High-throughput development and characterization of new functional nanobodies for gene regulation and epigenetic control in human cells

Reviewer #1 (Public review):

Summary:

This study presents a high-throughput screening platform to identify nanobodies capable of recruiting chromatin regulators and modulating gene expression. The authors utilize a yeast display system paired with mammalian reporter assays to validate candidate nanobodies, aiming to create a modular resource for synthetic epigenetic control.

Strengths:

(1) The overall screening design combining yeast display with mammalian functional assays is innovative and scalable.

(2) The authors demonstrate proof-of-concept that nanobody-based recruitment can repress or activate reporter expression.

(3) The manuscript contributes to the growing toolkit for epigenome engineering.

Weaknesses:

(1) The manuscript does not investigate which endogenous factors are recruited by the nanobodies. While repression activity is demonstrated at the reporter level, there is no mechanistic insight into what proteins are being brought to the target site by each nanobody. This limits the interpretability and generalizability of the findings. Related to this, Figure S1B reports sequence similarity among complementarity-determining regions (CDRs) of nanobodies that scored highly in the DNMT3A screen. However, it remains unclear whether this similarity reflects convergence on a common molecular target or is coincidental. Without functional or proteomic validation, the relationship between sequence motifs and effector recruitment remains speculative.

(2) The epigenetic consequences of nanobody recruitment are also left unexplored. Despite targeting epigenetic regulators, the study does not assess changes such as DNA methylation or histone modifications. This makes it difficult to interpret whether the observed reporter repression is due to true chromatin remodeling or secondary effects.

Reviewer #2 (Public review):

Summary:

Wan, Thurm et al. use a yeast nanobody library that is thought to have diverse binders to isolate those that specifically bind to proteins of their interest. The yeast nanobody library collection in general carries enormous potential, but the challenge is to isolate binders that have specific activity. The authors posit that one reason for this isolation challenge is that the negative binders, in general, dampen the signal from the positive binders. This is a classic screening problem (one that geneticists have faced over decades) and, in general, underscores the value of developing a good secondary screen. Over many years, the authors have developed an elegant platform to carry out high-throughput silencing-based assays, thus creating the perfect secondary screen platform to isolate nanobodies that bind to chromatin regulators.

Strengths:

Highlights the enormous value of a strong secondary screen when identifying binders that can be isolated from the yeast nanobody library. This insight is generalizable, and I expect that this manuscript should help inspire many others to design such approaches.

Provides new cell-based reagents that can be used to recruit epigenetic activators or repressors to modulate gene expression at target loci.

Weaknesses:

The authors isolate DNMT3A and TET1/2 enzymes directly from cell lysates and bind these proteins to beads. It is not clear what proteins are, in fact, bound to beads at the end of the IP. Epigenetic repressors are part of complexes, and it would be helpful to know if the IP is specific and whether the IP pulls down only DNMT3A or other factors. While this does not change the underlying assumptions about the screen, it does alter the authors' conclusions about whether the nanobody exclusively recruits DNMT3A or potentially binds to other co-factors.

Using IP-MS to validate the pull-down would be a helpful addition to the manuscript, although one could very reasonably make the case that other co-factors get washed away during the course of the selection assay. Nevertheless, if there are co-factors that are structural and remain bound, these are likely to show up in the MS experiment.