The Arginine Methyltransferase PRMT6 Regulates DNA Methylation and Contributes to Global DNA Hypomethylation in Cancer

Summary: DNA methylation plays crucial roles in chromatin structure and gene expression. Aberrant DNA methylation patterns, including global hypomethylation and regional hypermethylation, are associated with cancer and implicated in oncogenic events. How DNA methylation is regulated in developmental and cellular processes and dysregulated in cancer is poorly understood. Here, we show that PRMT6, a protein arginine methyltransferase responsible for asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a), negatively regulates DNA methylation and that PRMT6 upregulation contributes to global DNA hypomethylation in cancer. Mechanistically, PRMT6 overexpression impairs chromatin association of UHRF1, an accessory factor of DNMT1, resulting in passive DNA demethylation. The effect is likely due to elevated H3R2me2a, which inhibits the interaction between UHRF1 and histone H3. Our work identifies a mechanistic link between protein arginine methylation and DNA methylation, which is disrupted in cancer. : Veland et al. find that PRMT6, an arginine methyltransferase responsible for histone H3 arginine 2 (H3R2) methylation, negatively regulates maintenance DNA methylation by impairing UHRF1 recruitment to chromatin. The authors also find that PRMT6 upregulation contributes to global DNA hypomethylation in cancer cells. Keywords: PRMT6, arginine methylation, UHRF1, DNMT1, DNA methylation, cance

Maternal Setdb1 Is Required for Meiotic Progression and Preimplantation Development in Mouse

<div><p>Oocyte meiotic progression and maternal-to-zygote transition are accompanied by dynamic epigenetic changes. The functional significance of these changes and the key epigenetic regulators involved are largely unknown. Here we show that Setdb1, a lysine methyltransferase, controls the global level of histone H3 lysine 9 di-methyl (H3K9me2) mark in growing oocytes. Conditional deletion of <i>Setdb1</i> in developing oocytes leads to meiotic arrest at the germinal vesicle and meiosis I stages, resulting in substantially fewer mature eggs. Embryos derived from these eggs exhibit severe defects in cell cycle progression, progressive delays in preimplantation development, and degeneration before reaching the blastocyst stage. Rescue experiments by expressing wild-type or inactive Setdb1 in <i>Setdb1</i>-deficient oocytes suggest that the catalytic activity of Setdb1 is essential for meiotic progression and early embryogenesis. Mechanistically, up-regulation of Cdc14b, a dual-specificity phosphatase that inhibits meiotic progression, greatly contributes to the meiotic arrest phenotype. <i>Setdb1</i> deficiency also leads to derepression of transposons and increased DNA damage in oocytes, which likely also contribute to meiotic defects. Thus, <i>Setdb1</i> is a maternal-effect gene that controls meiotic progression and is essential for early embryogenesis. Our results uncover an important link between the epigenetic machinery and the major signaling pathway governing meiotic progression.</p></div

Proposed effects of Setdb1 depletion on meiotic progression and preimplantation development.

<p>Setdb1 depletion in growing oocytes leads to decreases in H3K9-methyl marks. One consequence is elevated expression of Cdc14b, which leads to Cyclin B1 degradation and inhibition of meiotic resumption. Cdc14b upregulation also contributes to subsequent spindle and chromosomal abnormalities and meiotic arrest at meiosis I. Other consequences of decreases in H3K9 methylation, including derepression of retrotransposons, DNA damage, altered gene expression, and chromatin defects, likely also play important roles in inducing spindle and chromosome defects at meiosis I. The mechanisms by which Setdb1 depletion leads to preimplantation development defects remain to be determined. The consequences of Setdb1 depletion (e.g. H3K9 methylation decreases, altered gene expression, and genomic and chromosomal instability) and/or the lack of maternal Setdb1 itself may affect essential cellular processes during preimplantation development.</p

Expression of WT, but not inactive, Setdb1 in Setdb1 KO GV oocytes partially rescues the meiotic and embryonic defects.

<p><b>(A)</b> Structure of the Setdb1 protein showing the major functional domains. Tud and Tud, tandem Tudor domain; MBD, methyl-CpG-binding domain; pre-S, pre-SET domain; S and ET, bifurcated SET domain. The location of the point mutation altering cysteine 1243 to alanine (C1243A) is indicated. The WT and mutant Setdb1 proteins expressed in oocytes have an N-terminal Flag tag. <b>(B-D)</b> Fully-grown GV oocytes were harvested from control and <i>Setdb1</i> KO mice. <i>Setdb1</i> KO oocytes were injected with mRNAs encoding Flag-tagged WT Setdb1 (Flag-Setdb1) or catalytically inactive Setdb1 (Flag-C1243A). The injected oocytes, as well as uninjected control and <i>Setdb1</i> KO oocytes, were incubated in IBMX-containing medium for 2 hours to allow the expression of Flag-tagged Setdb1 proteins, followed by <i>in vitro</i> maturation in IBMX-free medium for 18 hours. <b>(B)</b> Representative IF images showing expression of Flag-Setdb1 proteins (green) 2 hours after mRNA injection. The nuclei were stained with DAPI (blue). The boundaries of the oocytes are defined by circles. Scale bars, 35 μm. <b>(C)</b> Representative bright-field microscope images of uninjected oocytes and oocytes expressing Flag-Setdb1 or Flag-C1243A following 18 hours of <i>in vitro</i> maturation. Arrowheads and arrows indicate the polar bodies (characteristic of Met II oocytes) and the prominent nucleoli (characteristic of GV oocytes), respectively. Scale bars, 50 μm. <b>(D)</b> Percentages of oocytes at different meiotic stages (GV arrested, MI, Met II, and abnormal) following 18 hours of <i>in vitro</i> maturation. The total numbers of oocytes examined are indicated. <b>(E, F)</b> Met II oocytes derived from <i>in vitro</i> maturation (described above) were inseminated with sperm from WT mice and then cultured for 48 hours. <b>(E)</b> Representative images of embryos derived from the indicated Met II oocytes. Scale bars, 50 μm. <b>(F)</b> Percentages of different stages of preimplantation embryos, as well unfertilized oocytes (Met II), are shown. The total numbers of embryos/oocytes examined are indicated.</p

<i>Setdb1</i>^<i>m-z+</i> zygotes show severe delays in pronuclear maturation and entry into the first mitosis.

<p><b>(A)</b> Schematic representation of the timing of cell cycle phases of 1-cell and 2-cell stage embryos. (<b>B, C</b>) Determination of pronuclear (PN) stages after <i>in vitro</i> fertilization. Met II oocytes from <i>Setdb1</i> KO and control mice were fertilized <i>in vitro</i> with sperm from WT mice, and at 5 hours post-fertilization (hpf), the zygotes (<i>Setdb1</i>^<i>m-z+</i> and <i>Setdb1</i>^<i>m+z+</i>, respectively) were stained with DAPI (blue) to determine their PN stages. <b>(B)</b> Representative zygotes at PN1, PN2, and PN3 stages. The boundaries of the zygotes are defined by circles, and the male and female pronuclei are indicated. Pb, polar body. Scale bars, 30 μm. <b>(C)</b> The percentages of PN1, PN2-3, and abnormal zygotes. The total numbers of embryos examined are indicated. (<b>D, E</b>) <i>Setdb1</i> KO and control females were mated with WT males, zygotes (<i>Setdb1</i>^<i>m-z+</i> and <i>Setdb1</i>^<i>m+z+</i>, respectively) collected at E0.5 were incubated in the presence of colcemid for 18 hours and then stained with DAPI to determine the cell cycle stages. <b>(D)</b> Representative zygotes at interphase and M phase. The boundaries of the zygotes are defined by circles. Pb, polar body. Scale bars, 30 μm. <b>(E)</b> The percentages of interphase, M phase, and abnormal zygotes. The total numbers of embryos examined are indicated.</p

<i>Setdb1</i> KO oocytes show derepression of retrotransposons and increased DNA double-strand breaks.

<p><b>(A)</b> qRT-PCR analysis of retrotransposon transcripts in control and <i>Setdb1</i> KO GV oocytes. The data are presented as the mean ± SEM of triplicate assays. <i>IAP</i>, intracisternal A particles; <i>Line1</i>, long interspersed nuclear element 1; <i>MTA</i>, mouse transposon A; <i>MusD</i>, the type D murine LTR retrotransposon. <b>(B, C)</b> GV oocytes were examined for DNA double-strand breaks (DSBs) with anti-γ-H2AX staining. <b>(B)</b> Representative γ-H2AX (red) and DAPI (blue) images of control and <i>Setdb1</i> KO oocytes. The nuclei of oocytes are circled, and the γ-H2AX foci are indicated by arrowheads. Scale bars: 10 μm. <b>(C)</b> The proportions of oocytes with various numbers of γ-H2AX foci in the nuclei. In total, 178 control oocytes and 194 KO oocytes were examined.</p

<i>Setdb1</i> KO oocytes show severe meiotic arrest.

<p><b>(A, B)</b> Oocytes harvested from the oviducts of control and <i>Setdb1</i> KO mice at 16 hours post-hCG injection. <b>(A)</b> Representative bright-field microscope images of control and <i>Setdb1</i> KO oocytes. Arrowheads and arrows indicate the polar bodies (characteristic of Met II oocytes) and the prominent nucleoli (characteristic of GV oocytes), respectively. Scale bars, 50 μm. <b>(B)</b> Quantification of oocytes at different meiotic stages. Oocytes were classified as being GV arrested (GV), meiosis I (MI), metaphase II (Met II), or abnormal (Abnl, including those showing abnormal morphologies or undergoing degeneration). A representative oocyte of each type is shown at the bottom. Scale bars, 50 μm. Plotted are the average numbers of oocytes (mean ± SEM) at different stages from 12 control mice (306 oocytes in total) and 13 KO mice (364 oocytes in total). Statistical comparisons were made using unpaired t-test. **P < 0.01; ***P < 0.001. <b>(C, D)</b> <i>In vitro</i> oocyte maturation assays. Fully-grown GV oocytes from 6-week-old control and <i>Setdb1</i> KO mice were collected in M2 medium containing IBMX (200 μM) and, following IBMX washout, cultured in IBMX-free M16 medium. <b>(C)</b> The GVBD rates at 2 hours and 5 hours after IBMX removal. Plotted are data from 4 control mice (142 oocytes in total) and 4 KO mice (160 oocytes in total). Statistical comparisons were made using unpaired t-test. ***P < 0.001. <b>(D)</b> The percentages of oocytes at different meiotic stages after 20 hours of culture. Plotted are data from 6 control mice (238 oocytes in total) and 6 KO mice (265 oocytes in total). <b>(E, F)</b> Spindle and chromosome defects in <i>Setdb1</i> KO MI oocytes. Fully-grown GV oocytes harvested from control and <i>Setdb1</i> KO mice were cultured in maturation medium for 5 hours, and the oocytes were immunostained with α-tubulin antibody and DAPI to examine spindle and chromosome structures, respectively. <b>(E)</b> Representative IF images showing normal spindle (α-tubulin, green) and chromosome (DAPI, blue) morphologies in control MI oocytes and common abnormalities in KO MI oocytes. Spindle defects include dispersed, tread-like, non-bipolar, and multiple spindles. Chromosome defects include decondensed, lagging, and misaligned chromosomes. Scale bars, 25 μm. <b>(F)</b> Frequencies of spindle and chromosome defects (mean ± SEM) in MI oocytes. In total, 253 control MI oocytes and 232 KO MI oocytes were examined. Statistical comparisons were made using unpaired t-test. **P < 0.01; ***P < 0.001.</p

<i>Cdc14b</i> depletion in <i>Setdb1</i> KO oocytes alleviates meiotic arrest.

<p>GV oocytes were harvested from control and <i>Setdb1</i> KO mice. <i>Setdb1</i> KO oocytes were microinjected with either control siRNA or <i>Cdc14b</i> siRNA. These oocytes, as well as control GV oocytes, were incubated in IBMX-containing medium for 24 hours to allow siRNA-mediated <i>Cdc14b</i> depletion to occur, followed by <i>in vitro</i> maturation in IBMX-free medium for 20 hours. <b>(A)</b> qRT-PCR analysis of <i>Cdc14b</i> mRNA 24 hours after microinjection. Shown are relative <i>Cdc14b</i> mRNA levels in oocytes injected with control siRNA and <i>Cc14b</i> siRNA (mean ± SEM of duplicate assays). (<b>B, C)</b> IF analysis of Cdc14b 24 hours after microinjection. <b>(B)</b> Representative IF images of oocytes injected with control siRNA or <i>Cdc14b</i> siRNA. Scale bars, 35 μm. <b>(C)</b> Quantification of fluorescence intensity of Cdc14b. Twenty oocytes injected with control siRNA and 20 oocytes injected with <i>Cdc14b</i> siRNA were analyzed, and the data are presented as the mean ± SEM. Statistical comparisons were made using unpaired t-test. **P < 0.01. <b>(D, E)</b> Determination of meiotic stages after 20 hours of <i>in vitro</i> maturation. <b>(D)</b> Representative bright-field microscope images of control oocytes and <i>Setdb1</i> KO oocytes injected with control siRNA or <i>Cdc14b</i> siRNA. Arrowheads and arrows indicate the polar bodies (characteristic of Met II oocytes) and the prominent nucleoli (characteristic of GV oocytes), respectively. Scale bars, 50 μm. <b>(E)</b> Percentages of oocytes at different meiotic stages (GV arrested, MI, Met II, and abnormal). In total, 200 KO oocytes were injected with control siRNA and another 200 with Cdc14b siRNA, and 172 and 181 of them, respectively, survived.</p

Setdb1 is expressed and controls global H3K9me2 in growing oocytes.

<p><b>(A)</b> X-gal staining of paraffin-embedded sections of ovaries from 4-week-old <i>Setdb1</i>^{<i>3lox/+</i>} or wild-type (WT) mice. One follicle is enlarged, and the oocyte nucleus is indicated by an arrow. Scale bars, 50 μm. <b>(B)</b> qRT-PCR analysis of <i>Setdb1</i> transcript. Shown are relative levels of <i>Setdb1</i> mRNA in control and <i>Setdb1</i> KO GV oocytes (mean ± SEM of triplicate assays). <b>(C)</b> Western blot analysis of Setdb1 in control and <i>Setdb1</i> KO GV oocytes, with α-tubulin as a loading control. Each lane contains 150 GV oocytes. <b>(D, E)</b> IF analysis of H3K9 methylation in oocytes. Fully-grown GV oocytes harvested from control and <i>Setdb1</i> KO mice were immunostained with antibodies specific for H3K9 mono-, di- and tri-methyl (H3K9me1, H3K9me2, and H3K9me3) marks and counterstained with DAPI. <b>(D)</b> Representative images of H3K9me1, H3K9me2, and H3K9me3 marks (green) and DAPI (blue). The nuclei of the oocytes are circled. Note that H3K9me1 is enriched in the nucleoli, H3K9me2 exhibits a punctate staining pattern throughout the nuclei, and H3K9me3 is enriched in constitutive heterochromatin. Scale bars, 10 μm. <b>(E)</b> Quantification of fluorescence intensity of H3K9me1, H3K9me2, and H3K9me3 marks in oocytes. Fifteen oocytes for each genotype were stained by each antibody, and the data are presented as the mean ± SEM. Statistical comparisons were made using unpaired t-test. ** P < 0.01.</p