Nanobody-mediated control of gene expression and epigenetic memory

Mike V Van,Lacramioara Bintu,Taihei Fujimori

doi:10.1038/s41467-020-20757-1

Abstract

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. However, many chromatin regulators are large, making them difficult to deliver and combine in mammalian cells. Here, we develop a strategy for gene control using small nanobodies that bind and recruit endogenous chromatin regulators to a gene. We show that an antiGFP nanobody can be used to simultaneously visualize GFP-tagged chromatin regulators and control gene expression, and that nanobodies against HP1 and DNMT1 can silence a reporter gene. Moreover, combining nanobodies together or with other regulators, such as DNMT3A or KRAB, can enhance silencing speed and epigenetic memory. Finally, we use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder. These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory.

Highlights

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology
The Krüppelassociated box (KRAB), a strong repressive domain commonly used in gene circuits and CRISPR interference (CRISPRi) applications[1], recruits KAP1, which in turn interacts with other repressors such as histone deacetylases (HDACs) via the NuRD complex[2], histone methylase SETDB1, and heterochromatic protein HP13
We fused this nanobody to a reverse tetracycline repressor DNA-binding domain and used it to recruit various publicly available green fluorescent protein (GFP)-tagged chromatin regulators (CRs) (GFP-CR) to a TagRFP fluorescent reporter gene located at the AAVS1 locus in HEK293T cells (Fig. 1a)

Summary

Introduction

Targeting chromatin regulators to specific genomic locations for gene control is emerging as a powerful method in basic research and synthetic biology. We use the slow silencing speed and high memory of antiDNMT1 to build a signal duration timer and recorder These results set the basis for using nanobodies against chromatin regulators for controlling gene expression and epigenetic memory. The development of tools that can target a specific gene and alter its expression in a controlled manner is crucial for the advancement of this field These tools consist of one or more effector domains from transcription factors or chromatin regulators (CRs), fused to a DNA-binding domain that can bind to a specific sequence (e.g., Tetracycline Repressor or Gal[4], mostly used as components of synthetic genetic circuits) or be engineered to target any sequence in the genome (e.g., Zinc Fingers, TALEs, or dCas[9]).

Methods

Results

Conclusion