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. 2020 Jun 16;21(1):144.
doi: 10.1186/s13059-020-02062-8.

Removal of H2Aub1 by ubiquitin-specific proteases 12 and 13 is required for stable Polycomb-mediated gene repression in Arabidopsis

Lejon E M Kralemann  1 , Shujing Liu  1 , Minerva S Trejo-Arellano  1 , Rafael Muñoz-Viana  2 , Claudia Köhler  3 , Lars Hennig  1
Affiliations

Removal of H2Aub1 by ubiquitin-specific proteases 12 and 13 is required for stable Polycomb-mediated gene repression in Arabidopsis

Lejon E M Kralemann et al. Genome Biol. .

Abstract

Background: Stable gene repression is essential for normal growth and development. Polycomb repressive complexes 1 and 2 (PRC1&2) are involved in this process by establishing monoubiquitination of histone 2A (H2Aub1) and subsequent trimethylation of lysine 27 of histone 3 (H3K27me3). Previous work proposed that H2Aub1 removal by the ubiquitin-specific proteases 12 and 13 (UBP12 and UBP13) is part of the repressive PRC1&2 system, but its functional role remains elusive.

Results: We show that UBP12 and UBP13 work together with PRC1, PRC2, and EMF1 to repress genes involved in stimulus response. We find that PRC1-mediated H2Aub1 is associated with gene responsiveness, and its repressive function requires PRC2 recruitment. We further show that the requirement of PRC1 for PRC2 recruitment depends on the initial expression status of genes. Lastly, we demonstrate that removal of H2Aub1 by UBP12/13 prevents loss of H3K27me3, consistent with our finding that the H3K27me3 demethylase REF6 is positively associated with H2Aub1.

Conclusions: Our data allow us to propose a model in which deposition of H2Aub1 permits genes to switch between repression and activation by H3K27me3 deposition and removal. Removal of H2Aub1 by UBP12/13 is required to achieve stable PRC2-mediated repression.

Keywords: DUB; EMF1; H2AK121ub; H2Aub; H3K27me3; LHP1; PRC1; PRC2; REF6; Responsiveness.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
UBP12/13 directly repress PRC2 target genes. a Fold enrichment of the overlap of lists of genes upregulated in ubp12/13 (1150 genes, yellow circle) and genes upregulated in PRC1 (bmi1a/b, 387 genes, blue circle) [19], PRC2 (clf swn, 2824 genes, red circle) [19], and EMF1c (emf1, 2140 genes, purple circle) [9] mutants. The dashed line indicates the expected overlap based on chance. p values are based on a hypergeometric test for significant over-enrichment. b Waffle plot showing the presence of the H2Aub1 and H3K27me3 marks in wild type on UBP12/13 target genes. The black border indicates those UBP12/13 targets that are marked with H3K27me3 (and hence PRC2 targets). Each square corresponds to about 10 genes. c Boxplots showing differential expression of all expressed UBP12/13 targets (green) and all other protein-coding genes (grey). Test for significance was done by the Mann-Whitney U test; ns p ≥ 0.05, *p < 0.05, **p < 0.01,***p < 0.001. d Metagene profiles showing the median of H2Aub1 and H3K27me3 enrichment for gene categories defined by the presence of significant H2Aub1 and H3K27me3 peaks. The darker grey box indicates the first 1 kb of the gene body, and both grey boxes together indicate the mean gene body size within that category. e Pie charts indicating the percentage of genes in the indicated category corresponding to UBP12/13 targets. Note that above “UBP12/13 targets” refers to genes gaining H2Aub1 in ubp12/13 mutants. The biological material underlying the RNA-seq data in this figure is 2-week-old whole plants (bmi1a/b, clf swn, and ubp12/13) and 3-week-old whole plants (emf1). ChIP-seq data was obtained from 33 DAG rosettes
Fig. 2
Fig. 2
H2Aub1 is not a repressive mark per se. a Mean wild-type (wt) expression in the absence (−) or presence (+) of H2Aub1 or H3K27me3 after correction for the presence of the other mark (ANCOVA estimated marginal means, in fpkm). b Percentage of upregulated and downregulated genes in PRC1 mutants among the four categories defined by the presence of H2Aub1 and H3K27me3 and dependency of H3K27me3 on PRC1. Asterisks indicate the |log2FC| cut-off used to call significant deregulation: *≥ 4 or **≥ 1; the p value cut-off was 0.05 for both. Small circles indicate the mean of the four mutants. c wt expression level for genes defined by the presence of H3K27me3 and PRC1 dependency. d Expression change in a PRC2 (clf swn) mutant compared to wt for genes defined by the presence of H3K27me3 and PRC1 dependency. e Metagene profiles of H3K27me3 and H2Aub1 in PRC1-dependent and PRC1-independent gene categories. Based on re-analysed ChIP-seq data from [6] (a–e) and RNA-seq data from [20] (a, b*) and [19] (b**, c, d). An ANCOVA post hoc test was used to test for significance in a, and a Mann-Whitney U test for c and d; ns p ≥ 0.05, *p < 0.05, **p < 0.01,***p < 0.001; Bonferroni correction was applied with m = 6. The biological material underlying this figure is whole plants of 7 DAG (ChIP-seq data), 10 DAG (RNA-seq data in a and b*), and 14 DAG (RNA-seq data in b**, c, d)
Fig. 3
Fig. 3
UBP12/13 protect against H3K27me3 removal. a Differential H3K27me3 enrichment in the ubp12/13 mutant minus wild type, for UBP12/13 targets and non-targets (left), for UBP12/13 targets and non-targets marked with H3K27me3 (middle), and for UBP12/13 targets with H3K27me3 on the first 20% or the last 20% of gene bodies (right). Tests for significance (for a–c) were done by Mann-Whitney U test; ns p ≥ 0.05, *p < 0.05, **p < 0.01,***p < 0.001; Bonferroni correction was applied with m = 6. b Metagene plot showing the median H3K27me3 coverage-scaled fragment counts in the ubp12/13 mutant (dashed line) and wild type (solid line), for UBP12/13 targets with H3K27me3 (green) and non-targets with H3K27me3 (grey). c REF6 enrichment (median coverage-scaled fragment count) at narrow H2Aub1 peaks (left) and at CTCTGYTY motifs (right). Depicted in the left panel are all significant H2Aub1 peaks (purple), all peaks with at least one CTCTGYTY motif (red) and all peaks that do not possess at least one CTCTGYTY motif (blue). Depicted in the right panel are all CTCTGYTY motifs (purple), all motifs with H2Aub1 (red), and all motifs without H2Aub1 (blue). REF6-HA binding sites and CTCTGYTY data obtained from [25]. d Differential coverage-scaled REF6 fragment counts (mutant-wt) for genes that lose H2Aub1 (“Down”), genes without a change in H2Aub1 (“Stable”), and genes gaining H2Aub1 (“Up”). e Scatterplots made with the coverage-scaled fragment counts of H2Aub1 and REF6 in 50-bp windows (genome-wide). CTCTGYTY motifs were also counted in 600-bp windows around the centre of the windows, and windows were split over separate plots depending on the number of motifs. The x- and y-axes are logarithmic (Log10). Statistics: 0 motifs (m = 0.33, R2 = 0.17, p < 2.2E−16), 1 motif (m = 0.27, R2 = 0.09, p < 2.2E−16), 2 motifs (m = 0.39, R2 = 0.11, p < 2.2E−16), 3 motifs (m = 0.47, R2 = 0.10, p < 2.2E−16), 4 motifs (m = 0.47, R2 = 0.07, p < 2.2E−16), and 5+ motifs (m = 0.41, R2 = 0.05, p < 3.7E−9). The biological material used to generate the data in this figure was 33 DAG rosettes, apart from the REF6-HA data, which was obtained from 12 DAG seedlings
Fig. 4
Fig. 4
Model of PRC1/PRC2-mediated gene regulation. Before PRC1/PRC2 recruitment, PRC1-dependent genes are active, while PRC1-independent genes are already pre-repressed. The pre-repressed genes can be directly targeted by PRC2, while PRC2 targeting to active genes is inhibited, possibly by nascent RNA. PRC1 presence mitigates this inhibition, allowing PRC2 to target and silence the genes. However, PRC1-mediated H2Aub1 enhances REF6 binding and H3K27 demethylation, allowing genes marked by H2Aub1, to be rapidly reactivated in response to a stimulus. Therefore, removal of H2Aub1 by UBP12/13 is necessary for stable repression
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