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. 2017 May 8:10:24.
doi: 10.1186/s13072-017-0129-1. eCollection 2017.

Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells

Masahiro Okada  1 , Mitsuhiro Kanamori  1 , Kazue Someya  1 , Hiroko Nakatsukasa  1 , Akihiko Yoshimura  1
Affiliations

Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells

Masahiro Okada et al. Epigenetics Chromatin. .

Abstract

Background: Epigenome 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.

Results: In 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.

Conclusions: Our 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.

Keywords: CRISPR; Epigenome editing; Foxp3; TET1; Treg; dCas9; p300.

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Figures

Fig. 1
Fig. 1
CRISPR-dCas9-based epigenome editing for primary T cells. a A retroviral vector for the expression of dCas9-epigenome regulator fusion proteins from Moloney murine leukemia virus promoter long terminal repeats (ΔLTRs) and green fluorescent protein (GFP) from an internal ribosomal entry site (IRES). Retroviral and lentiviral vector for bicistronic expression of the gRNA from a U6 promoter (U6) and DsRed from a short EF1a promoter (EFS). b Protein expression of dCas9-epigenome regulator fusion proteins in transfected HEK293T cells was detected by western blot against anti-Flag antibody. Anti α-tubulin antibody was used for loading control
Fig. 2
Fig. 2
dCas9-TET1CD-mediated demethylation of the Foxp3 CNS2 locus. a Sequence at the Foxp3 CNS2 locus is shown. Each gRNA sequence is underlined and numbered #C2-1 to #C2-10. Specific CpG sites are lettered red. b and c The methylation status of CpG sites at the Foxp3 CNS2 locus in dCas9-TET1CD and each gRNA-expressing 68-41 cells (b) and dCas9-TET1CD or the TET1CD catalytic mutant and gRNA #C2-7 expressing 68-41 cells (c) was determined by bisulfite sequence analysis. The 68-41 cells stably expressing dCas9-TET1CD were transduced with each gRNA expression lentivirus and sorted (b). The 68-41 cells were co-transduced with dCas9-TET1CD or TET1CD mutant and gRNA CNS2#C2-7 and sorted (c). A horizontal row depicts one sequenced clone in which CpGs was methylated (black) or demethylated (white). Data are pooled from two independent experiments
Fig. 3
Fig. 3
Maintenance of Foxp3 expression induced by dCas9-TET1CD-mediated demethylation of the Foxp3 CNS2 locus. a The methylation status of CpG sites at the Foxp3 CNS2 locus in untransduced, dCas9-TET1CD single, with #C2-1, and with #C2-7 transduced iTregs was determined by bisulfite sequence analysis. GFP/DsRed(−/−), (+/−), and (+/+) cells are sorted, respectively. A horizontal row depicts one sequence clone in which CpGs was methylated (black) or demethylated (white). The number below bisulfite sequences indicates demethylated CpG sites. Data are pooled from two independent experiments. (b and c) Flow cytometry analysis of Foxp3(hCD2) expression in iTregs co-transduced with dCas9-TET1CD and gRNA CNS2 #C2-1 (blue) or #C2-7 (red) (b), dCas9-TET1CD (red), or TET1CD mutant (blue) and gRNA #C2-7 (c) under inflammatory cytokine conditions. Percentages of Foxp3(+) and mean fluorescence intensity (MFI) relative value to iTregs co-transduced with dCas9-TET1CD and #C2-1 (b), dCas9-TET1CD and #C2-7 (c) were plotted. Data are pooled from seven (b) or four (c) independent experiments and represent the means ± SDs. *p < 0.05
Fig. 4
Fig. 4
dCas9-p300CD-mediated Foxp3 promoter acetylation and transcriptional activation. a Sequence at the Foxp3 promoter locus is shown. Each gRNA sequence is underlined and numbered #P-1 to #P-10. Transcription start sites are lettered bold red. b Foxp3 mRNA expression in each gRNA-transduced 68-41 cell stably expresses dCas9-p300CD relative to control 68-41 cells (control). Data are pooled from three independent experiments and represent the means ± SDs. c Flow cytometry analysis of Foxp3 expression in each gRNA-transduced 68-41 cell stably expresses dCas9-p300CD. Foxp3 MFI relative value to control 68-41 cells was plotted. Data are pooled from three independent experiments and represent the means ± SDs. d Flow cytometry analysis of Foxp3 expression in each gRNA-transduced 68-41 cell stably expresses dCas9-p300CD or p300 mutant. e Enrichment of acetyl histone H3 at Foxp3 TSS locus in #P-3 or #P-4 transduced 68-41 cell stably expresses dCas9-p300CD. f Flow cytometry analysis of Foxp3 expression in each clone isolated by limiting the dilution from dCas9-p300CD and #P-4 co-transduced 68-41 cells
Fig. 5
Fig. 5
dCas9-p300CD-mediated Foxp3 transcriptional activation is strengthened by TGF-β signal. a Upper, experimental scheme. Magnetic-activated cell sorting (MACS)-sorted naïve CD4+ T cells were cultured under Th1, Th2, and Th17 skewing conditions. On day 2, dCas9-p300CD and gRNA #P-3 or #P-4 were transduced with polybrene and further cultured for 2 days. Lower, flow cytometry plots of Th subsets co-transduced with dCas9-p300 and gRNAs show expression of CD4 and Foxp3 (hCD2). Percentages of Foxp3(+) cells and subtraction of #P-3 from #P-4 were plotted. Data are pooled from three independent experiments and represent the means ± SDs. *p < 0.05; **p < 0.01. b Upper, experimental scheme. MACS-sorted naïve CD4+ T cells were cultured under Th1 skewing conditions, and on day 2, dCas9-p300CD and gRNAs were transduced with polybrene. The next day, Th1 was harvested and further cultured under Th1 or iTreg skewing conditions without T cell receptor (TCR) stimulation. Lower, flow cytometry plots of Th1 co-transduced with dCas9-p300CD and gRNAs show expression of CD4 and Foxp3 (hCD2). Percentages of Foxp3(+) cells were plotted. Data are pooled from three independent experiments and represent the means ± SDs. *p < 0.05; **p < 0.01
Fig. 6
Fig. 6
dCas9-p300CD-applied iTregs showed higher suppressive activity. a and b Flow cytometry analysis of Foxp3 (hCD2) expression in iTregs co-transduced with dCas9-p300CD and gRNA #P-3 (blue) or #P-4 (red) (a), dCas9-p300CD (red) or p300CD mutant (blue) and #P-4 (B). Percentages of Foxp3(+) cells and MFI relative value to iTregs co-transduced with dCas9-p300CD and #P-3 (a) or dCas9-p300CD and #P-4 (b) were plotted. Data are pooled from four independent experiments and represent the means ± SDs. *p < 0.05; **p < 0.01. b Relative expression of CD25 or CTLA-4 of IL-12-treated iTregs co-transduced with dCas9-p300CD and gRNAs. Relative MFI values to #P-3 were plotted. Data represent each experimental value of eight independent experiments. c Treg suppression activity was measured by CFSE dilution in labeled Teff. Flow cytometry analysis of CFSE dilution in labeled Teff, co-cultured with or without iTregs co-transduced with dCas9-p300CD and gRNA #P-3 (blue) or #P-4 (red). Foxp3 (hCD2) expression in iTregs co-transduced with dCas9-p300CD and gRNA #P-3 (blue) or #P-4 (red) was also analyzed. Data are pooled from three independent experiments and represent the means ± SDs. *p < 0.05; **p < 0.01
Fig. 7
Fig. 7
Model of epigenome editing in primary T cells. a dCas9-TET1CD demethylates the Foxp3 CNS2 locus and enhances Foxp3 expression weakly. b dCas9-p300CD acetylates the Foxp3 promoter locus, activates transcription in coordination with the TGF-β signal, and promotes immunosuppressive function
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References

    1. Thakore PI, Black JB, Hilton IB, Gersbach CA. Editing the epigenome: technologies for programmable transcription and epigenetic modulation. Nat Methods. 2016;13(2):127–137. doi: 10.1038/nmeth.3733. - DOI - PMC - PubMed
    1. Liu PQ, Rebar EJ, Zhang L, Liu Q, Jamieson AC, Liang Y, Qi H, Li PX, Chen B, Mendel MC, et al. Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J Biol Chem. 2001;276(14):11323–11334. doi: 10.1074/jbc.M011172200. - DOI - PubMed
    1. Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M, Cong L, Platt RJ, Scott DA, Church GM, Zhang F. Optical control of mammalian endogenous transcription and epigenetic states. Nature. 2013;500(7463):472–476. - PMC - PubMed
    1. Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol. 2014;32(4):347–355. doi: 10.1038/nbt.2842. - DOI - PMC - PubMed
    1. Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK. CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013;10(10):977–979. doi: 10.1038/nmeth.2598. - DOI - PMC - PubMed

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