High-throughput discovery and characterization of compact transcriptional effectors

Human gene expression is regulated by thousands of proteins that can activate or repress transcription. To model and manipulate gene expression, we need to measure the strength with which effectors regulate transcription when recruited to a promoter and the dynamics of transcription after they are released (i.e. epigenetic memory). To systematically measure the function of transcriptional effector domains in human cells, we developed a high-throughput assay (HT-recruit) in which pooled libraries of thousands of domains are recruited individually to a reporter gene. Cells are then separated by reporter expression level, and the library of protein domains is sequenced to determine the frequency of each domain in silenced versus active cell populations.

We used this method to:

1) quantify the activation, silencing, and epigenetic memory capability of most of the nuclear protein domains annotated in Pfam, including the KRAB family of over 300 domains. We find that while evolutionary young KRABs are strong repressors, some of the old KRABs are activators.

2) characterize the amino acids responsible for effector function via deep mutational scanning. We applied it to the KRAB domain used in CRISPRi to map the co-repressor binding surface and identify substitutions that improve stability, silencing, and epigenetic memory.

3) discover novel functional domains in unannotated regions of large chromatin regulators, including repressors as short as 10 amino acids.

The compact effectors we have identified so far can be used in synthetic biology and therapeutic applications, where small size is essential for efficient delivery. Our high-throughput method can be easily extended to answer a range of biological questions in processes that already have established fluorescent reporters: DNA repair, RNA splicing, protein degradation, and cell signaling.