Engineering the Epigenetic Regulatory Networks of T Cell Exhaustion and Embryonic Stem Cell Differentiation with Transcription Factor Perturbations

Abstract

The complex interplay of epigenetic factors including chromatin remodeling, histone modifications, and transcription factor binding regulate gene expression and therefore give rise to the diversity of cells within the human body from a common genome. Transcription factors are master regulators of these gene programs and are an attractive lever to manipulate the 1000’s of regulatory elements at the end of a signal cascade with a single perturbation. Here, we screen transcription factor perturbations and characterize their ability to engineer epigenetic regulatory networks in the context of T cell exhaustion or embryonic stem cell differentiation.

First, we develop a new in vitro model to study T cell exhaustion suitable for high-throughput screens. We use single-cell genomics to demonstrate our T cells recapitulate the exhaustion seen in human tumor infiltrating lymphocytes functionally, transcriptionally, and epigenetically.

Second, we identify the transcription factor Ikaros as a regulator of T cell exhaustion with a CRISPR knockout screen. We determine that knockout of Ikaros is sufficient to prevent the establishment of exhaustion in our model and preserve effector function. We use the clinically approved Ikaros degrader iberdomide to phenocopy our knockout and characterize its genome-wide effects on the transcriptome and epigenome. Then, we determine its mechanism of action, demonstrating that Ikaros represses enhancers of key effector genes by recruiting nucleosomes and preventing the binding of the activation transcription factor families AP-1, NFAT, ETS, and NF-kB/REL.

Third, we develop a new single-cell, multi-omic screening method termed Perturb SHARE-seq to enable high-throughput perturbations paired with rich readouts. We use this method both for CRISPR knockout screens in hematopoietic differentiation and transcription factor overexpression to guide differentiation of embryonic stem cells.

Collectively, our results highlight the utility of transcription factor perturbations and necessity of single cell epigenomics for understanding and manipulating cell state. Our workflow can be generalized to map the epigenetic regulatory networks in a variety of biological contexts and perturb each network member to investigate its role in controlling gene regulation and cell state.