Abstract
BackgroundEpigenome editing is expected to manipulate transcription and cell fates and to elucidate the gene expression mechanisms in various cell types. For functional epigenome editing, assessing the chromatin context-dependent activity of artificial epigenetic modifier is required.ResultsIn this study, we applied clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9-based epigenome editing to mouse primary T cells, focusing on the Forkhead box P3 (Foxp3) gene locus, a master transcription factor of regulatory T cells (Tregs). The Foxp3 gene locus is regulated by combinatorial epigenetic modifications, which determine the Foxp3 expression. Foxp3 expression is unstable in transforming growth factor beta (TGF-β)-induced Tregs (iTregs), while stable in thymus-derived Tregs (tTregs). To stabilize Foxp3 expression in iTregs, we introduced dCas9-TET1CD (dCas9 fused to the catalytic domain (CD) of ten-eleven translocation dioxygenase 1 (TET1), methylcytosine dioxygenase) and dCas9-p300CD (dCas9 fused to the CD of p300, histone acetyltransferase) with guide RNAs (gRNAs) targeted to the Foxp3 gene locus. Although dCas9-TET1CD induced partial demethylation in enhancer region called conserved non-coding DNA sequences 2 (CNS2), robust Foxp3 stabilization was not observed. In contrast, dCas9-p300CD targeted to the promoter locus partly maintained Foxp3 transcription in cultured and primary T cells even under inflammatory conditions in vitro. Furthermore, dCas9-p300CD promoted expression of Treg signature genes and enhanced suppression activity in vitro.ConclusionsOur results showed that artificial epigenome editing modified the epigenetic status and gene expression of the targeted loci, and engineered cellular functions in conjunction with endogenous epigenetic modification, suggesting effective usage of these technologies, which help elucidate the relationship between chromatin states and gene expression.
Highlights
Epigenome editing is expected to manipulate transcription and cell fates and to elucidate the gene expression mechanisms in various cell types
Mouse TET1 catalytic domain (TET1CD) H1620Y, D1622A, and mouse p300CD D1398Y were mutated for catalytic inactive mutants
We examined the promotive effect of dCas9-TET1CD on Forkhead box P3 (Foxp3) stability in induced Tregs (iTregs)
Summary
Epigenome editing is expected to manipulate transcription and cell fates and to elucidate the gene expression mechanisms in various cell types. Artificial epigenome editing is a novel strategy for manipulating cell fate by altering the specific epigenomic landscape and can help elucidate the mechanisms between chromatin states and gene expression [1]. DCas fusion with p300, lysine-specific demethylase 1 (LSD1), Krüppel-associated box (KRAB), DNA methyltransferase 3a (DNMT3a), and ten-eleven translocation (TET) dioxygenase 1 (TET1) enable gene expression regulation by modifying epigenetic states [7,8,9,10,11]. These biological devices were developed by using cultured cell lines and clearly proposed their versatile performance. Little is known about the relationship between artificial epigenome editing and endogenous epigenetic modifications in immune cells
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