NF-κB p65 (RelA) subunit transcriptional activation domain (p65)

Characteristics

More than 20 candidate effectors with known transcriptional roles were fused to the C terminus of S. pyogenes dCas9 and ranked by a fluorescent reporter assay in HEK293T cells. dCas9-VP64, dCas9-p65 and dCas9-Rta gave the most meaningful reporter induction of the panel, but neither the p65 nor the Rta fusion activated more strongly than dCas9-VP64. Adding p65 to dCas9-VP64 raised activity further, and adding both p65 and Rta in tandem produced VPR. Replacing p65 in VPR with mCherry confirmed that the domain contributes to VPR activity, and testing permutations confirmed that the VP64-p65-Rta order is optimal.

Applications: Middle domain of the VPR activator in dCas9-VPR, used to activate endogenous coding and noncoding genes in human cells, to activate several genes at once, and to differentiate human iPS cells into neurons. Also recruited in multiple copies by two-component activation systems, where repeated recruitment of a modest activation domain raises transcriptional output substantially.

Limitations: As a lone dCas9 fusion, dCas9-p65 is no stronger an activator than dCas9-VP64, so p65 earns its place in combination rather than on its own. Domain order matters: VP64-p65-Rta outperforms the other permutations tested, so p65 should sit between VP64 and Rta.

Mechanism: Transcriptional activation domain of the NF-κB p65 subunit. In VPR it adds a second activation domain between VP64 and Rta, where the three domains together activate more strongly than any of them alone.

References

1. Chavez et al. (2015). Highly efficient Cas9-mediated transcriptional programming. Nat Methods - Chavez 2015 VPR