1. Volume 15, Issue 3, Pages 281-294 (September 2014)
Histone Variant H2A.X Deposition Pattern Serves as a Functional Epigenetic Mark for Distinguishing the Developmental Potentials of iPSCs 
Tao Wu, Yifei Liu, Duancheng Wen, Zito Tseng, Martik Tahmasian, Mei Zhong, Shahin Rafii, Matthias Stadtfeld, Konrad Hochedlinger, Andrew Xiao 
Cell Stem Cell 
Volume 15, Issue 3, Pages (September 2014)
DOI: /j.stem
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2. Cell Stem Cell 2014 15, 281-294DOI: (10.1016/j.stem.2014.06.004)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 H2A.X Plays a Role in Targeting Extraembryonic Genes
(A) Schematics of H2A.X ChIP-seq and bioinformatic analyses. First, ESC and MEF H2A.X peaks were determined against H2A.X KO ESC or MEF controls, IgG control, and DNA input. Then, an established hidden Markov model (RSEG) was used to obtain ESC-specific H2A.X deposition regions.
(B) The specificity of H2A.X ChIP method. DNA sample libraries copurified with H2A.X antibodies or isolated from input were amplified by 1,000-fold. Note that no signals were detected in the H2A.X KO DNA sample library.
(C) A locus on Chr18 (30–32 Mb) that is specifically enriched for H2A.X deposition in ESCs. Y axis: normalized H2A.X ChIP-seq signals (reads per million) in ESCs (orange) and MEFs (gray).
(D) The normalized H2A.X ChIP-seq signals (reads per million) in ESC-specific H2A.X deposition regions (orange) and Cdx2-bound enhancers (purple) at the Dab2 and Gata3 loci.
(E) At the genome-wide level, 63.5% of ESC-specific H2A.X deposition regions are overlapped with 44% of Cdx2 binding sites (p < 1.0 × 10−6 versus genome random), whereas MEF-H2A.X peaks covered only 1.5% of Cdx2 targets (see Figure S1).
(F) One of the DNA binding motifs of H2A.X (see Figure S1) is similar to the major Cdx2 motif (analyzed by “STAMP,” E-value = 1.66 × 10−16).
(G) Comparative H2A.X deposition in TSCs on whole Chr12. Y axis: relative H2A.X deposition levels (TSCs versus ESCs) are shown in enrichment scores determined with RSEG. Positive value: regions enriched for H2A.X deposition over ESCs (gray); negative values: regions devoid of H2A.X deposition over ESC (green bars).
(H) At the genome-wide level, 38% of the regions devoid of H2A.X deposition in TSCs are overlapped with Cdx2 binding sites (p < 1.0 × 10−5 versus genome random).
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4. Figure 2
H2A.X Plays a Critical Role in Repressing Extraembryonic Gene Expression
(A) The p value distribution of all genes in RNA-seq analysis of H2A.X KO ESCs (versus control ESCs). y axis: number of genes associated with a specified p value; x axis: significance scores derived from p value (−log10 [p value]). Several TE lineage markers are labeled in red.
(B) Gene ontology term analysis of H2A.X-repressed genes. Shown are the top ten biological processes.
(C) Upregulated genes identified in RNA-seq (H2A.X−/− ESCs versus control ESCs) are significantly overlapped (48.3%) with TE lineage genes (regulated by Cdx2, Gata3, or Eomes; p = 5.17 × 10−46, hypergeometric test).
(D) Consistent with the RNA-seq results, the qRT-PCR approach confirmed that only extraembryonic genes were upregulated in H2A.X KO ESCs. Extra-embryonic, extraembryonic lineage genes; pluripotency, ESC pluripotency genes; germ layer, three germ layer (ecto-, meso-, and endoderm) lineage genes; Ctrl, GAPDH and H2A.X. The average of triplicate experiments (∗p < 0.05, t test) is shown. Error bars represent SEM.
(E) ChIP-qPCR approach demonstrated that H2A.X deficiency significantly increased Cdx2 binding to the targeted sites. Top: schematic of H2A.X knockdown and induction of Cdx2 expression. Bottom: Cdx2 binding (relative ChIP signals versus input) at eight Cdx2-targeted enhancers. The average of triplicate experiments (p < 0.05, t test) is shown. Error bars represent SEM.
(F) ChIP-qPCR approach demonstrated that H2A.X deficiency significantly reduced the levels of H3K9Me3, but not H3K27Me3 levels (see Figure S2) at the extraembryonic gene loci. The average of triplicate experiments (∗p < 0.05, t test) is shown. Error bars represent SEM.
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5. Figure 3
H2A.X Depositions Are Strongly Correlated with the Developmental Potentials of the iPSC Lines
(A) Unsupervised hierarchical cluster analysis using H2A.X ChIP-seq signals (shown in heatmap) successfully distinguished the 4N− iPSCs (n = 6) from 4N+ iPSCs (n = 3) and ESCs (n = 3); only 4N+ iPSC lines (n = 3) were grouped into the same clade as ESCs (n = 3). Rows: ESC and iPSC lines as labeled. Columns: genomic regions with ESC-specific H2A.X deposition. Blue, decreased; green, similar or no change.
(B) A box plot illustrated that the 4N− iPSC lines (n = 3) had significantly more genomic regions devoid of H2A.X deposition than those in 4N+ iPSC lines (n = 3) or ESC lines (n = 3; p < 0.001, t test). 4N+ iPSC lines were similar to ESC lines (no p value).
(C) Comparative H2A.X depositions in 4N+ iPSCs and 4N− iPSCs on whole Chr14. Shown are the H2A.X ChIP signals in iPSCs relative to ESCs. Y axis: enrichment scores determined with RSEG. Positive value: regions enriched for H2A.X deposition over control ESCs (gray); negative values: regions devoid of H2A.X deposition over control ESCs (blue bars).
(D) Immunblotting demonstrated that the total H2A.X protein levels in ESCs, 4N+ iPSCs, and 4N− iPSCs (n = 2 for each group) were similar.
(E) Venn diagram showed that genome regions devoid of H2A.X deposition in all 4N− iPSC lines are significantly overlapped with the Cdx2-targeted enhancer sites (48%, p < 1.0 × 10−6 versus genome random).
(F) The relative H2A.X deposition levels (versus ESC control) are shown in five 4N− iPSC lines and two 4N+ iPSC lines at a critical extraembryonic gene locus, Fgfr2. Y axis: relative H2A.X deposition levels (iPSC versus ESC) are shown in enrichment scores determined with RSEG. No signals are displayed in 4N+ iPSC lines because H2A.X depositions are identical to those of the ESC control.
See also Figure S3 and Table S1.
Cell Stem Cell  , DOI: ( /j.stem )
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6. Figure 4
H2A.X Deposition Defects in 4N− iPSCs Result in Aberrant Upregulation of Extraembryonic Genes
(A) Comparative RNA-seq results: 4N− iPSCs versus 4N+ iPSCs. Y axis: the relative enrichment in 4N−iPSCs (versus 4N+ iPSCs, log2); x axis: normalized expression level of individual genes. Red dots: FDR < 0.01.
(B) In 4N− iPSCs, 68% of the upregulated genes are located in the genomic regions devoid of H2A.X deposition (p < versus genome random).
(C) The upregulated genes in 4N− iPSCs are significantly overlapped with those in H2A.X KO ESCs (p = 2.75 × 10−22, hypergeometric test).
(D) The upregulated genes in 4N− iPSCs are significantly overlapped (45%) with TE lineage genes (regulated by Cdx2, Gata3, or Eomes; p = 1.61 × 10−7, hypergeometric test).
(E) The qRT-PCR approach confirmed that only extraembryonic genes were upregulated in 4N− iPSCs. Extra-embryonic, extraembryonic lineage genes; pluripotency, ESC pluripotency genes; Germ layer, three germ layer (ectoderm, mesoderm, and endoderm) lineages genes; Ctrl, GAPDH and H2A.X. Error bars represent SEM.
(F)A ChIP-qPCR approach demonstrated that H2A.X deficiency significantly reduced the levels of H3K9Me3 at the extraembryonic gene loci. H3K27Me3 levels were unchanged (see Figure S4). Error bars represent SEM.
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7. Figure 5
In Vitro TE Lineage Differentiation Assays Distinguish the Developmental Potentials of iPSC Clones
(A) An ESC line in which Oct4 expression was controlled by the “Tet-On” system (Ivanova et al., 2006) was used in TE lineage differentiation. H2A.X was first depleted by an RNAi approach. Then, the H2A.X knockdown or control knockdown cells were cultured without Oct4 expression for 5 days (by removing doxycycline). Cdx2, Eomes, and Elf5 expressions were examined with qRT-PCR. Y axis: relative gene expression level (Oct4− versus Oct4+ control). The TE markers were further increased in H2A.X knockdown cells after shutting down Oct4 expression (∗p < 0.05; ∗∗p < 0.01, t test). Error bars represent SEM.
(B) Oct4 was depleted by RNAi approach in 4N+ or 4N− iPSC lines. qRT-PCR approaches demonstrated that the extraembryonic lineage markers in 4N− iPSCs were significantly higher than those in the 4N+ iPSC. y axis: the relative enrichment (Oct4 knockdown versus Control knockdown). Error bars represent SEM.
(C) H2A.X−/− and control ESCs were cultured under ES or TS culture condition for 96 hr without feeder cells. qRT-PCR approaches demonstrated that the extraembryonic lineage marker expressions in H2A.X−/− ESCs were significantly higher than those in control ESCs. Y axis: the relative expression level (log2, TS media culture versus ES media culture control). Error bars represent SEM.
(D) Similar experiments were performed in 4N− iPSCs and 4N+ iPSCs under ES or TS culture condition. Error bars represent SEM. See also Figure S5.
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8. Figure 6
In Vivo Assays Distinguish the Developmental Potentials of iPSC Clones
(A) The control H2A.XFloxed/Floxed ESCs (H2A.X [f/f]) and H2A.X−/− ESCs (generated from H2A.X [f/f] + Cre) were labeled with H2B-GFP, cultured in TS media for 5 days, then injected into four-cell embryos and examined at the blastocyst stage. The engrafted H2A.X−/− ESCs contributed to the developing trophectoderm layer (white arrows) at a much higher frequency than the engrafted H2A.X (f/f) ESCs (p = 0.036, Chi-square test).
(B) The 4N+ iPSCs and 4N− iPSCs were cultured in TS media for 5 days (labeled with a red fluorescent dye, pKH26, Sigma), then injected into four-cell embryos, and examined at the blastocyst stage. The engrafted 4N− iPSCs contributed to the developing trophectoderm layer (white arrows) at a much higher frequency than the engrafted 4N+ iPSCs (p = 0.01, Chi-square test).
(C) Top: engrafted H2A.X (f/f) ESCs that contributed to ICM (white arrows) were negative for Cdx2 staining. Bottom: engrafted H2A.X−/− ESCs that contributed to the trophectoderm were costained with Cdx2 (white arrowheads).
(D) Top: engrafted 4N+ iPSCs that contributed to ICM were negative for Cdx2 staining (white arrows). Bottom: engrafted 4N− iPSCs that contributed to the trophectoderm were costained with Cdx2 (white arrowheads). See also Figure S6.
Cell Stem Cell  , DOI: ( /j.stem )
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9. Figure 7 A Model Illustrating the Functions of H2A.X in ESCs and iPSCs
(A) In wild-type ESCs and 4N+ iPSCs, H2A.X is deposited to extraembryonic genes, most of which are downstream targets of Cdx2. H2A.X negatively regulates Cdx2 binding via controlling chromatin structures at enhancers and therefore, represses the expression of these genes.
(B) In H2A.X-deficient ESCs, extraembryonic genes are aberrantly regulated due to the lack of H2A.X-mediated repression.
(C) In 4N− iPSCs, H2A.X is not redistributed to the extraembryonic genes. Because of this epigenetic mutation, extraembryonic genes are aberrantly regulated. Thus, these cells are primed to extraembryonic lineage differentiation.
Cell Stem Cell  , DOI: ( /j.stem )
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