuronal development. One issue in studying the mouse retina is that it is dominated by rod photoreceptors, leading to bulk studies that overlook

less abundant cell types. The Blackshaw Lab and others have used single cell technologies to develop transcriptomic (scRNA-seq) and epigenomic (scATAC-seq) atlases of mouse retinogenesis. These studies have revealed cell-type-specific patterns of transcription factor (TF) mediated remodeling of chromatin that

occurs during developmental cell state transcriptions and are predicted to influence the 3D chromatin architecture; however, the direct link has not been established. Previous studies on genome-wide chromatin interactions using Hi-C have been conducted at the bulk level, are sparse, or focused only on rod

photoreceptors. A higher-resolution single-cell Hi-C (scHi-C) study is needed to understand changes in chromatin organization associated with the specification of different retinal cell types. Through the integration of multi-omic data at single cell resolution, I aim to identify how transcription factors manipulate genomic structure to control cell fate specification. During retinal development, expression

of the NFI family of TFs (NFIA/B/X) controls the competence of late-stage RPCs and drives specification of late-born retinal cell types (late-born rods, bipolar cells, and Muller glia). The Blackshaw lab’s previous scRNA- seq and scATAC-seq studies have found many thousands of genes and cis-regulatory-elements that are

differentially regulated upon Nfia/b/x loss-of-function, which suggests that the NFI family TFs affect chromatin state. Other studies have also found that the NFI family TFs affect chromatin compartmentalization and promote the formation of super-enhancers. However, little is known about whether the NFI family TFs directly

regulate chromatin structure to activate downstream genes. In this proposed research, I will capture the 3D chromatin interactions of mouse retinal cells at the single-cell level and at key developmental stages, using droplet microfluidic scHi-C (Aim 1). ScHi-C will be adapted for the 10x Genomics droplet single-cell system and applied to the mouse retina at key developmental

stages. I will then use scHi-C to capture changes in chromatin organization associated with retinal progenitor- specific loss-of-function of Nfia/b/x (Aim 2). To do this, I will first combine scHi-C with the Blackshaw Lab’s previously obtained scATAC-seq, scRNA-seq, and Nfia/b/x ChIP-seq datasets to form a multiomic dataset of

retinal cells. I will then use the dataset to identify chromatin interactions associated with NFIA/B/X that drive specification of late-stage RPCs. The droplet microfluidic scHi-C protocol will provide a generalizable method that can be used for other genomic studies. The single-cell multi-omic dataset of retinal development will allow

improved construction of retinal gene regulatory networks and novel targets for treating retinal disorders.