H4K20 methylation regulates quiescence and chromatin compaction

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA, 02129, USA. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA. Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles and Department of Biological Chemistry, David Geffen School of Medicine, Los Angeles, CA 90095, USA.
Molecular biology of the cell (Impact Factor: 4.47). 08/2013; 24(19). DOI: 10.1091/mbc.E12-07-0529
Source: PubMed


The transition between proliferation and quiescence is frequently associated with changes in gene expression, in the extent of chromatin compaction and in histone modifications, but whether changes in chromatin state actually regulate cell cycle exit with quiescence is unclear. We discovered that primary human fibroblasts induced into quiescence exhibited tighter chromatin compaction. Mass spectrometry analysis of histone modifications revealed that H4K20me2 and -me3 are increased in quiescence and that other histone modifications are present at similar levels in proliferating and quiescent cells. Analysis of cells in S, G2/M, and G1 phases shows that H4K20me1 increases after S phase and is converted to -me2 and -me3 in quiescence. Knockdown of the enzymes that create H4K20me2 and -me3 resulted in an increased fraction of cells in S phase, a defect in exiting the cell cycle, and decreased chromatin compaction. Overexpression of Suv4-20h1, the enzyme that creates H4K20me2 from H4K20me1, resulted in G2 arrest, consistent with a role for H4K20me1 in mitosis. The results suggest that the same lysine on H4K20 may, in its different methylation states, facilitate mitotic functions in M phase and promote chromatin compaction and cell cycle exit in quiescent cells.

36 Reads
  • Source
    • "For measurement of proliferation by Click-iT analysis, MEFs (lines WT 3 and KO 3) were incubated for 1 h in 10 µM EdU, and labeled as previously described (Evertts et al., 2013). Briefly, 1 to 2×105 cells were pelleted, fixed with 4% paraformaldehyde, permeabilized and treated with Alexa 488 azide (Invitrogen). "
    [Show abstract] [Hide abstract]
    ABSTRACT: PDCD2 (programmed cell death domain 2) is a highly conserved, zinc finger MYND domain-containing protein essential for normal development in the fly, zebrafish and mouse. The molecular functions and cellular activities of PDCD2 remain unclear. In order to better understand the functions of PDCD2 in mammalian development, we have examined PDCD2 activity in mouse blastocyst embryos, as well as in mouse embryonic stem cells (ESCs) and embryonic fibroblasts (MEFs). We have studied mice bearing a targeted PDCD2 locus functioning as a null allele through a splicing gene trap, or as a conditional knockout, by deletion of exon2 containing the MYND domain. Tamoxifen-induced knockout of PDCD2 in MEFs, as well as in ESCs, leads to defects in progression from the G1 to the S phase of cell cycle, associated with increased levels of p53 protein and p53 target genes. G1 prolongation in ESCs was not associated with induction of differentiation. Loss of entry into S phase of the cell cycle and marked induction of nuclear p53 were also observed in PDCD2 knockout blastocysts. These results demonstrate a unique role for PDCD2 in regulating the cell cycle and p53 activation during early embryonic development of the mouse.
    Biology Open 08/2014; 3(9). DOI:10.1242/bio.20148326 · 2.42 Impact Factor
  • Source
    • "Increased chromatin compaction was confirmed by probing chromatin accessibility by micrococcal nuclease (MNase) digestion . In accord with a recent study showing global chromatin compaction and upregulation of H4K20me3 in contact-inhibited human fibroblasts (Evertts et al., 2013), quiescent NIH 3T3 cells and wild-type MEFs exhibited a chromatin structure that is less accessible to MNase digestion (Figures 3C, 3D, and S3A). In contrast, chromatin from Suv4-20h À/À MEFs was readily digested , regardless of whether cells were cultured in 10% or 0.1% serum, indicating that H4K20me3-mediated chromatin compaction alters global chromatin structure in response to growth arrest. "
    [Show abstract] [Hide abstract]
    ABSTRACT: A complex network of regulatory pathways links transcription to cell growth and proliferation. Here we show that cellular quiescence alters chromatin structure by promoting trimethylation of histone H4 at lysine 20 (H4K20me3). In contrast to pericentric or telomeric regions, recruitment of the H4K20 methyltransferase Suv4-20h2 to rRNA genes and IAP elements requires neither trimethylation of H3K9 nor interaction with HP1 proteins but depends on long noncoding RNAs (lncRNAs) that interact with Suv4-20h2. Growth factor deprivation and terminal differentiation lead to upregulation of these lncRNAs, increase in H4K20me3, and chromatin compaction. The results uncover a lncRNA-mediated mechanism that guides Suv4-20h2 to specific genomic loci to establish a more compact chromatin structure in growth-arrested cells.
    Molecular cell 04/2014; 54(4). DOI:10.1016/j.molcel.2014.03.032 · 14.02 Impact Factor
  • Source
    • "It has a distinctive transcriptional profile, with overall changes in the level of transcription and chromatin organisation [13]. Entry into quiescence is generally associated with increased chromatin compaction [14] and it is therefore perhaps surprising that this was accompanied by reduced trypsin-sensitive masking of 5meC. However, this observation seems consistent with results of staining in the zygote where the level of 5meC epitope masking was greatest in the late zygote stage where chromatin structure is generally considered least compacted. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The methylation of CpG dinucleotides is a pervasive epigenetic signature with critical roles governing genomic stability and lineage-specific patterns of gene expression. Reprogramming the patterns of CpG methylation accompanies key developmental transitions and the onset of some pathologies, such as cancer. In this study we show that levels of immuno-detectable 5meC decreased as mouse embryonic fibroblasts withdraw from the cell-cycle (became mitotically quiescent), but increased as they aged in culture. Two pools of 5meC epitope were found to exist, one solvent exposed after acid-induced denaturation of chromatin and another that required the additional step of tryptic digestion for detection. Proliferative cells displayed a relatively greater accumulation of detectable 5meC within the trypsin-sensitive pool than did quiescent cells. A substantial proportion of the 5meC was associated with a large number of heterochromatic foci scattered throughout nuclei, yet much of this was masked in a trypsin-sensitive manner, particularly in young proliferative cells. This study showed that the growth status of cells changed the level of solvent exposure of 5meC in fibroblasts and the long-accepted conventional methods of immunolocalization underestimate the level of 5meC in cells. This resulted in an artefactual assessment of the levels and patterns of nuclear localization of the antigen. The use of an additional tryptic digestion step improved antigen retrieval and revealed a more dynamic response of 5meC levels and distribution patterns to changes in the cell's growth state. This discovery will provide a basis for investigating the role of changes in chromatin structure that underlie this dynamism.
    PLoS ONE 04/2014; 9(4):e92523. DOI:10.1371/journal.pone.0092523 · 3.23 Impact Factor
Show more

Similar Publications


36 Reads
Available from