TRACE generates fluorescent human reporter cell lines to characterize epigenetic pathways

Iva A Tchasovnikarova,Sharon K Marr,Manashree Damle,Robert E Kingston

doi:10.1016/j.molcel.2021.11.035

Iva A Tchasovnikarova, Sharon K Marr + Show 2 more

Open Access

https://doi.org/10.1016/j.molcel.2021.11.035

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Abstract

Genetically encoded biosensors are powerful tools to monitor cellular behavior, but the difficulty in generating appropriate reporters for chromatin factors hampers our ability to dissect epigenetic pathways. Here, we present TRACE (transgene reporters across chromatin environments), a high-throughput, genome-wide technique to generate fluorescent human reporter cell lines responsive to manipulation of epigenetic factors. By profiling GFP expression from a large pool of individually barcoded lentiviral integrants in the presence and absence of a perturbation, we identify reporters responsive to pharmacological inhibition of the histone lysine demethylase LSD1 and genetic ablation of the PRC2 subunit SUZ12. Furthermore, by manipulating the HIV-1 host factor LEDGF through targeted deletion or fusion to chromatin reader domains, we alter lentiviral integration site preferences, thus broadening the types of chromatin examined by TRACE. The phenotypic reporters generated through TRACE will allow the genetic interrogation of a broad range of epigenetic pathways, furthering our mechanistic understanding of chromatin biology.

Highlights

Encoded biosensors are powerful tools to dissect the molecular mechanisms underlying complex cellular pathways
Integrate into the bodies of actively transcribed genes; we demonstrate that ablation of the HIV-1 integrase-binding cellular factor lens epithelium-derived growth factor (LEDGF) redirects lentiviral integrations away from gene bodies, thereby allowing TRACE to sample a wider range of chromatin environments
The green fluorescent protein (GFP)+ population was isolated by fluorescenceactivated cell sorting (FACS) to generate a TRACE library, which we estimated to contain $100,000 unique integrations based on the starting proportion of GFP+ cells

Summary

Introduction

Encoded biosensors are powerful tools to dissect the molecular mechanisms underlying complex cellular pathways. Existing methods to generate chromatin reporter lines involve either knockin of a fluorescent protein into an endogenous gene whose transcription is affected by a perturbation of interest or targeted knockin of an artificial expression cassette into a genomic locus thought to be regulated by the epigenetic pathway under investigation. A broadly applicable method capable of generating chromatin reporter cell lines would unleash the power of mammalian genetics to study epigenetic processes in human cells

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