Reassessing the contribution of the histone H3 tail to KRAB-DNMT3L-mediated epigenetic silencing

Abstract

Neumann et al. introduced CHARM, a compact epigenetic silencer in which a histone H3 tail fused to DNMT3L was proposed to recruit and stimulate endogenous DNMT3A, enabling durable gene repression without a fused DNMT3A catalytic domain. Here, we evaluated the contribution of the H3 tail in independent reporter and endogenous-gene contexts. In an SNRPN reporter system, a KRAB-DNMT3L-dCas9 construct lacking the H3 tail displayed silencing kinetics comparable to CRISPRcharm Kv2, and mutating the critical H3K4 residue to alanine in CRISPRcharm Kv2 did not compromise this silencing. Similarly, after transient delivery of editor mRNAs to HEK293T cells, CRISPRcharm Kv2 did not consistently outperform the corresponding H3-tail-free construct at three endogenous loci, and mutating the critical H3K4 residue to alanine in CRISPRcharm Kv2 did not compromise this activity. These observations suggest that the engineered H3 tail does not confer a general functional advantage within the KRAB-DNMT3L-dCas9 architecture under the conditions tested.