1. Volume 20, Issue 5, Pages 689-705.e9 (May 2017)
PRC2 Facilitates the Regulatory Topology Required for Poised Enhancer Function during Pluripotent Stem Cell Differentiation 
Sara Cruz-Molina, Patricia Respuela, Christina Tebartz, Petros Kolovos, Milos Nikolic, Raquel Fueyo, Wilfred F.J. van Ijcken, Frank Grosveld, Peter Frommolt, Hisham Bazzi, Alvaro Rada-Iglesias 
Cell Stem Cell 
Volume 20, Issue 5, Pages e9 (May 2017)
DOI: /j.stem
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2. Cell Stem Cell 2017 20, 689-705.e9DOI: (10.1016/j.stem.2017.02.004)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1
Epigenomic Identification of mESC PEs that Become Active in Anterior Neural Progenitors
(A) ChIP-seq data for p300, H3K27me3, H3K27ac, and H3K4me3 were used to identify 1,015 PEs in mESCs. Average mESC ChIP-seq signal profiles for H3K27ac, H3K27me3, H3K4me1, and H3K4me3 are shown around the central position of the p300 sites defining PEs.
(B) p300, H3K4me1, H3K27ac, H3K27me3, and H3K4me3 ChIP-seq profiles in mESCs are shown around a representative PE (PE Lhx5(−109), green shade) located 109 kb upstream of Lhx5.
(C) In silico functional annotation of PEs was performed using GREAT (McLean et al., 2010). To illustrate the gene categories with which PEs were preferentially associated, five of the ten most overrepresented terms belonging to three major gene ontologies are shown: Gene Ontology (GO) Biological Process (red), Mouse Genome Informatics (MGI) expression: detected (blue), and Mouse Phenotype (green).
(D) H3K27ac peaks identified in pairs of mouse embryonic tissues at E11.5 and E14.5 were combined (top: forebrain and liver; bottom: forebrain and limb). The percentages of H3K27ac peaks occurring in only one tissue or in both are shown for all H3K27ac peaks (left) or for H3K27ac peaks overlapping with PEs (right).
(E) Vista Enhancer Browser sequences were classified in five different groups depending on their in vivo enhancer activity in mouse E11.5 embryos (Brain includes sequences with enhancer activity in forebrain, midbrain, hindbrain or eye). The relative abundance of each Vista enhancer category was compared between all Vista sequences (n = 2,274) and those overlapping with PEs (n = 25). p values were calculated using hypergeometric tests.
(F) Strategy used to identify PEs that became active in anterior neural progenitors (AntNPCs).
(G) p300, H3K27me3, and H3K27ac ChIP-seq profiles in mESCs and AntNPCs are shown around PE Lhx5(−109) (red shade).
(H) ChIP-seq data for H3K27ac in AntNPCs was used to identify PEs that became active in AntNPCs (PoiAct enhancers). Average H3K27ac ChIP-seq signals in mESCs and AntNPCs are shown around the central position of the p300 sites defining PoiAct enhancers.
In (A), (B), (G), and (H), the ChIP-seq signals were normalized as RPGC (reads per genome coverage) using 5-bp bins.
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4. Figure 2
PoiAct Enhancers Display In Vivo Enhancer Activity and Physically Interact with Their Target Genes in mESCs and AntNPCs
(A) RNA-seq data from mESCs and AntNPCs were used to calculate the expression values (as fragments per kilobase of transcript per million mapped reads [FPKM]) for all genes (All), genes linked to PEs (Poised), and genes linked to PoiAct enhancers. p values were calculated using paired Wilcoxon tests.
(B) H3K27ac peaks identified in mouse embryonic forebrain and liver at E11.5 and E14.5 were combined. The percentages of H3K27ac peaks occurring in only one tissue or in both are shown for all H3K27ac peaks (left) or for H3K27ac peaks overlapping with PoiAct enhancers (right).
(C) Vista Enhancer Browser sequences were categorized into five different groups as in Figure 1E. The relative abundance of each Vista enhancer category was compared between all Vista sequences (n = 2,274) and those overlapping with PoiAct enhancers (n = 10). p values were calculated using hypergeometric tests.
(D) Representative E11.5 mouse embryos in which Vista sequences overlapping PoiAct enhancers were used for in vivo reporter assays (Visel et al., 2007).
(E–H) 4C-seq and H3K27ac ChIP-seq profiles generated in mESCs and AntNPCs are shown around PE Lhx5(−109) (E), PE Six3(−133) (F), PE Sox1(+35) (G), and PE Lmx1b(+59) (H). The PEs and their putative target genes are shaded in green and yellow, respectively. 4C-seq experiments were performed using PEs as viewpoints (red triangles).
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5. Figure 3
PEs Are Necessary for the Induction of Major Anterior Neural Regulators
(A) CRISPR/Cas9 approach used to delete PEs in mESCs. Two guide RNAs (gRNAs) flanking each PE candidate region were used to generate the deletions.
(B) List of the five PE candidates for which deletions in mESC were generated. PEs were named based on the distance (in Kb) and relative location (upstream [−] or downstream [+]) with respect to their putative target genes.
(C, E, G, and I) p300, H3K27me3, and H3K27ac ChIP-seq profiles in mESCs and of H3K27ac ChIP-seq profiles in AntNPCs are shown around PE Lhx5(−109) (C), PE Six3(−133) (E), PE Sox1(+35) (G), and PE Wnt8b(+21) (I). The deleted regions are shaded in green.
(D, F, H, and J) The expression of four PE putative target genes (Lhx5 in D, Six3 in F, Sox1 in H, and Wnt8b in J) and of the pluripotency marker Zfp42 was investigated by qRT-PCR in mESCs and AntNPCs that were either WT (blue) or homozygous for a deletion of the indicated PE. The results obtained in two biologically independent differentiation experiments are presented in each panel (Rep1 and Rep2). Expression values were normalized to two housekeeping genes (Eef1a1 and Tbp), and error bars represent standard deviations (SDs) from technical triplicates. The WT AntNPCs used in each panel were independently derived, as mESC lines with PE deletions were always compared to WT mESCs in differentiations performed in parallel.
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6. Figure 4
Functional and Mechanistic Characterization of a Representative Poised Enhancer Controlling Lhx5 Induction in AntNPCs
(A) LHX5 and α-TUBULIN levels were investigated by western blot in mESCs and AntNPCs that were either WT or homozygous for a deletion of the PE Lhx5(−109) (PE Lhx5(−109)−/−).
(B) LHX5 protein levels were investigated by immunofluorescence in mESCs and AntNPCs that were either WT (top) or homozygous for a deletion of the PE Lhx5(−109) (bottom).
(C) RNA-seq experiments in AntNPC derived from WT mESCs (WT AntNPCs) or mESCs with a PE Lhx5(−109) homozygous deletion (PE Lhx5(−109)−/− AntNPC). Mouse genes were plotted according to the average normalized RNA-seq read counts in WT AntNPCs and PE Lhx5(−109)−/− AntNPCs. Genes considered as significantly up and downregulated in PE Lhx5(−109)−/− AntNPCs are shown in red and blue, respectively.
(D) Genes significantly downregulated in PE Lhx5(−109)−/− AntNPCs were annotated according to Gene Ontology Biological Process terms. Ten terms among the 20 most significantly overrepresented ones are shown.
(E) ChIP-qPCR primers were designed in proximity to the deleted PE regions to investigate how the PE deletions affected the binding of H3K27ac (F) or RNA Pol2 (G and H).
(F) H3K27ac ChIPs were performed in mESCs and AntNPCs that were either WT (blue) or homozygous for a deletion of the PE Lhx5(−109) (red). H3K27ac fold-enrichment levels were investigated by ChIP-qPCR at the PE Lhx5(−109) (green shade) and the Lhx5 promoter and two other putative Lhx5 enhancers active in AntNPCs (yellow shades). H3K27ac ChIP-qPCR signals in each ChIP sample were normalized to the average signals obtained in the same sample when using two different control intergenic regions (“Chr6_neg” and “Chr2_neg”). SDs were calculated from technical triplicate reactions and are represented as error bars.
(G and H) Pol2 8WG16 (G) and Pol2 S5P (H) ChIPs were performed in mESCs and AntNPCs that were either WT (dark and light blue) or homozygous for a deletion of the PE Lhx5(−109) (red and orange). Pol2 8WG16 and Pol2 S5P fold-enrichment levels were investigated at the Lhx5 promoter, the PE Lhx5(−109) and the Zfp42 promoter as described in (F).
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7. Figure 5
The Interactions between PEs and Their Target Genes in mESCs Are PRC2 Dependent
(A–D) H3K27me3 ChIP-seq profiles generated in WT mESCs and 4C-seq profiles generated in WT and EED−/− mESCs are shown around PE Lhx5(−109) (A), PE Six3(−133) (B), PE Sox1(+35) (C), and PE Lmx1b(+59) (D). The PEs are shaded in green, while the H3K27me3-marked regions covering the PE target genes are shaded in yellow. 4C-seq experiments were performed using PEs as viewpoints (red triangles).
(E) The 4C signals presented in (A)–(D) were quantified in two regions (R1 and R2) within each of the four investigated loci. R1 corresponds to a 20-kb window centered on the transcription start site (TSS) of each PE putative target gene (R1 dashed line); R2 corresponds to a 20-kb window centered on an intermediate position between the PEs and their putative target genes (R2 dashed line). p values were calculated using paired Wilcoxon tests.
(F) Interactions between PEs and their target gene promoters were validated by PCR analysis of 3C and 4C samples generated in WT and EED−/− mESCs. A ∼200-bp fragment at the TBP locus was used as a loading control, while the Sox1 PE-Six3 promoter primer combination was used as a negative control.
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8. Figure 6
PRC2 as a Potential Facilitator of PEs' Regulatory Activity during AntNPC Differentiation
(A) H3K27me3 and H3K27ac ChIP-seq profiles generated in WT and EED−/− mESCs and H3K27ac ChIP-seq profiles generated in WT and EED−/− AntNPCs are shown around a representative PE (PE Lhx5(−109)).
(B and C) Average H3K27me3 ChIP-seq signals in WT and EED−/− mESC (B) and average H3K27ac ChIP-seq signal profiles in WT and EED−/− mESCs and WT and EED−/− AntNPCs (C) are shown around the central position of the p300 sites defining PoiAct enhancers.
(D) Gene expression levels measured by RNA-seq as FPKM in WT mESCs, EED−/− mESCs, WT AntNPCs, and EED−/− AntNPCs are shown for selected genes associated with PoiAct enhancers as well as for markers of pluripotent cells, mesoderm/endoderm, and hindbrain/spinal cord. Error bars represent the SDs of either two (WT mESCs and EED−/− mESCs) or four (WT AntNPCs and EED−/− AntNPCs) biological replicates.
(E) RNA-seq data from WT mESCs, EED−/− mESCs, WT AntNPCs, and EED−/− AntNPCs were used to calculate the expression values (as FPKM) for all genes (All), genes linked to PEs (Poised), genes linked to PoiAct enhancers (PoiAct), and genes linked to PoiAct enhancers that were induced ≥2-fold in WT AntNPCs compared to WT ESCs (indPoiAct). p values were calculated using paired Wilcoxon tests.
(F) Fold-change expression differences between WT AntNPCs and EED−/− AntNPCs are shown for indPoiAct genes, all genes induced ≥2-fold in WT AntNPCs compared to WT ESC after excluding indPoiAct genes (indAll), and genes whose promoters are marked by H3K27me3 in mESCs (H3K27me3). p values were calculated using unpaired Wilcoxon tests.
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9. Figure 7 PEs' Sequence Features Enable H3K27me3 Deposition
(A) Motifs significantly enriched in mESC PEs with respect to mESC active enhancers were identified with analysis of motif enrichment (AME) (McLeay and Bailey, 2010).
(B) CpG dinucleotide frequencies plotted from 1 kb upstream (−1 kb) to 1 kb downstream (+1 kb) from the center of active (purple) and poised (green) enhancers.
(C) Fraction of active (purple) and poised (green) enhancers overlapping classical (left) or weak (right) CpG islands (see STAR Methods for details) upon extension of the enhancer sequences by 0.25 kb or 3 kb in both directions. The numbers on top of the green bars indicate the fold-change increase in the fraction of PEs overlapping CpG islands compared to active enhancers.
(D and E) ChIP-qPCR primers were designed in proximity to the deleted PE regions to investigate how the PE deletions affected H3K27me3 deposition. H3K27me3 ChIPs were performed in WT mESCs and in mESCs in which the deleted PEs were either not overlapping (D) or overlapping (E) classical CpG islands. For each region, H3K27me3 ChIP-qPCR signals in each ChIP sample were normalized to the average signals obtained in the same sample when using two different control regions (“Chr6_neg” and “Sox2_prom” [Sox2 promoter]). The error bars represent the SDs of six different ChIP-qPCR measurements (two ChIP biological replicate samples and three technical replicate reactions for each sample).
(F and G) H3K27me3 ChIP-seq profiles generated in WT mESCs, PE Lhx5(−109)−/− mESCs, and PE Sox1(+35)−/− mESCs are shown around PE Lhx5(−109) (not overlapping a CpG island) (F) and PE Sox1(+35) (overlapping a CpG island) (G). CpG islands (CGI) are shown as green rectangles.
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