Nanobody-mediated control of gene expression and epigenetic memory

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 developed a new 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.

Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing

“…Recent work has demonstrated that it is possible for epigenome editing to write a stable transcriptional program that is remembered and propagated by human cells without constitutive expression of the programmable epigenetic modulators (Amabile et al, 2016;Bintu et al, 2016;Park et al, 2019;Van et al, 2021). In particular, Amabile et al (2016) showed that it was possible to heritably silence human genes by recruitment of a cocktail of DNA methyltransferase and KRAB domains.…”

Nanobodies identify an activated state of the TRIB2 pseudokinase

“…Reagents that can differentiate between the two most closely-related Tribbles proteins will be key to fully understanding the different functions of TRIB1 and TRIB2, and eventually to their targetability in disease. While nanobodies seem unlikely to be therapeutically viable option for Tribbles proteins, which are intracellular and expressed during differentiation—they could be extremely valuable as research reagents (Cheloha et al, 2020; Van et al, 2021). The three nanobodies we report here exhibit specificity for TRIB2 over both TRIB1 and TRIB3, and also differ in solution behaviour.…”

Novel synthetic biology approaches for developmental systems

“…Although small-molecule modifiers of epigenetic silencing have been used successfully in vitro, it is likely that these would affect the developmental potential of stem cells also thereby altering efforts to mimic developmental transitions if applied to embryo models. Another possibility for controlling epigenetic silencing could lie in synthetic factors that could insulate or control the epigenetic status of user-defined genetic loci (Bintu et al, 2016;Park et al, 2016Park et al, , 2019Van et al, 2021). In the future, complex synthetic circuits could contain sensors for epigenetic silencing that trigger synthetic reversion of silencing itself.…”