1. Volume 51, Issue 5, Pages 647-661 (September 2013)
The Aurora B Kinase and the Polycomb Protein Ring1B Combine to Regulate Active Promoters in Quiescent Lymphocytes 
Alberto Frangini, Marcela Sjöberg, Monica Roman-Trufero, Gopuraja Dharmalingam, Vanja Haberle, Till Bartke, Boris Lenhard, Marcos Malumbres, Miguel Vidal, Niall Dillon 
Molecular Cell 
Volume 51, Issue 5, Pages (September 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 51, 647-661DOI: (10.1016/j.molcel.2013.08.022)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1
Aurora B and PRC1 Proteins Bind to Active Promoters in Resting B Cells
(A) Comparison of the transcriptional levels of active genes in total RNA from resting and G1-elutriated LPS-activated B cells. Levels were calculated relative to a spike-in RNA (see the Experimental Procedures). Bars show mean ± SD, n = 3.
(B) Aurora B coIPs with Ring1B, Bmi1, and Cbx7, but not with Suz12 in resting B cells. Input lanes, 1% of input. Asterisk indicates Ig bands.
(C) ChIP analysis of Aurora B at gene promoters in resting and activated B cells. The values on the y axis represent the ratio of enrichment of immunoprecipitated samples relative to input. Bars show mean ± SD, n = 4. A ratio of 1 indicates no enrichment of IP over input, and analysis of Aurora B and Ring1B knockout cells confirmed that this represents the baseline where no significant binding is observed (data not shown). Values obtained for ChIP with nonspecific IgG (not shown) were always significantly lower than for specific antibodies.
(D) ChIP analysis of Ring1B and Suz12 in resting B cells. Bars show mean ± SD, n = 4.
(E) ChIP analysis of Ring1B in G0 resting B cells and in G1 cells purified by elutriation following activation for 3 days with LPS or anti-CD40. Bars show mean ± SD, n = 4.
(F) ChIP analysis of Cbx7 and Bmi1 in resting and purified G1-activated B cells. Bars show mean ± SD, n = 4.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Requirement of Aurora B and Ring1B for Transcription of a Range of Active Genes in Resting B Cells
(A) Western blot analysis of wild-type (WT) resting B cells and cells in which Aurora B (Aurkb) and Ring1B have been conditionally knocked out by 48 hr incubation with tamoxifen.
(B) ChIP-seq analysis of binding of Aurora B and Ring1B in WT and knockout resting B cells. FPKM values representing the expression of each gene are shown in brackets next to the gene name.
(C) Analysis of primary transcripts in nuclear RNA isolated from WT and Aurkb KO cells using primer pairs that span intron-exon junctions of selected genes. RNA levels were calculated relative to a spike-in RNA. Transcript levels are expressed as log2 fold gene expression changes in the knockout relative to WT. Bars show mean ± SD, n = 3.
(D) Comparison of transcript levels in total RNA from Aurkb and Ring1B KO resting B cells with levels in WT cells. Measurements were carried out as in (C) using within-exon primer pairs. Bars show mean ± SD, n = 4.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
ChIP-seq Analysis of the Genome-wide Distribution of Aurora B, PRC1, and PRC2 in Resting B Cells
(A) Examples from ChIP-seq analysis of Aurora B, Ring1B, Cbx7, H3K27me3, and Ezh2 in resting B cells. FPKM values from mRNA-seq analysis are shown in brackets after each gene name.
(B) Heatmaps showing the distribution of Aurora B, Ring1B, Cbx7, H3K27me3, and Ezh2 within 3 kb of transcriptional start sites (TSSs) in resting B cells. Active genes were ranked based on their transcriptional level (FPKM values) as assessed by mRNA-seq analysis (Table S6). For the analysis of PRC2-repressed genes, a total of 2,000 genes with the highest density of tags for H3K27me3 around the TSS were selected and ranked in decreasing order of H3K27me3 tag-density. Another 2,000 genes (silent) were randomly selected from among the silent genes that showed low H3K27me3 tag density.
(C) Correlation plots for enrichment levels of Aurora B and Ring1B at active promoters in resting B cells. RPM (reads per million of mapped reads) for each data set were calculated from the number of reads overlapping within the TSS (±1 kb) of active genes (FPKM > 0.5). A total of 13,264 genes were used for the analysis (see the Experimental Procedures).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Analysis of RNA Pol II Binding to Gene Promoters in AurkB and Ring1B Knockout Resting B Cells
(A) qPCR-ChIP analysis comparing binding of unphosphorylated RNAPII (8WG16) and RNAPII-S5ph in WT resting B cells with Aurora B KO cells (left panels) and Ring1B KO cells (right panels). Bars show mean ± SD, n = 2.
(B) ChIP-seq was used to analyze binding of unphosphorylated RNAPII (8WG16) and RNAPII-S5ph in Aurkb KO and Ring1B KO resting B cells. Examples are shown of the binding profiles at individual genes. RPKM values representing the expression of each gene are shown in brackets next to the gene name.
(C) Line plots depicting the average reduction in binding of unphosphorylated RNAPII and RNAPII-S5ph in Ring1B KO cells (left) and in Aurkb KO cells (right). The binding is assessed in a window covering 6 kb centered on the TSS (based on RefSeq data).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
A Switch Occurs between Binding of Aurora B and MSK1 at Active Promoters during B Cell Activation
(A) Time course of binding of Aurora B and MSK1 to the promoters of the transcriptionally active Fcrl1, Mapbpip, and Gba genes. ChIP experiments were performed on resting B cells treated for 0, 30, 60, and 120 min with LPS. Points on the graphs represent mean ± SD, n = 2.
(B) ChIP analysis of H3S28ph at active promoters in WT and Aurkb KO cells. Bars show mean ± SD, n = 2.
(C) ChIP-seq analysis of H3S28ph in wild-type and Aurkb knockout resting B cells. Examples are shown for the binding profiles of individual expressed genes. The bottom panel shows a gene-dense region of mouse chromosome 3 where a cluster of silent genes is flanked by active genes.
(D) Line plots depicting the average reduction in H3S28ph in in Aurkb KO cells. The binding is assessed in a window covering 6 kb centered on the TSS (based on RefSeq data).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Aurora B Inhibits H2A Ubiquitination and Promotes Deubiquitination at Active Promoters in Resting B Cells
(A) ChIP analysis of H2Aub1 at gene promoters in WT and Aurkb KO resting B cells. Bars show mean ± SD, n = 4. Fdx1 and Atp5s are not bound by Aurora B, and their transcription levels are not reduced in the Aurora B KO cells (Figure 1C).
(B) ChIP analysis of binding of the USP16 deubiquitinase to gene promoters in WT and AurkB KO cells. Bars show mean ± SD, n = 2.
(C) ChIP-seq analysis of H2Aub1 in wild-type and AurkB KO cells. Examples are shown for the binding profiles of individual expressed genes and compared with the profiles for Ring1B.
(D) Heatmaps of H2A ubiquitination within 3 kb of TSSs of expressed genes in WT and Aurora B KO resting B cells in WT and Aurora B KO cells. Active genes were ranked based on their transcriptional level (FPKM values) as assessed by mRNA-seq analysis (Table S6).
(E) In vitro deubiquitination by USP16. HEK293T-purified nucleosomes were deubiquitnated by USP16 in the presence or absence of Aurora B and the Aurora B inhibitor AZD1152 as indicated above the figure. Levels of H2Aub1 and H2A were measured by western blot (top and middle panels). The lower panel shows quantification of H2Aub1 levels relative to H2A. Bars show mean ± SD, n = 3. Asterisk indicates that difference is statistically significant (Student’s t test, p < 0.05).
(F) In vitro ubiqutination of recombinant nucleosomes in the presence and absence of Aurora B (top panel) and comparing wild-type (WT) UBE2D3 and UBE2D3-S138A (bottom panel).
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 Aurora B and Ring1B Regulate Transcription in Resting T Cells
(A) ChIP analysis of binding of Aurora B and Ring1B to gene promoters in quiescent resting CD4+ T cells. Bars show mean ± SD, n = 2.
(B) Transcriptional analysis of total RNA from Aurkb and Ring1B KO resting CD4+ T cells. Measurements were carried out using within-exon primer pairs as described for Figure 2D. Bars show mean ± SD, n = 3.
(C) Resting B and T cells display reduced viability following knockout of Aurora B or Ring1B. Viable cells were scored by trypan blue exclusion. WT, Aurkb KO, and Ring1B KO resting B or T cells were cultured for the indicated times in the presence of 250 nM tamoxifen (B cells, left panel) or 500 nM tamoxifen (T cells, right panel). Bars show mean ± SD, n = 3. Values for means and SDs and the p values obtained using a two-tailed homoscedastic t test are shown in Table S5.
(D) Model to explain the role of Aurora B and Ring1B in regulating transcription at active genes in resting B cells. Aurora B phosphorylates the USP16 deubiquitinase and the UBE2D3 ligase. These phosphorylation events enhance the deubiquitinatation activity of USP16 and block the E2 ligase activity of UBE2D3. Ring1B and other PRC1 proteins are recruited to promoters independently of PRC2 through interaction with DNA binding transcription factors (TF1 and TF2). The PRC1 proteins interact with and recruit unphosphorylated RNAPII, and inhibition of PRC1-mediated H2A ubiquitination allows the PRC1 complex to function as a coactivator. Aurora B also phosphorylates H3S28 and through this modification promotes binding of RNAP and transcriptional initiation.
Molecular Cell  , DOI: ( /j.molcel )
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