pubmed.ncbi.nlm.nih.gov

PRMT5-mediated histone H4 arginine-3 symmetrical dimethylation marks chromatin at G + C-rich regions of the mouse genome - PubMed

PRMT5-mediated histone H4 arginine-3 symmetrical dimethylation marks chromatin at G + C-rich regions of the mouse genome

Michael Girardot et al. Nucleic Acids Res. 2014 Jan.

Abstract

Symmetrical dimethylation on arginine-3 of histone H4 (H4R3me2s) has been reported to occur at several repressed genes, but its specific regulation and genomic distribution remained unclear. Here, we show that the type-II protein arginine methyltransferase PRMT5 controls H4R3me2s in mouse embryonic fibroblasts (MEFs). In these differentiated cells, we find that the genome-wide pattern of H4R3me2s is highly similar to that in embryonic stem cells. In both the cell types, H4R3me2s peaks are detected predominantly at G + C-rich regions. Promoters are consistently marked by H4R3me2s, independently of transcriptional activity. Remarkably, H4R3me2s is mono-allelic at imprinting control regions (ICRs), at which it marks the same parental allele as H3K9me3, H4K20me3 and DNA methylation. These repressive chromatin modifications are regulated independently, however, since PRMT5-depletion in MEFs resulted in loss of H4R3me2s, without affecting H3K9me3, H4K20me3 or DNA methylation. Conversely, depletion of ESET (KMT1E) or SUV420H1/H2 (KMT5B/C) affected H3K9me3 and H4K20me3, respectively, without altering H4R3me2s at ICRs. Combined, our data indicate that PRMT5-mediated H4R3me2s uniquely marks the mammalian genome, mostly at G + C-rich regions, and independently from transcriptional activity or chromatin repression. Furthermore, comparative bioinformatics analyses suggest a putative role of PRMT5-mediated H4R3me2s in chromatin configuration in the nucleus.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.

PRMT5 expression, localization and histone methylation activity in embryonic cells. (A) RT-PCR analysis of Prmt5 and Prmt7 expression in primary MEFs and ES cells. RT+ and RT– indicate presence and absence of reverse transcriptase (RT), respectively. (B) Western blot analysis of total protein extracts. Lanes ‘PRMT5’ and ‘PRMT7’ were loaded with diluted protein samples of 293T cells in which the PRMT5 and PRMT7 proteins were over-expressed. β-Actin (ACTB) is used as a loading control. (C) Immunofluorescence staining of PRMT5 in MEFs and ES cells (upper panels). Nuclei were counter-stained with DAPI (middle panels). (D) Western blot analysis of cytoplasmic (Cyt) and nuclear (Nuc) protein fractions of MEFs and ES cells. Tubulin and H3K9me3 constitute cytoplasmic and nuclear controls, respectively. (E) Strongly reduced PRMT5 protein expression in MEFs stably infected with lentiviral shRNA constructs (sh-Prmt5-1 and sh-Prmt5-2) directed against Prmt5. As a negative control, MEFs stably expressing a scrambled shRNA (Sh-Ctrl) were analysed. PRMT5, H4R3me2s (anti-serum 2) and H2A/H4R3me2s levels (anti-serum 1) were assessed by western blotting of total protein extracts; ACTB provides a loading control. (F) Strongly reduced Prmt5 gene expression in sh-Prmt5-1 and sh-Prmt5-2 cells. cDNA was made from total RNAs using random oligonucleotides. Expression levels were determined relative to Gapdh by real-time PCR amplification, and were put arbitrarily at 100% in the control cells (Sh-Ctrl).

Figure 2.
Figure 2.

Genome-wide analyses of H4R3me2s in mouse ES cells and MEFs. (A) Representative ChIP-Seq data along a 65-kb window on mouse chromosome 15; normalized tag densities for H4R3me2s in ES and MEF are represented. Gene positions are indicated below. (B) Genome-wide correlation between H4R3me2s-enriched regions in ES (y-axis) and MEF (x-axis) cells. The scales of the x- and y-axis are transformed by the following formula: Log10(1 + x). Genomic regions (100-bp windows) with defined degrees of ES and MEF H4R3me2s enrichments are plotted as hexagonal tiles. The number of genomic regions in each hexagon is depicted by a grey colour scale of increasing darkness, according to the count scale reported on the right. (C) Venn diagram representing the number of MACS-predicted peaks (P < 1e-5) of H4R3me2s in ES and MEF cells. The intersection of the areas representing ES and MEF cells indicates peaks common to both cell types. (D) Comparisons of the localizations of ES and MEF’s H4R3me2s enrichments peaks. In both cell types, most peaks are located at genes.

Figure 3.
Figure 3.

H4R3me2s accumulates at promoters and other G + C-rich regions, generally irrespective of transcriptional activity. (A) The unsupervised clustering of transcriptional start sites (TSSs) is represented as a heatmap of tag densities (0–60) per 100-bp bins, from 5 kb upstream to 5 kb downstream of mouse TSS (n = 33 876) for the indicated ChIP-seq/RNA-seq experiments in ES cells. ChIP-seq profiles for H3K4me3, H3K27me3 and RNA-seq from ES are from Marks et al. (25). The non-expressed class of TSS marked by an asterisk contains genes expressed in differentiated cells such as genes belonging to the immune response, inflammatory response or locomotory behaviour Gene Ontology terms. (B) The two class of TSS (groups 2 and 3) indicated in (A) were analysed for their G + C ratio. The two classes of promoters are represented as density plot and box-and-whisker plots in red and blue, for the groups 2 and 3, respectively. (*) Indicates Wilcoxon rank sum test with a P < 2.2e-16. (C) H4R3me2s enrichment around TSSs grouped into four different classes according to the genes’ log expression values. (D) Mean H4R3me2s enrichment levels in ES cells are represented as a function of their distance from the peak summits of H3K4me3 or H3K4me1 peaks. H4R3me2s is enriched around H3K4me3 peaks, but not around H3K4me1 peaks. However, H4R3me2s enrichment occurs both at H3K4me3-enriched regions with high H3K4me3 levels (high H3K4me3) and those with lower H3K4me3 enrichment (low H3K4me2). (E) H4R3me2s enrichment according to the G + C content of 100-bp genomic windows in ES and MEF cells showing the absence of H4R3me2s in regions with less than 50% G + C ratio. (F) Hexagonal binning of H4R3me2s-enriched regions according to their DNA methylation level assessed by MeDIP, or hydroxymethylation level assessed by hydroxyMeDIP (hMeDIP), in ES cells (24). The scales of the x- and y-axis are transformed by the following formula: asinh-1(x). Darker shades of grey indicate higher densities of bins as reported on the count scale.

Figure 4.
Figure 4.

Allelic H4R3me2s enrichment at ICRs. (A) The unsupervised clustering of non-oriented CpG islands (n = 16 026) is represented as a heatmap of H4R3me2s enrichment levels in mouse ES cells in a window of 5 kb around the centre of each CGIs (the minimum to maximum lengths of CGIs are 201–5129 bp with an average length of 655 bp). (B) Heatmap representation of H4R3me2s enrichments in MEF and ES cells at ICRs, which are DNA-methylated on the maternal (‘maternal ICRs’) or the paternal (‘paternal ICRs’) chromosome. (C–F) ChIP-seq profiles for H4R3me2s, in ES cells and MEFs at the KvDMR1 (C), Snrpn (D), IG-DMR (E) and Rasgrf1 (F) ICRs. (G) Sequence profiles around single nucleotide polymorphisms (SNPs) at the indicated ICRs. The parental alleles are equally present in the input chromatin used for ChIP (Input). H4R3me2s, H3K9me3 and H3K4me3 are preferentially enriched at one parental allele indicated by an arrow and the identified parental allele at ICRs. H3K4me3 is present on non-methylated alleles, while H4R3me2s and H3K9me3 are present on DNA-methylated alleles of ICRs.

Figure 5.
Figure 5.

PRMT5 recruitment correlates with H4R3me2s enrichment at specific target loci. (A) ChIP of PRMT5 on cross-linked chromatin from MEFs. The CyclinE1 (Ccne1) promoter is bound by PRMT5 and enriched for H4R3me2s in MEF cells, in agreement with previous studies (10). Shown are real-time PCR quantifications of PRMT5 association at Cyclin E1 (Ccne1), the KvDMR1 ICR and IAP elements. (B) Direct DNA sequencing of ChIP samples shows PRMT5 binding to the H4R3me2s-marked (and DNA-methylated) allele of the KvDMR1 and Snrpn ICRs. (C) Real-time PCR amplification shows reduced H4R3me2s at ICRs and at IAP elements in PRMT5-depleted MEFs (sh-Prmt5-1 cells). ChIP was performed on native chromatin. In a mock ChIP experiment (Mock), an unrelated control IgG (against chicken IgY) was used. Experiments were performed in triplicate; bars indicate standard deviation.

Figure 6.
Figure 6.

H4R3me2s is regulated independently from H3K9me3 and H4K20me3 at imprinted loci. (A) Knockdown of ESET by retroviral shRNA in MEF cells (line sh-Eset-1). Levels of ESET, H3K9me3, PRMT5 and H4R3me2s were assessed by western blotting; ACTB and H4 are included as internal controls. As a negative control, MEFs stably expressing a scrambled shRNA were analysed (line sh-Ctrl). (B) ChIP-qPCR shows loss of H3K9me3 at the H19 and KvDMR1 ICRs in the sh-Eset-1 MEFs. H4R3me2s levels are unaltered. Experiments were performed in triplicate; bars indicate standard deviation. (C) In PRMT5-depleted MEFs (line sh-Prmt5-1), H3K9me3 levels are unaltered at H19 and KvDMR1. (D) Conditional knockout of Eset in ES cells through a Cre-recombinase and loxP system (17). Levels of ESET, H3K9me3, PRMT5 and H4R3me2s were assessed by western blotting. (E) ChIP-qPCR shows loss of H3K9me3 at the H19 and KvDMR1 ICRs in ESET knockout ES cells. H4R3me2s levels are unaffected. Mock ChIPs were as described for Figure 5G. (F) In Suv4-20h1/h2 double knockout MEFs, H4R3me2s levels assessed by ChIP-qPCR are not affected at the H19 and KvDMR1 ICRs. (G) H4K20me3 levels are unaltered at ICRs in PRMT5-depleted MEFs (line sh-Prmt5-1). Experiments were performed in triplicate; bars indicate standard deviation. Mock ChIPs were as described for Figure 5G.

Similar articles

Cited by

References

    1. Law JA, Jacobsen SE. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat. Rev. Genet. 2010;11:204–220. - PMC - PubMed
    1. Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat. Genet. 2003;33(Suppl.):245–254. - PubMed
    1. Barlow DP. Genomic imprinting: a mammalian epigenetic discovery model. Ann. Rev. Genet. 2011;45:379–403. - PubMed
    1. Dambacher S, Hahn M, Schotta G. Epigenetic regulation of development by histone lysine methylation. Heredity. 2010;105:24–37. - PubMed
    1. Di Lorenzo A, Bedford MT. Histone arginine methylation. FEBS Lett. 2011;585:2024–2031. - PMC - PubMed

Publication types

MeSH terms

Substances