1. NoRC Recruitment by H2A.X Deposition at rRNA Gene Promoter Limits Embryonic Stem Cell Proliferation 
Boris Eleuteri, Sergi Aranda, Patrik Ernfors 
Cell Reports 
Volume 23, Issue 6, Pages (May 2018)
DOI: /j.celrep
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2. Cell Reports 2018 23, 1853-1866DOI: (10.1016/j.celrep.2018.04.023)
Copyright © 2018 The Author(s) Terms and Conditions

3. Figure 1 H2A.X-Dependent Modulation of ESC Proliferation
(A) Cell growth analyses of H2A.X-WT and -KO ESCs at days 1, 2, and 3 in vitro (n = 3 in triplicate).
(B) Cell number recovery after 48 hr by H2A.X re-introduction (n = 3).
(C) Proliferation rate by 10-min BrdU pulse (n = 5).
(D and E) Cell cycle distribution was analyzed by FACS. (D) is a representative cell cycle profile and (E) is a quantitative analysis (n = 4).
(F) Pluripotency analysis (H3 as loading control).
(G–I) Quantification of Oct 3/4 (G), Sox2 (H), and Nanog (I) protein levels in H2A.X-WT and -KO ESCs (n = 4). Values are represented as the mean ± SEM. Two-tailed t test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
See also Figure S1.
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4. Figure 2
H2A.X Acts on Proliferation Independently of H2A.X S139 and Y142 Phosphorylation
(A) H2A.X phosphorylation status in ESCs. H2A.X KO was used as negative control and H3 as loading control.
(B) The expression and the phosphorylation state of re-introduced H2A.X, H2A.X S139A, H2A.X Y142F, and H2A.X S139A:Y142F in H2A.X-KO ESCs analyzed by WB. Vinculin is the loading control. Exogenous (exo) and endogenous (endo) levels of H2A.X are shown.
(C) Rescue of proliferation by various H2A.X mutants (n = 5 in triplicate; mean ± SEM; 1-way ANOVA Bonferroni, ∗∗∗p < 0.001).
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5. Figure 3 Proteomic Analyses of H2A.X Protein Interactome
(A) Outline of experimental strategy for mass spectrometric analyses and representative silver-stained gel.
(B) Gene ontology (GO) for cellular compartments of 92 identified proteins. Bars indicate the log10 (p value) for each group (p values, modified Fisher’s exact test, Expression Analysis Systematic Explorer [EASE] score).
(C) GO analysis for biological processes. Bars indicate the log10 (p value).
(D) H2A.X protein interaction network by STRING analysis. Proteins shared between processes are colored half blue and half red. Proteins not included in any cluster are in white. DSB repairs have a thick black ring.
(E) Number of proteins in each cluster highlighted with STRING.
(F) Protein network of chromatin-associated proteins by STRING. H2A.X is highlighted in pink. Proteins not included in the three main clusters are in white. DSB repair proteins have a thick black ring.
See also Figures S2–S4 and Table S1.
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6. Figure 4
The NoRC Is Critical for ESC Proliferation and Interacts with H2A.X
(A) Co-immunoprecipitation (coIP) of endogenous H2A.X and BRG1 in H2A.X-WT cells and H2A.X-KO cells.
(B) Effects of BRG1 on cell numbers using siRNA against Brg1 (siBrg1) or control siRNA (siCTRL). Quantification of cell numbers at 48 hr is shown (n = 3, in triplicate).
(C) CoIP of endogenous H2A.X with FACT subunit SPT16 was performed as in (A).
(D) Effects of Spt16 on cell numbers using siRNA against Spt16 and analyzed as in (B) (n = 3, in triplicate).
(E) CoIP of endogenous H2A.X with NoRC subunit TIP5 performed as in (A).
(F) Effects of Tip5 on cell numbers using siRNA against Tip5 and analyzed as in (B) (n = 3, in triplicate).
(G) H2A.X interacts with the NoRC and co-factors as determined by coIP.
(H) H2A.X interaction with the NoRC and co-factors is S139 and Y142 independent. FLAG H2A.X WT or FLAG-H2A.X S139A:Y142F in H2A.X-KO ESCs were expressed for 48 hr. Horizontal lane IgGs shows IP efficiency. Values are represented as the mean ± SEM (two-tailed t test, ∗p < 0.05). Vertical lines indicate splicing of blots.
See also Figures S5 and S6.
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7. Figure 5 H2A.X-Dependent Recruitment of the NoRC to rDNA Promoters
(A) Scheme of ribosomal genes. The view includes a segment (13.8 kb) containing the promoter and 18S, 5.8S, and 28S genes next to the transcriptional start site. The upstream promoter element (blue) and the core element (red) of the promoter are highlighted. The regions amplified in the qPCR after ChIPs are indicated with a black line.
(B) Occupancy of H2A.X at different regions of rDNA in H2A.X-WT and -KO ESCs (normal rabbit IgGs, set as 1, gray line in graphs; n = 3, in triplicates).
(C) Occupancy of H2A.X at different regions of ribosomal genes indicated in (A) and performed as in (B) (n = 3, in triplicates).
(D) NoRC (SNF2H) occupancy in the presence or absence of H2A.X (normal rabbit IgGs, set as 1, gray line; n = 3, in triplicates).
(E) Analysis of NoRC (TIP5) recruitment at the promoter of ribosomal genes. (Left) ChIP of endogenous TIP5 was performed as in (D) (n = 5, in triplicates). (Right) Analysis of NoRC (TIP5) recruitment at the promoter of ribosomal genes occupied by H2A.X was by re-ChIP analysis (ChIP on ChIP). The amplification for the coding region (8 kb) was used as a negative control and qPCRs gave a value below what could be determined (n = 3, in triplicate).
(F) Rescue of NoRC recruitment (TIP5) by H2A.X at ribosomal promoters. ChIP of endogenous TIP5 used H2A.X-KO ESCs transfected with a plasmid expressing H2A.X (FLAG H2A.X) or with an empty plasmid (FLAG) used as negative control and to normalize the quantification (n = 3, in triplicate).
(G) CoIP of endogenous SNF2H with H2A.X using H2A.X-WT and -KO ESCs.
(H) CoIP of endogenous TIP5 with H2A.X using H2A.X-WT and -KO ESCs. Values are represented as the mean ± SEM (1-way ANOVA Bonferroni, B–E, two-tailed t test, F, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).
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8. Figure 6
rRNA Repression by H2A.X Recruitment of the NoRC Affects Proliferation
(A) Quantification of rRNA synthesis in the presence or absence of H2A.X or after H2A.X re-expression in H2A.X-KO cells. Mature (5.8S, 18S, and 28S) and precursor (45S) rRNA content per cell was quantified, and rRNAs in H2A.X-WT ESCs were set as 100% (n = 6, in triplicates; H2A.X re-expression n = 3, in triplicates).
(B and C) Quantification of protein synthesis in ESCs in the presence or absence of H2A.X or after H2A.X re-expression in H2A.X-KO cells. The presence of nascent polypeptides is shown in (B) and quantified in (C) (n = 4 for H2A.X-WT and -KO cells; n = 3 for re-expression in H2A.X-KO cells).
(D and E) The NoRC is a critical component limiting the proliferation of ESCs and requires H2A.X. Tip5 expression was silenced in H2A.X-WT ESCs (D) or H2A.X-KO cells (E) by RNAi (siTip5) or a scramble control siRNA (siCTRL). Quantification of mature (5.8S, 18S, and 28S) and precursor (45S) rRNAs and cell numbers is shown. rRNA levels in siTip5 were normalized to rRNAs transfected with siCTRL (n = biological/technical, n = 2/3 rRNAs, n = 2/3 cell number except 48 hr where n = 7/3).
(F) Transcriptional activation of ribosomal genes enhances ESC proliferation. Ribosomal genes were activated in H2A.X-WT ESCs using CRISPR/Cas9 activation. Sg-UPE, guide RNA for upstream promoter element; sg-Gal4, control guide RNA (n = 3, in triplicates).
(G and H) Repression of ribosomal genes limits proliferation of ESCs. (G) Ribosomal genes were repressed by CRISPR/Cas9 interface in WT ESCs and H2A.X-KO cells and the effect was quantitated by measuring 45S pre-rRNA. (H) Effect of CRIPSR/Cas9 interference on cell number. sg-rRNA, guide for promoter; sg-Gal4, control guide RNA (n = 3, in triplicates). Values are represented as the mean ± SEM (two-tailed t test, ∗p < 0.05 and ∗∗p < 0.01).
See also Figure S6.
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9. Figure 7
H2A.X Participates in the Formation of Heterochromatin-like Structure at the rDNA Promoter
Epigenetic state at the promoter of ribosomal genes in the presence or absence of H2A.X. ChIP of indicated histone modifications at the ribosomal promoter is shown as the percentage of H2A.X-WT control ESCs (n = 3, in triplicates). Values are represented as the mean ± SEM (two-tailed t test, ∗∗p < 0.01 and ∗∗∗p < 0.001).
Cell Reports  , DOI: ( /j.celrep )
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