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Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation - PubMed

. 2013 Oct;23(10):1563-79.

doi: 10.1101/gr.154872.113. Epub 2013 Jul 26.

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Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation

Hélène Neyret-Kahn et al. Genome Res. 2013 Oct.

Abstract

Despite numerous studies on specific sumoylated transcriptional regulators, the global role of SUMO on chromatin in relation to transcription regulation remains largely unknown. Here, we determined the genome-wide localization of SUMO1 and SUMO2/3, as well as of UBC9 (encoded by UBE2I) and PIASY (encoded by PIAS4), two markers for active sumoylation, along with Pol II and histone marks in proliferating versus senescent human fibroblasts together with gene expression profiling. We found that, whereas SUMO alone is widely distributed over the genome with strong association at active promoters, active sumoylation occurs most prominently at promoters of histone and protein biogenesis genes, as well as Pol I rRNAs and Pol III tRNAs. Remarkably, these four classes of genes are up-regulated by inhibition of sumoylation, indicating that SUMO normally acts to restrain their expression. In line with this finding, sumoylation-deficient cells show an increase in both cell size and global protein levels. Strikingly, we found that in senescent cells, the SUMO machinery is selectively retained at histone and tRNA gene clusters, whereas it is massively released from all other unique chromatin regions. These data, which reveal the highly dynamic nature of the SUMO landscape, suggest that maintenance of a repressive environment at histone and tRNA loci is a hallmark of the senescent state. The approach taken in our study thus permitted the identification of a common biological output and uncovered hitherto unknown functions for active sumoylation at chromatin as a key mechanism that, in dynamically marking chromatin by a simple modifier, orchestrates concerted transcriptional regulation of a network of genes essential for cell growth and proliferation.

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Figures

Figure 1.
Figure 1.

Chromatin profiles of SUMO1 and SUMO2. (A) Association of SUMO1 (left) and SUMO2 (right) with Pol II, H3K4me3, H3K27me3, and H3K9me3 in WI38 cells. Comparison of tag density in the region of ±5 kb around the SUMO-occupied loci. Clustering identifies five classes as indicated. (B) A pie chart representation of the distribution of SUMO sites in five different genomic regions. The definition of each region is described below. (C) Frequency of SUMO1 and SUMO2 site localization with respect to TSS. (D) Comparison of SUMO2 tag density around the SUMO1 peaks (left) and of SUMO1 tag density around the SUMO2 peaks (right). (E) Histogram representing the percentage of the H3K4me3, H3K27me3, or H3K9me3 peaks with an overlapping SUMO1 and/or SUMO2 peak.

Figure 2.
Figure 2.

SUMO is highly enriched at promoters of actively transcribed genes. (A) Clustering comparison of SUMO1, SUMO2, Pol II, and RNA-seq reads on TSS. (B) Expression status of SUMO-bound TSS (expression defined by a minimum of two RPKM in three mRNA-seq replicates). (C) Merged profiles of SUMO1, SUMO2, Pol II, and H3K4me3 read density with respect to distance from active TSS. (D) Mean profile of SUMO1, SUMO2, and Pol II read density with respect to distance from TSS of genes showing low, middle, and high levels of expression. (E) Venn diagram representing overlap between SUMO1- and SUMO2-marked TSS. (F) Functional annotation of TSS commonly occupied by SUMO1 and SUMO2 using GREAT. The top overrepresented categories belonging to three different ontologies are shown. (G) Scatterplot comparison of common SUMO1- and SUMO2-marked TSS. In red, TSS with a SUMO1/SUMO2 read ratio ≤0.5 or ≥2. (H) IPA upstream regulator analysis of SUMO1-enriched TSS (≥ twofold). (I) Same as in H but for SUMO2.

Figure 3.
Figure 3.

SUMO is highly enriched at histone, ribosomal protein, and tRNA genes. (A,B) Distribution of (A) SUMO1- and (B) SUMO2-marked TSS by peak height and TSS density showing the top quartile corresponding to the highest SUMO peaks in gray. Functional annotation of genes associated to the top quartile TSS using DAVID. The top overrepresented categories are shown. Table below summarizes the statistical enrichment of SUMO-bound TSS for histone, tRNA, and ribosomal protein annotations. (C) A Genome Browser view of ChIP-seq data across the histone and tRNA gene cluster in chromosome 6p. (D) Comparison of SUMO1, SUMO2, and H3K4me3 tag density at tRNA loci (upper) and their merged profiles restricted to the H3K4me3-positive subcluster with respect to distance from TSS (lower). (E) Relative enrichment of SUMO1, SUMO2, and H3K9me3 reads at the major satellite repeat (GSAT_MM), a tRNA, RN7SL1 (7SL), RNU6 (U6), and rRNA loci in the ChIP-seq compared to input. (F) ChIP-qPCR for SUMO1 and SUMO2 on three regions of the rRNA gene amplified with specific primers (Guetg et al. 2012). FOS promoter is used here as a positive control.

Figure 4.
Figure 4.

Chromatin profiles of UBC9 and PIASY. (A) Association of UBC9 (left) and PIASY (right) with SUMO1, SUMO2, Pol II, H3K4me3, H3K27me3, and H3K9me3 in WI38 proliferating cells. Comparison of the tag density in the ±5-kb region around UBC9- or PIASY-bound loci. Clustering identifies four classes as indicated. (B) Pie chart representation of UBC9 and PIASY binding site distribution in five different genomic regions as described in Fig. 1B. (C) Venn diagram representing overlap between SUMO1- and/or SUMO2-, UBC9- and PIASY-marked TSS. (D) Merged profiles of the SUMO machinery read density with respect to distance from TSS. (E,F) A Genome Browser view of the indicated ChIP-seq data at the FOS (E) and HRAS loci (F). (G) DAVID functional annotation of TSS marked by SUMO, UBC9, and PIASY (left) or SUMO, UBC9, but no PIASY (right). The top overrepresented categories are shown.

Figure 5.
Figure 5.

Sumoylation controls expression of histone and growth control genes. (A) WI38 cells were infected with lentiviruses expressing control shCt or shUBC9 shRNAs. Five days post-selection, the expression of the indicated genes was analyzed by RT-qPCR. (B) Western blot analysis of WI38 cells expressing shCt or shUBC9 showing expression of histones H3, H2A, H2B, and H4. Tubulin and Ponceau were used as loading controls. Depletion of UBC9 and concomitant loss of global sumoylation and sumoylated SP100 are shown as controls for knockdown efficiency. (C) WI38 cells were transfected with a control siCt or siPIASY siRNAs, and the expression of the indicated genes was analyzed by RT-qPCR. (D) Affymetrix analysis of histone mRNA differential expression in retrovirally infected WI38 cells overexpressing PIASY or a control vector (WT). (E–G) As in A. (H) Forward scatter analysis (FSC) of Ubc9+/+ (WT) and Ubc9fl/-;T2 (KO) MEFs treated for 7 d by tamoxifen (Demarque et al. 2011) (four embryos/genotype); mean size (FSC units): 628 ± 17.5 (Ubc9−/−) versus 594.3 ± 7.1 (Ubc9+/+); P-value = 0.006; one representative example is shown. (I) Total protein levels as measured by OD normalized to cell number of Ubc9+/+ (WT) and Ubc9fl/-;T2 (KO) MEFs treated for 7 d by tamoxifen (four embryos/genotype); mean amount (μg/mL): 219.3 ± 32.9 (Ubc9−/−) versus 193.5 ± 63.2 (Ubc9+/+); P-value = 0.019. (J) As in A. For all RT-qPCR, experiments were carried out in triplicate and data are represented as mean ±SEM (n = 3).

Figure 6.
Figure 6.

Depletion of UBC9 induces altered gene expression program together with a senescence-related phenotype. (A,B) Top selected categories identified by DAVID ontology analysis of up-regulated (A) and down-regulated (B) genes marked by SUMO1 and/or SUMO2 in their promoters in shUBC9 WI38 cells. (C) Growth curve of WI38 cells following infection with lentiviruses expressing shCt or shUBC9 shRNAs. (D) Percentage of EdU and SA-β-Gal positive WI38 cells at 4 d (early) and 8 d (late) post-infection. (E) Representative micrographs showing SA-β-Gal staining. (F) Western blot analysis of shCt or shUBC9 WI38 extracts for the indicated senescence markers. Actin and GAPDH were used as loading controls. (G) WI38 cells infected with lentiviruses expressing shCt, shUBC9, GFP, or HRASG12V were stained with propidium iodide and subjected to cell cycle analysis by flow cytometry. (H) Venn diagram showing overlap between Affymetrix gene expression profiles of HRASG12V-induced senescent and shUBC9 WI38 cells. Heat map below represents fold changes (FC) of selected genes in the two data sets. (I) Immunostaining of WI38 infected cells with control shCt, shUBC9, or HRASG12V as indicated and stained for PML (red), H3K9me3 (green), and DAPI for SAHF visualization. The unique PML aggregate in the shUBC9 cells is used as a positive control for knockdown efficiency (Zhong et al. 2000). (Right) Graph presenting the associated percentages of SAHF positive nuclei.

Figure 7.
Figure 7.

The SUMO machinery is released from chromatin in senescent cells. (A) Comparison of SUMO1 association with Pol II, H3K4me3, H3K27me3, and H3K9me3 in proliferating (P) and HRASG12V-induced senescent (S) WI38 cells. (B) ChIP-qPCR for SUMO1 in proliferating (P), HRASG12V-induced senescent (RAS), and replicative senescent (RS) WI38 cells on MTBP/MRPL13 and FOS promoters. (C) Proliferating and senescent cells were fractionated, and the presence of the indicated proteins in each fraction was quantitated by Western blot. Equal amounts of proteins were loaded for total lysate, Cyt (cytoplasmic fraction), SNE (soluble nuclear extract), and pellet (insoluble fraction). (D) Plots showing peak density in proliferating (red) and senescent (blue) cells over chromosome 6. (E) Scatter plot comparison of differential gene expression as measured by RNA-seq and differential occupancy of SUMO1 (left) or SUMO2 (right) (peak height fold change) in proliferating versus senescent cells. Differential expression is shown as log2 of reads per RPKM.

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