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. 2021 Aug 5;11(1):15912.
doi: 10.1038/s41598-021-95398-5.

Investigating crosstalk between H3K27 acetylation and H3K4 trimethylation in CRISPR/dCas-based epigenome editing and gene activation

Weiye Zhao #  1 , Ying Xu #  1 , Yufan Wang  1 , Dan Gao  1 , Jasmine King  1 , Yajie Xu  1 , Fu-Sen Liang  2
Affiliations

Investigating crosstalk between H3K27 acetylation and H3K4 trimethylation in CRISPR/dCas-based epigenome editing and gene activation

Weiye Zhao et al. Sci Rep. .

Abstract

Epigenome editing methods enable the precise manipulation of epigenetic modifications, such as histone posttranscriptional modifications (PTMs), for uncovering their biological functions. While histone PTMs have been correlated with certain gene expression status, the causalities remain elusive. Histone H3 Lysine 27 acetylation (H3K27ac) and histone H3 Lysine 4 trimethylation (H3K4me3) are both associated with active genes, and located at active promoters and enhancers or around transcriptional start sites (TSSs). Although crosstalk between histone lysine acetylation and H3K4me3 has been reported, relationships between specific epigenetic marks during transcriptional activation remain largely unclear. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/dCas-based epigenome editing methods, we discovered that the ectopic introduction of H3K27ac in the promoter region lead to H3K4me3 enrichment around TSS and transcriptional activation, while H3K4me3 installation at the promoter cannot induce H3K27ac increase and failed to activate gene expression. Blocking the reading of H3K27ac by BRD proteins using inhibitor JQ1 abolished H3K27ac-induced H3K4me3 installation and downstream gene activation. Furthermore, we uncovered that BRD2, not BRD4, mediated H3K4me3 installation and gene activation upon H3K27ac writing. Our studies revealed the relationships between H3K27ac and H3K4me3 in gene activation process and demonstrated the application of CRISPR/dCas-based epigenome editing methods in elucidating the crosstalk between epigenetic mechanisms.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Investigating the H3K27ac/H3K4me3 crosstalk and functions through CRISPR/dCas9 based epigenome editing approaches.
Figure 2
Figure 2
Locus-specific writing of H3K27ac induced by dCas9-p300. (a) and (b) The sites targeted by sgRNAs (red stars) and locations probed by qPCR amplicon (grey solid circles) at the IL1RN and GRM2 loci. Enrichment of H3K27ac written induced by dCas9-p300 targeting at (c) IL1RN and (f) GRM2 loci. Enrichment of H3K4me3 at (d) IL1RN and (g) GRM2 loci upon targeted dCas9-p300 editing of H3K27ac. Changes in mRNA expression levels of (e) IL1RN and (h) GRM2 upon induced H3K27ac writing. Fold changes of H3K27ac or H3K4me3 enrichment and mRNA level changes were calculated by normalizing results to those from samples of dCas9-PYL. Error bars represent ± s.e.m. from biological replicates (n = 6 for c and d, n = 3 for e–h). The p value less than 0.05 was marked as *, less than 0.01 as ** and less than 0.001 as ***.
Figure 3
Figure 3
Locus-specific writing of H3K4me3 induced by dCas9-SET(CD). Enrichment of H3K4me3 at (a) IL1RN and (d) GRM2 loci upon dCas9-SET(CD) (or dCas9-SET*) targeted H3K4me3 editing. Enrichment of H3K27ac at (b) IL1RN and (e) GRM2 loci upon targeted dCas9-SET editing of H3K4me3. Changes in mRNA expression levels of (c) IL1RN and (f) GRM2 upon induced H3K4me3 writing. Fold changes of H3K27ac and H3K4me3 enrichment as well as mRNA level changes were calculated by normalizing results to those from samples of dCas9-PYL. Error bars represent ± s.e.m. from biological replicates (n = 6 for a,b; n = 3 for c, f; n = 2 for d,e). The p value less than 0.05 was marked as *, less than 0.01 as ** and less than 0.001 as ***.
Figure 4
Figure 4
Effects of JQ1 treatment in dCas9-p300-induced H3K27ac writing and gene activation. The mRNA level changes of (a) IL1RN and (b) GRM2 under dCas9-p300 targeting with or without JQ1 treatment. The enrichment fold changes of RNA polymerase II at (c) IL1RN and (d) GRM2 loci upon JQ1 treatment. The enrichment fold changes of H3K27ac at (e) IL1RN and (f) GRM2 loci upon JQ1 treatment. The enrichment fold changes of H3K4me3 at (g) IL1RN and (h) GRM2 loci upon JQ1 treatment. Fold changes of Pol II, H3K27ac and H3K4me3 enrichment as well as the mRNA level changes were calculated by normalizing results to those from samples of DMSO treatment. Error bars represent ± s.e.m. from biological replicates (n = 3 for a–d; n = 4 for e,f; n = 5 for g,h). The p value less than 0.05 was marked as *, less than 0.01 as ** and less than 0.001 as ***.
Figure 5
Figure 5
Recruitment of BRD proteins to dCas9-p300 targeted genome loci. Enrichment of BRD4 at (a) IL1RN and (c) GRM2 loci upon dCas9-p300 targeting. Enrichment of BRD2 at (b) IL1RN and (d) GRM2 loci upon dCas9-p300 targeting. Fold changes of BRD2 and BRD4 enrichment were calculated by normalizing results to those from samples of dCas9-PYL. Error bars represent ± s.e.m. from biological replicates (n = 3; a,b; n = 5 for c; n = 4 for d). The p value less than 0.05 was marked as *, less than 0.01 as ** and less than 0.001 as ***.
Figure 6
Figure 6
The effects of JQ1 on BRD recruitment upon dCas9-p300 targeting. Enrichment of BRD2 at (a) IL1RN and (c) GRM2 loci upon JQ1 treatment under dCas9-p300 targeting. Enrichment of BRD4 at (b) IL1RN and (d) GRM2 loci upon JQ1 treatment under dCas9-p300 targeting. Fold changes of BRD2 and BRD4 enrichment were calculated by normalizing results to those from DMSO-treated samples. Error bars represent ± s.e.m. from biological replicates (n = 3 for a; n = 5 for b,d; n = 4 for c). The p value less than 0.05 was marked as *, less than 0.01 as ** and less than 0.001 as ***.
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