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.
Highlights
Epigenome editing methods enable the precise manipulation of epigenetic modifications, such as histone posttranscriptional modifications (PTMs), for uncovering their biological functions
H3K4me[3] at promoters has been positively correlated with active g enes[10] and shown to reactivate gene expression[35], we found that H3K4me[3] is not sufficient for gene activation at the gene loci targeted by clustered regularly interspaced short palindromic repeats (CRISPR)/ dCas9-based editing methods in this study
We transfected HEK293T cells with the dCas9-p300 or dCas9-PYL plasmids along with reported sgRNAs to target the promoters of IL1RN33,34 (Fig. 2a) or GRM234 gene locus (Fig. 2b), which has been studied previously and validated for H3K27ac editing by CRISPR/dCas9-based methods. 72 h after transfection, cells were harvested and analyzed by chromatin immunoprecipitation (ChIP) assays using anti-H3K27ac or anti-H3K4me[3] antibodies followed by quantitative polymerase chain reaction assays using primers designed to determine the levels of enrichment at indicated genome loci surrounding the promoter region and transcriptional start sites (TSSs) under each condition (Fig. 2a,b)
Summary
Epigenome editing methods enable the precise manipulation of epigenetic modifications, such as histone posttranscriptional modifications (PTMs), for uncovering their biological functions. Our studies revealed the relationships between H3K27ac and H3K4me[3] in gene activation process and demonstrated the application of CRISPR/dCas-based epigenome editing methods in elucidating the crosstalk between epigenetic mechanisms. Clustered regularly interspaced short palindromic repeats (CRIPSR)/dCas9-based epigenome editing technologies allow programmable locus-specific manipulation of epigenetic environment without global disruption, which is advantageous in investigating epigenetic regulation in gene a ctivities[27,28,29,30,31,32] Using this approach, p300 acetyltransferase have been targeted locus to install H3K27ac at promoters and enhancers leading to transcriptional activation of targeted genes[33]. We uncovered the crosstalk between H3K27ac and H3K4me[3] and their roles in transcriptional activation
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