Long non-coding RNA PARTICLE bridges histone and DNA methylation

Abstract

PARTICLE (Gene PARTICL- 'Promoter of MAT2A-Antisense RadiaTion Induced Circulating LncRNA) expression is transiently elevated following low dose irradiation typically encountered in the workplace and from natural sources. This long non-coding RNA recruits epigenetic silencers for cis-acting repression of its neighbouring Methionine adenosyltransferase 2A gene. It now emerges that PARTICLE operates as a trans-acting mediator of DNA and histone lysine methylation. Chromatin immunoprecipitation sequencing (ChIP-seq) and immunological evidence established elevated PARTICLE expression linked to increased histone 3 lysine 27 trimethylation. Live-imaging of dbroccoli-PARTICLE revealing its dynamic association with DNA methyltransferase 1 was confirmed by flow cytometry, immunoprecipitation and direct competitive binding interaction through electrophoretic mobility shift assay. Acting as a regulatory docking platform, the long non-coding RNA PARTICLE serves to interlink epigenetic modification machineries and represents a compelling innovative component necessary for gene silencing on a global scale.

Figure 1

PARTICLE and low dose irradiation act synergistically to enhance the H3K27me3 modification. (A) Absolute quantification of H3K27me3 per histone extraction concentration (ng/mg His. Ex.) as determined by ELISA 2 hr, 24 hr or 48 hr in MDA-MB-361 transfected with lipofectamine (LF) or PARTICLE transcript (over-expression: OE) plus or minus exposure to 0.025 Gy. Data are represented as mean ± SEM and the asterisk(s) represent significant values (p < 0.05). (B) Representative Western blots of Histone 3 (H3) and H3K27me3 (upper) in MDA-MB-361 (WT) in LF, NC1 control (NCTL) transfected, OE or OE exposed to 0.025 Gy (OE + I). Cropped images of Western blots shown. (C) Epifluorescence micrographs of immunofluoresently detected H3K27me3 (green) in MDA-MB-361 at the 24 hr time point in WT, OE or OE exposed to 0.025 Gy (OE + I). DAPI stained nuclei (blue; merged images below). Scale bar 5 μm. (D) Peak correlation scatterplot generated by pairwise comparison of H3K27me3 in irradiated MDA-MB-361 (WT + I) versus irradiated MDA-MB-361 with PARTICLE overexpression (OE + I). (E) Heatmaps of H3K27me3 distributions across five clustered differentially active regions (C1–C5) in irradiated MDA-MB-361 (WT + I) versus irradiated MDA-MB-361 with PARTICLE overexpression (OE + I). (F) Pie charts illustrating the genome-wide distribution patterns of H3K27me3 in irradiated MDA-MB-361 (WT + I) and irradiated MDA-MB-361 with PARTICLE overexpression (OE + I). DP: Distal Promoter (1–5 Kb); PP: Proximal Promoter (0–1 Kb); 5′UTR: 5′ untranslated region; EX: Exon; INT: Intron; 3′UTR: 3′ untranslated region; PD: Promoter Downstream (0–1 Kb); DD: Distal Downstream Promoter (1–5 Kb); DI: Distal Intergenic Region.

Figure 2

(A) Integrative Genomics Viewer screenshot (http://software.broadinstitute.org/software/igv/) of H3K27me3 ChIP-seq track peaks across the human genome (hg 19 assembly) in MDA-MB-361 cells exposed 24 hr previously to 0.025 Gy (WT + I, lower) and over-expressing PARTICLE (OE + I, upper). Chromosomal numbers are displayed. (B,C) Chromosomal location (red rectangle; top) and integrated genomics viewer screenshots for ChIP-seq data of OE + I (upper) and WT + I (lower). Genomic regions displayed represent MAT2A (B) and WWOX (C) loci plus 1.5 kb and 50 kb upstream respectively. Y-axis indicates the read per million (RPM) height of the ChIP-seq readout. CpG island in MAT2A (Chr. 2: 85765695–85766983) and WWOX (Chr. 16: 78133076–78134066) are depicted as green boxes. (D,E) Consensus motif logos within WWOX in WT + I (D) and OE + I (E).

Figure 3

In vivo PARTICLE and DNA methyltransferase 1 (DNMT1) interaction. (A) Linearized dbroccoli in pUC57 (2956 bp; p.db.) after EcoRI/HindIII restriction enzyme digestion resolved on a 1.8% agarose gel (left). Plasmid pGEM-T encoding dbroccoli-PARTICLE (p.db.PT) restriction digested with Nco I/HindIII to produce 3728 bp and 950 bp products (upper and lower arrows). DL3000 (Genscript) and Kb ladder (Ld. left and right gels respectively). (B) dbroccoli-PARTICLE (1678 bp; db.PT) in vitro transcript resolved through a 12% NuPAGE gel alongside a high range RNA ladder (Ld. Thermo Fisher RiboRuler) before and after staining in DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4H)-one; left and right gels respectively). (A,B) Cropped images of gels shown. (C) Time-lapse fluorescence images of a U2OS cell transfected with db.PT and expressing a chromobody to DNMT1-V_HH fused to TagRFP (DNMT1_RFP) before (minus time point) and after (plus time points) DFHBI-1T (20 μM) addition. Scale bar 2 μm. (D) Histograms of arbitrary units (AU) of overall fluorescence intensity with time in U2OS transfected with db.PT dose range in the presence of DFHBI-1T (20 μM). Data are represented as mean ± SEM from n = 3 experiments per dose. (E) Summary co-localization plots for DNMT_RFP (ChR = red channel) and db.PT (ChG = green channel) from ROIs in the nucleus with time. Data are represented as mean ± SEM. (F,G) Plots of AU of fluorescence intensity with time from DNMT_RFP (F) and in db.PT (G) in ROIs (n = 10) within nuclear (encircling dashed line) and extranuclear (Extranuc.) cellular compartments.

Figure 4

PARTICLE is implicated in global methylome enhancement, WWOX CpG island methylation and enzyme activity via DNMT1 interaction. (A) Histograms of global methylome percentage (5-mC ng/100ng genomic DNA input) from MDA-MD-361 post 24 hr sham irradiation lipofectamine control (LF) with PARTICLE over-expression (O) or knockdown (K) and/or 0.025 Gy exposure (I). (B) Histograms showing percentage hypermethylation of the CpG island within the WWOX promoter in MDA-MB-361 as indicated in (Fig. 2C) above. (C) Histograms showing DNMT activity in MDA-MB-361 (nomenclature as indicated in (A) above) and with PARTICLE (P) and dbroccoli-PARTICLE (dbP) overexpression. Data are represented as mean ± SEM (n = 3) and the asterisk(s) represent significant values (p < 0.05). (D) Flow cytometry scatter plots of MDA-MB-361 transfected with lipofectamine (LF), DNMT_RFP (DNMT1) and dbroccoli-PARTICLE in the absence or presence of DFHBI-1T (20 μM). Time points since DFHBI-1T addition or comparative interval. (E,F) Representative nucleotide retardation gels (6%) of electrophoretic mobility shift assay involving binding reactions containing negative in vitro transcript control (NC1; E) or biotinylated dbroccoli-PARTICLE (b-db.PT., 10 nM; F), ±human DNMT1 peptide (2.5 μM) and increasing concentrations of unlabeled PARTICLE. (G) PARTICLE (PT.) or dbroccoli-PARTICLE (dbPT.) pulldown in U2OS and immunodetection with anti-DNA methyltransferases. (E,G) Cropped images of gels shown.