Noncoding RNAs prevent spreading of a repressive histone mark

Journal name:
Nature Structural & Molecular Biology
Volume:
20,
Pages:
994–1000
Year published:
DOI:
doi:10.1038/nsmb.2619
Received
Accepted
Published online
Corrected online

Abstract

Transcription of eukaryotic genomes is more widespread than was previously anticipated and results in the production of many non–protein-coding RNAs (ncRNAs) whose functional relevance is poorly understood. Here we demonstrate that ncRNAs can counteract the encroachment of heterochromatin into neighboring euchromatin. We have identified a long ncRNA (termed BORDERLINE) that prevents spreading of the HP1 protein Swi6 and histone H3 Lys9 methylation beyond the pericentromeric repeat region of Schizosaccharomyces pombe chromosome 1. BORDERLINE RNAs act in a sequence-independent but locus-dependent manner and are processed by Dicer into short RNAs referred to as brdrRNAs. In contrast to canonical centromeric short interfering RNAs, brdrRNAs are rarely loaded onto Argonaute. Our analyses reveal an unexpected regulatory activity of ncRNAs in demarcating an epigenetically distinct chromosomal domain that could also be operational in other eukaryotes.

At a glance

Figures

  1. The ability of Swi6 to bind RNA is a factor restricting heterochromatin to pericentromeric repeats on chromosome 1.
    Figure 1: The ability of Swi6 to bind RNA is a factor restricting heterochromatin to pericentromeric repeats on chromosome 1.

    (a) Physical map showing the organization of the three S. pombe centromeres. The pericentromeric dg and dh tandem repeats are assembled into heterochromatin. cnt, central core domain; imr, innermost repeats; cen1-R, heterochromatin-euchromatin boundary region to the right of centromere 1; blue lines, tRNA genes. (b,c) ChIP-PCR experiments for Swi6 (b) and H3K9me2 (c) showing dependence of heterochromatin boundary formation at cen1-R on the RNA affinity of Swi6. Percentage DNA precipitation is shown relative to input DNA. Enrichment of H3K9me2 on dg and dh (dg/dh) repeats was set at 100% for each sample. Error bars, s.e.m.; n ≥ 3 independent biological replicates. Nucleotide positions along chromosome are shown at bottom of each figure.

  2. Spreading of heterochromatin into neighboring euchromatin in swi6* cells occurs specifically on centromere 1 but not on centromere 2 or at the mating-type locus.
    Figure 2: Spreading of heterochromatin into neighboring euchromatin in swi6* cells occurs specifically on centromere 1 but not on centromere 2 or at the mating-type locus.

    (a) H3K9me2 ChIP-seq enrichment profiles for swi6+ and swi6* cells on centromere 1. The region on the right arm of chromosome 1 (cen1-R), where barrier activity is impaired in swi6* cells, is shown in gray. (b) H3K9me2 ChIP-seq enrichment profiles for swi6+ and swi6* cells on centromere 2. Supplementary Figure 4 shows ChIP-PCR for the same region. (c) Distribution of H3K9me2 at the silent mating-type locus. Green bars represent the distribution of TFIIIC (Sfc6), as determined in ref. 18. The y axes in ac represent log2 ChIP-seq enrichments in 200-base-pair genomically tiled windows calculated over clr4Δ cells.

  3. lncRNAs are produced at the heterochromatin boundary of cen1-R.
    Figure 3: lncRNAs are produced at the heterochromatin boundary of cen1-R.

    (a) Physical map of the right arm of chromosome 1 indicating the heterochromatic and euchromatic regions. Orange, BORDERLINE lncRNAs. (b) Northern blot showing BORDERLINE lncRNAs produced from cen1-R. Strand-specific 32P-labeled oligonucleotide probes were used. Supplementary Figures 1 and 7 show the loading control and uncropped images, respectively. (c) Sequence of BORDERLINE as determined by RACE-PCR and Sanger sequencing. The different 5′ ends are shown in green. The 3′ ends can differ in two Us (red) and are followed by an oligo(A) tail.

  4. Production of RNA, irrespective of the underlying DNA sequence, is sufficient for heterochromatin boundary formation at cen1-R.
    Figure 4: Production of RNA, irrespective of the underlying DNA sequence, is sufficient for heterochromatin boundary formation at cen1-R.

    (a) Spreading of H3K9me2 beyond BORDERLINE or borderlineΔ∷ura3+, assessed by ChIP-PCR. The BORDERLINE DNA sequence was replaced with a heterologous ura3+ expression cassette from C. albicans (red), for which expression is enhanced in uracil-depleted medium. Enrichments over input values normalized to adh1+ are shown relative to enrichment of H3K9me2 on dg and dh repeats. Average fold enrichment ± s.d. is shown for at least three independent experiments. (b) ura3+, adh1+ and U6 RNA levels, determined by quantitative real-time PCR. Fold-change RNA levels are shown relative to those for cells grown in pombe minimal glutamate (PMG) medium with uracil and normalized to act1+ RNA. *P < 0.05. (c) As in a, with the same cell cultures from b used in H3K9me2 ChIP analysis. **P < 0.01. P values in ac were generated by Student's t test (two-tailed distribution; two-sample, unequal variance). Error bars, s.e.m. for four independent biological replicates.

  5. RNAs produced at the border of heterochromatin on centromere 1 are processed into siRNAs that fail to load onto Ago1.
    Figure 5: RNAs produced at the border of heterochromatin on centromere 1 are processed into siRNAs that fail to load onto Ago1.

    (a) Small-RNA northern blot showing siRNAs derived from centromeric-repeat lncRNAs (top, cen siRNAs) or BORDERLINE lncRNAs (bottom, brdrRNA) produced from cen1-R. Strand-specific 32P-labeled oligonucleotide probes were used. Bp, base pairs. Uncropped images are shown in Supplementary Figure 7. (b) Small-RNA reads obtained by deep sequencing of total small-RNA libraries (18-nt to 28-nt PAGE-purified small RNAs) or libraries generated from Ago1-bound siRNAs26, mapped to the cen1-R region. Red, brdrRNAs; + and − denote orientation of the RNA. Supplementary Figure 6 shows the entire centromeric region of chromosome 1. (c,d) Small-RNA reads obtained by deep sequencing of total small-RNA libraries, mapped to the ura3+ coding sequence. The ura3+ gene was inserted into the borderline+ locus (borderlineΔ∷ura3+) or euchromatin (euchromatic ura3+). Color code denotes nucleotides found at the 5′ end of the small-RNA reads. Nt, nucleotides.

  6. Involvement of opposing activities of ncRNAs in the formation of a distinct heterochromatin domain.
    Figure 6: Involvement of opposing activities of ncRNAs in the formation of a distinct heterochromatin domain.

    (a) Chromatin-associated lncRNAs act as assembly platforms recruiting the H3K9 methyltransferase Clr4 to heterochromatin nucleation sites. Centromeric repeat–derived siRNA guide molecules that associate with the RNAi factor Ago1 provide specificity. Iterative HP1 binding to methylated H3K9 and recruitment of Clr4 results in heterochromatin spreading. This is likely to be reinforced by additional nucleation sites, thus resulting in the formation of an extended heterochromatic domain. Cotranscriptional siRNA biogenesis and direct transfer of siRNAs to Ago1 prevents unwanted Swi6 eviction within pericentromeric repeats. (b) Model for ncRNA function in boundary formation. BORDERLINE lncRNAs originating proximal to the centromeric repeats bind Swi6. This triggers a conformational change in the chromodomain that leads to eviction of Swi6 from chromatin and thus discontinuation of heterochromatin spreading. Subsequently, BORDERLINE lncRNAs are processed into brdrRNAs by the RNAi machinery. Unlike repeat-derived siRNAs, brdrRNAs fail to load onto Ago1. Whether brdrRNAs also participate in the eviction of Swi6 is not known.

Accession codes

Primary accessions

Gene Expression Omnibus

Change history

Corrected online 25 September 2013
In the original article, the authors identified a long noncoding RNA, termed BORDERLINE, that prevents spreading of pericentromeric heterochromatin. In this addendum, they note that the BORDERLINE-encoding sequence partially overlaps with the previously described element IRC1-R.

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Author information

  1. These authors contributed equally to this work.

    • Claudia Keller &
    • Raghavendran Kulasegaran-Shylini

Affiliations

  1. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.

    • Claudia Keller,
    • Raghavendran Kulasegaran-Shylini,
    • Yukiko Shimada,
    • Hans-Rudolf Hotz &
    • Marc Bühler
  2. University of Basel, Basel, Switzerland.

    • Claudia Keller,
    • Raghavendran Kulasegaran-Shylini,
    • Yukiko Shimada,
    • Hans-Rudolf Hotz &
    • Marc Bühler
  3. Swiss Institute of Bioinformatics, Basel, Switzerland.

    • Hans-Rudolf Hotz

Contributions

R.K.-S., C.K. and Y.S. performed ChIP experiments. R.K.-S. prepared libraries for ChIP-seq and analyzed the data and performed RACE experiments. C.K. created strains, performed Northern blotting and quantitative real-time PCR and prepared RNA for small-RNA deep sequencing. H.-R.H. conducted bioinformatic analysis of small-RNA deep-sequencing data. M.B., C.K. and R.K.-S. designed the study, analyzed the data and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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