Targeted epigenetic editing of SPDEF reduces mucus production in lung epithelial cells

Abstract

Airway mucus hypersecretion contributes to the morbidity and mortality in patients with chronic inflammatory lung diseases. Reducing mucus production is crucial for improving patients' quality of life. The transcription factor SAM-pointed domain-containing Ets-like factor () plays a critical role in the regulation of mucus production and, therefore, represents a potential therapeutic target. This study aims to reduce lung epithelial mucus production by targeted silencing using the novel strategy, epigenetic editing. Zinc fingers and CRISPR/dCas platforms were engineered to target repressors (KRAB, DNA methyltransferases, histone methyltransferases) to the promoter. All constructs were able to effectively suppress both mRNA and protein expression, which was accompanied by inhibition of downstream mucus-related genes [anterior gradient 2 (), mucin 5AC ()]. For the histone methyltransferase G9A, and not its mutant or other effectors, the obtained silencing was mitotically stable. These results indicate efficient silencing and downregulation of mucus-related gene expression by epigenetic editing, in human lung epithelial cells. This opens avenues for epigenetic editing as a novel therapeutic strategy to induce long-lasting mucus inhibition.

Cancer induction and suppression with transcriptional control and epigenome editing technologies

“…Cancer suppression with G9a was reported by Rots and Hylkema groups [50]. They introduced de novo histone methylation using dCas9-G9a (catalytic domain of G9a fused with dCas9) in A549 lung cancer cells.…”

“…For the suppressors, Krüppel-associated box (KRAB) domain [45] and mSin3 interaction domain (SID) [49] are often used. Of these, transcriptional suppression mediated by KRAB domain has frequently been reported in cancer modeling and therapeutic approaches, including the induction of breast cancer [31] and the suppression of several cancers including ovarian [33], lung [50], and monocytic leukemia [51].…”

“…DNMT3A is a DNA methyltransferase working in de novo methylation. DNMT3A-mediated tumor suppression was shown by introducing DNA methylation at the promoter regions of oncogenes in MCF7 cells [64], SKOV3 ovarian cancer cells [32], and A549 lung cancer cells [50]. One especially notable research is SOX2 silencing by Blancafort and colleagues [64], showing the tumor growth suppression in vivo by methylating DNA using ZF-DNMT3A (fusion protein of ZF and the catalytic domain of DNMT3A) targeting the SOX2 promoter.…”

“…Since there are a wide variety of histone modifications mediated by numerous effectors, programmable enzymes targeting histone tags are also variable compared to those for the DNA modification; e.g., histone demethylase (LSD1 [34,42]), histone methyltransferases (PRDM9 [40], G9a [30,33,50,65] and SUV39H1 [30,33]), and histone acetyltransferase (p300) [66].…”

“…Especially, G9a, also known as EHMT2, is a lysine 9 of histone H3 methyltransferase, which has been proven to be utilized in cancer modeling [65] and suppression [33,50] as a programmable enzyme. Kim and colleagues demonstrated the cancer modeling with the catalytic SET domain of G9a [65], by showing that cancer malignancy was enhanced in HeLa and HCT116 colon cancer cells.…”