RNAi promotes heterochromatic silencing through replication-coupled release of RNA Pol II

Journal name:
Nature
Volume:
479,
Pages:
135–138
Date published:
DOI:
doi:10.1038/nature10501
Received
Accepted
Published online

Heterochromatin comprises tightly compacted repetitive regions of eukaryotic chromosomes. The inheritance of heterochromatin through mitosis requires RNA interference (RNAi), which guides histone modification1 during the DNA replication phase of the cell cycle2. Here we show that the alternating arrangement of origins of replication and non-coding RNA in pericentromeric heterochromatin results in competition between transcription and replication in Schizosaccharomyces pombe. Co-transcriptional RNAi releases RNA polymerase II (Pol II), allowing completion of DNA replication by the leading strand DNA polymerase, and associated histone modifying enzymes3 that spread heterochromatin with the replication fork. In the absence of RNAi, stalled forks are repaired by homologous recombination without histone modification.

At a glance

Figures

  1. Transcription and replication of pericentromeric heterochromatin in fission yeast.
    Figure 1: Transcription and replication of pericentromeric heterochromatin in fission yeast.

    a, Pericentromeric heterochromatin on centromere 3. dh (red), dg (green) and imr (magenta) repeats are shown, bordered by tRNA genes (brown). Replication origins (yellow) are found in each repeat. b, Tiling microarrays of K9me2 ChIP (light blue) and clusters of small RNA sequences (dark blue) from wild-type cells. ChIP-seq reads corresponding to poised (S5-P) and elongating (S2-P) RNA polymerase II enriched in dcr1Δ cells relative to wild-type (WT) cells are in black. c, cDNA clones (beige) from dcr1Δ cells. PolyA sites are indicated as vertical lines and correspond to peaks of Pol II. Arrows indicate the direction of ‘Forward’ transcription. d, Alignment of probes used in previous studies indicates that regions enriched for Pol II11 (cen-dg) and transcriptional run-on probes1 (TRO) lie downstream of forward orientation polyA sites.

  2. RNA interference and DNA replication restrict RNA polymerase II accumulation and prevent DNA damage.
    Figure 2: RNA interference and DNA replication restrict RNA polymerase II accumulation and prevent DNA damage.

    a, Small RNA (blue) and Pol II ChIP-seq reads (black) and regions of significant enrichment (blue and red rectangles) from wild type and dcr1Δ on the right arm of centromere 1. b, A replication bubble is shown, initiated at one of the three origin homology regions at centromere 1 (yellow boxes). c, Chromatin immunoprecipitation for RNA Pol II and Rad22Rad52 from hydroxyurea-arrested and released wild-type (dashed lines) and dcr1Δ (solid lines). Cell cycle progression after release from hydroxyurea block is monitored by septation index, which peaks coincident with S phase. d, Representative parental and non-parental di-type tetrads from crosses between rhp51Δ cells, defective in homologous recombination, and dcr1Δ or ago1Δ.

  3. Replication fork stalling during heterochromatin replication.
    Figure 3: Replication fork stalling during heterochromatin replication.

    Replication intermediates in wild-type and mutant cells resolved by 2D gel electrophoresis and probed with the unique DS/E probe from the ura4 transgene within the dg repeat on chromosome 1 (Fig. 2a). a, Schematic of replication intermediates in 2D gels indicates joint molecules (X), and forks (Y). bf, Junction molecules indicate fork stalling in WT (b) and dcr1Δ mutant cells (c), and are reduced in mms19Δ (d), swi6Δ (e) and clr4Δ (f).

  4. Replication-coupled transcriptional silencing through histone modification and RNAi.
    Figure 4: Replication-coupled transcriptional silencing through histone modification and RNAi.

    a, The Rik1 complex (red hexagon) is recruited to heterochromatic replication forks by interactions with methylated histone H3K9me2 and with the leading strand DNA polymerase (Pol ε, green). Swi6 induces origin firing, but collision with RNA polymerase II (orange) stalls replication forks. RNAi releases Pol II by processing of pre-siRNA transcripts (red lines), allowing leading strand DNA polymerase to complete DNA replication and the associated Rik1 histone modification complex (red hexagon) to spread histone modification (black circles). b, In the absence of RNAi, origins fire but Pol II is not released, stalling replication forks. Stalled Pol II signals repair via homologous recombination instead. Recombination could in principle occur with sister chromatids (shown here) or with other copies of the same repeat (not shown). DNA polymerase ε and the associated Rik1 complex are lost along with the replisome, and fail to spread histone modification into neighbouring reporter genes.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Gene Expression Omnibus

References

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Author information

Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA

    • Mikel Zaratiegui,
    • Stephane E. Castel,
    • Danielle V. Irvine,
    • Anna Kloc,
    • Jie Ren,
    • An-Yun Chang,
    • Derek Goto,
    • Benoit Arcangioli &
    • Robert A. Martienssen
  2. Watson School of Biological Sciences, Cold Spring Harbor Laboratory, New York 11724, USA

    • Stephane E. Castel &
    • Robert A. Martienssen
  3. Molecular and Cellular Biology, University of California Berkeley, Berkeley 94720, USA

    • Fei Li &
    • W. Zacheus Cande
  4. Instituto de Biología Funcional y Genómica. CSIC/Universidad de Salamanca, Salamanca 37007, Spain

    • Elisa de Castro,
    • Laura Marín &
    • Francisco Antequera
  5. Molecular and Cell Biology program, Stony Brook University, Stony Brook, New York 11794, USA

    • An-Yun Chang
  6. Institut Pasteur, Paris, France

    • Benoit Arcangioli
  7. Present addresses: Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854, USA (M.Z.); Yale Stem Cell Center, Yale University, New Haven, Connecticut 06520, USA (A.K.); Creative Research Initiative Sousei, Hokkaido University, 001-0021 Sapporo, Japan (D.G.); Department of Biology, New York University, New York, New York 10003, USA (F.L.); Murdoch Children’s Research Institute, University of Melbourne, Melbourne, Victoria 3052, Australia (D.V.I.).

    • Mikel Zaratiegui,
    • Danielle V. Irvine,
    • Anna Kloc,
    • Fei Li &
    • Derek Goto

Contributions

S.C., D.V.I., A.K., J.R. contributed equally to this work and are listed in alphabetical order. M.Z., S.C., D.V.I., A.K., J.R., F.L., E.d.C., L.M., A.-Y.C. and D.G. performed experiments, and S.C. analysed the data. W.Z.C., F.A., B.A. and R.A.M. designed experiments and R.A.M. and M.Z. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Genomics data and analysis are available from the Gene Expression Omnibus accession number GSE30837. Individual cDNA sequences are available from GenBank with accession numbers JN388396–JN388565.

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    The file contains Supplementary Tables 1-4, Supplementary Figures 1-5 with legends and additional references.

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