High-Throughput Discovery and Characterization of Human Transcriptional Effectors

Cell. 2020 Dec 23;183(7):2020-2035.e16. doi: 10.1016/j.cell.2020.11.024. Epub 2020 Dec 15.


Thousands of proteins localize to the nucleus; however, it remains unclear which contain transcriptional effectors. Here, we develop HT-recruit, a pooled assay where protein libraries are recruited to a reporter, and their transcriptional effects are measured by sequencing. Using this approach, we measure gene silencing and activation for thousands of domains. We find a relationship between repressor function and evolutionary age for the KRAB domains, discover that Homeodomain repressor strength is collinear with Hox genetic organization, and identify activities for several domains of unknown function. Deep mutational scanning of the CRISPRi KRAB maps the co-repressor binding surface and identifies substitutions that improve stability/silencing. By tiling 238 proteins, we find repressors as short as ten amino acids. Finally, we report new activator domains, including a divergent KRAB. These results provide a resource of 600 human proteins containing effectors and demonstrate a scalable strategy for assigning functions to protein domains.

Keywords: CRISPRi; Hox; KRAB; chromatin regulation; deep mutational scan; domain of unknown function; high-throughput screening; mammalian synthetic biology; protein domains; transcriptional effectors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • CRISPR-Cas Systems / genetics
  • Female
  • Gene Silencing
  • Genes, Reporter
  • HEK293 Cells
  • High-Throughput Screening Assays*
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Humans
  • K562 Cells
  • Lentivirus / physiology
  • Molecular Sequence Annotation
  • Mutation / genetics
  • Nuclear Proteins / metabolism
  • Promoter Regions, Genetic / genetics
  • Protein Domains
  • Repressor Proteins / chemistry
  • Repressor Proteins / metabolism
  • Reproducibility of Results
  • Transcription Factors / metabolism*
  • Transcription, Genetic
  • Zinc Fingers


  • Homeodomain Proteins
  • Nuclear Proteins
  • Repressor Proteins
  • Transcription Factors