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Centromeric histone H2B monoubiquitination promotes noncoding transcription and chromatin integrity

Nature Structural & Molecular Biology volume 21, pages 236–243 (2014)Cite this article

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Abstract

Functional centromeres are essential for proper cell division. Centromeres are established largely by epigenetic processes resulting in incorporation of the histone H3 variant CENP-A. Here, we demonstrate the direct involvement of H2B monoubiquitination, mediated by RNF20 in humans or Brl1 in Schizosaccharomyces pombe, in centromeric chromatin maintenance. Monoubiquinated H2B (H2Bub1) is needed for this maintenance, promoting noncoding transcription, centromere integrity and accurate chromosomal segregation. A transient pulse of centromeric H2Bub1 leads to RNA polymerase II–mediated transcription of the centromere's central domain, coupled to decreased H3 stability. H2Bub1-deficient cells have centromere cores that, despite their intact centromeric heterochromatin barriers, exhibit characteristics of heterochromatin, such as silencing histone modifications, reduced nucleosome turnover and reduced levels of transcription. In the H2Bub1-deficient cells, centromere functionality is hampered, thus resulting in unequal chromosome segregation. Therefore, centromeric H2Bub1 is essential for maintaining active centromeric chromatin.

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Figure 1: Heterochromatin is formed in the centromere in H2Bub1-deficient cells despite the maintenance of a distinct central domain.
Figure 2: Heterochromatin boundaries are maintained without H2Bub1.
Figure 3: Cell-cycle regulation of H2Bub1 levels leads to chromatin transcription in M phase.
Figure 4: H2Bub1 is required for functional centromeres.
Figure 5: Human centromeres are associated with H2Bub1 and other factors required for transcription.
Figure 6: Decreased RNF20 leads to altered α-satellite chromatin in human centromeres.
Figure 7: Decreased RNF20 leads to DNA damage and chromosome segregation defects.
Figure 8: Cartoon depicting a model of the transcription-mediated role of H2Bub1 in centromere remodeling and maintenance as the dividing cell progresses through the cell cycle.

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Acknowledgements

Reagents were kindly provided by S. Grewal (US National Cancer Institute), K.C. Scott (Duke University), C. Norbury (University of Oxford), K. Gull (University of Oxford) and R.C. Allshire (University of Edinburgh). This study was supported by the Swedish Research Council (VR-M2579 and VR-NT4448) (K.E.) and the Swedish Cancer Society (CAN2012/238) (K.E.). The authors would like to thank S. Le Guyader at the Live Cell Imaging unit, Department of Biosciences and Nutrition, Karolinska Institutet, which is supported by grants from the Knut and Alice Wallenberg Foundation (KAW2006.0265) and the Swedish Research Council (822-2007-902 and K2008-661-21010-01-1). We are also grateful to P. Prasat for technical assistance.

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  1. Laia Sadeghi and Lee Siggens: These authors contributed equally to this work.

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  1. Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden

    Laia Sadeghi, Lee Siggens, J Peter Svensson & Karl Ekwall

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  1. Laia Sadeghi
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Contributions

L. Sadeghi performed all experiments in S. pombe, and L. Siggens performed all experiments in human cells. L. Sadeghi, L. Siggens, J.P.S. and K.E. designed the study, analyzed the data and wrote the manuscript.

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Correspondence to J Peter Svensson or Karl Ekwall.

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Integrated supplementary information

Supplementary Figure 1 Genome-browser views.

Genome browser view of (a) centromere 1 and (b) centromere 3 using anti-H3K9me2, anti-H3, and anti-CENP-ACnp1 relative to input; WT (top), htb1-K119R mutant (middle), and htb1-K119R/WT ratio (bottom). (c) ChIP levels of H3K9me2, H3, and CENP-ACnp1 at two centromeric loci (central core domain, cnt1; innermost repeat, imr2) in WT and htb1-K119R determined ChIP-qPCR relative to empty beads. Error bars indicate s.e.m. (n=3 independent experiments). (d) ChIP-qPCR of H3 and H3K9me2 at control locus LTR(n=3 independent experiments).

Supplementary Figure 2 Global effects of the htb1-K119R mutation on transcription and chromatin compaction.

H2Bub1-levels correlate with transcription (a) Scatterplot of H2Bub1 and RNA Pol II versus RNA Pol II and RNA expression. heterochromatin mark H3K9me2 in WT and H2Bub1-deficient cells (htb1-K119R). Pearson's correlation coefficient is indicated at the bottom right of each panel. (b) Transcripts have been divided into 4 categories depending on their expression level (high: 12–14, medium: 10–12, low: 8–10, background: 6–8). Transcripts were aligned after their transcription start site (TSS) and the levels of H2Bub1 and H3K4me2 (from ref. 35) were plotted as a function of position relative to TSS. (c) Scatterplot of RNA levels in WT and htb1-K119R. Grey line: unchanged between the samples, red dashed line: up-regulated in mutant >2-fold, green dashed line: down-regulated >2-fold. Genes encoding kinetochore proteins are marked (x) (d) MNase digestion of chromatin from WT and htb1-K119R cells. * marks oligonucleosomes present in WT but absent in htb1-K119R, arrow points at regions of undigested product in the htb1-K119Rextracts.

Supplementary Figure 3 No htb1-K119R effect on centromere transcript stability or nucleosome stability.

(a) Transcripts from the centromere core are rapidly degraded. Three primers pairs (1–3) were used in qRT-PCR to quantify the transcription of the pericentric (1) and central domain (2–3) of the centromere in WT and htb1-K119R cells. Error bars indicate s.e.m. (n=3 independent experiments) (b) (left) Nucleosomal turnover in WT and htb1-K119R cells as measured by ChIP-qPCR using antibody to hemagglutinin (HA) 2h and 0h after induction of H3-HA expression at two genes, sua1 and cdm1. Bars indicate range (n=2 independent experiments) (right) Expression levels of sua1 and cdm1 as determined by qRT-PCR. Error bars indicate s.e.m.(n=3 independent experiments).

Supplementary Figure 4 H2Bub1 and H4 levels in WT (lane 1), ubp8Δ (lane 3) andubp16Δ (lane 4) cells, as determined by immunoblotting of histone extracts.

Supplementary Figure 5 Colocalization of proteins with centromere-specific histone CENP-A in human cells.

Representative images of HeLa cells and in situ proximity ligation assay using one antibody against CENP-A and a second antibody against H2Bub1, RNF20 or RNA Pol II (n=2 independent experiments). Proximity between the two antibodies (<40 nm) result in a signal (red). Nuclei are counterstained with DAPI (blue).

Supplementary Figure 6 RNA-seq in human NCCIT cells afterRNF20siRNA knockdown (GSE25882).

(a) Repeat transcript levels (α-satellite: ALR, ALR1; Pol II-transcribed L1 repetitive sequences; and Pol III-transcribed ALU repeats). Reads from high-throughput sequencing were aligned to repetitive sequences from Repbase. (b) Genome wide correlation of mRNA expression in si-RNF20 cells compared to control. (c) Transcript expression correlation of centromeric genes only, identified as having one or more centromere or kinetochore associated functions in AMIGO (http://amigo.geneontology.org/cgi-bin/amigo/go.cgi).

Supplementary Figure 7 Full-length gel images and immunoblots.

(a) Agarose gel electrophoresis of RT-PCR products corresponding to Fig. 3f. (b) Immunoblot analysis of RNF20 expression in HEK293T cells stably expressing non-targeting and RNF20 targeting shRNA corresponding to Fig. 6a. H2B was used as a loading control. (c) Analysis of H2Bub1 in HEK293T cells expressing RNF20 targeting shRNA as in (b).

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Sadeghi, L., Siggens, L., Svensson, J. et al. Centromeric histone H2B monoubiquitination promotes noncoding transcription and chromatin integrity. Nat Struct Mol Biol 21, 236–243 (2014). https://doi.org/10.1038/nsmb.2776

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