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CTCF-binding element regulates ESC differentiation via orchestrating long-range chromatin interaction between enhancers and HoxA

Open AccessPublished:February 10, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100413
      Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer–enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure.

      Keywords

      Abbreviations:

      AP (alkaline phosphatase), CBE (CTCF-binding element), EEIC (enhancer–enhancer interaction complex), ESC (embryonic stem cell), HoxA (Homeobox A), ICM (inner cell mass), RA (retinoic acid), TAD (topologically associated domain)
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      ) by controlling target gene expression through organizing high-order chromatin structures. We and others have found that multiple enhancers are required for RA-induced HoxA expression and early ESC differentiation via long-range chromatin interactions with HoxA genes (
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      ). Previous studies have also reported many regulatory patterns for HoxA activation during ESC differentiation; for example, several CBEs (also known as CBS or CTCF-binding sites) within the HoxA locus can organize HoxA chromatin structures and play significant regulatory roles in ESC differentiation and leukemogenesis (
      • Luo H.
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      CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation.
      ). However, it is unknown whether CBEs function to regulate HoxA expression by orchestrating enhancer chromatin structures during RA-induced early ESC differentiation.
      Here, we identified a novel functional CBE+47 required for RA to activate HoxA expression and promote early ESC differentiation. Relevant to mechanism, we report that CBE+47 regulates HoxA expression by organizing precise chromatin interactions between its adjacent enhancers and HoxA. Our study increases understanding of mechanisms underlying RA-induced HoxA activation and early ESC differentiation and highlights the unique and precise regulatory roles of CBE in high-order chromatin structure.

      Results

      Identification of a CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same TAD in ESC

      TADs are basic structural units in high-order chromatin, and DNA functional elements such as enhancers or insulators in the same TAD likely regulate expression of neighboring genes (
      • Lupianez D.G.
      • Kraft K.
      • Heinrich V.
      • Krawitz P.
      • Brancati F.
      • Klopocki E.
      • Horn D.
      • Kayserili H.
      • Opitz J.M.
      • Laxova R.
      • Santos-Simarro F.
      • Gilbert-Dussardier B.
      • Wittler L.
      • Borschiwer M.
      • Haas S.A.
      • et al.
      Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions.
      ,
      • Ji X.
      • Dadon D.B.
      • Powell B.E.
      • Fan Z.P.
      • Borges-Rivera D.
      • Shachar S.
      • Weintraub A.S.
      • Hnisz D.
      • Pegoraro G.
      • Lee T.I.
      • Misteli T.
      • Jaenisch R.
      • Young R.A.
      3D chromosome regulatory landscape of human pluripotent cells.
      ). To identify functional elements that regulate HoxA expression, we first analyzed Hi-C data in ESC. We found that the HoxA 5'-end was located at a TAD boundary region, while the 3'-end was within the TAD (Fig. 1A). Others had shown that CBEs located at the HoxA locus regulate HoxA expression and ESC differentiation (
      • Narendra V.
      • Rocha P.P.
      • An D.
      • Raviram R.
      • Skok J.A.
      • Mazzoni E.O.
      • Reinberg D.
      CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation.
      ,
      • Rousseau M.
      • Crutchley J.L.
      • Miura H.
      • Suderman M.
      • Blanchette M.
      • Dostie J.
      Hox in motion: Tracking HoxA cluster conformation during differentiation.
      ). Interestingly, we identified many significant CBEs in the same TAD at the HoxA 3'-end (Fig. 1B), but their function was not previously characterized.
      Figure thumbnail gr1
      Figure 1Identification of a CBE (CBE+47) closest to the HoxA 3'-end within the same TAD in ESC. A, Hi-C interaction map of ∼0.5 Mb region surrounding the HoxA locus in undifferentiated ESC. Data were extracted from Bonev et al., 2017. B, IGV (Integrative Genomics Viewer) screenshots showing gene tracks of CTCF, MED1, MED12, and YY1 ChIP-seq signal occupancy at Skap2 and HoxA loci in ESC. CTCF ChIP-seq shows CTCF binding throughout the locus. Multiple CTCF sites are located downstream of the HoxA locus. C and D, IGV view of selected ChIP-seq tracks at Skap2 and HoxA loci in ESC. Shown are H3K27ac, H3K4me1, H3K4me2, H3K4me3 (C), and Pol ll, ATAC-seq and DNase (D). Vertical blue line indicates the CBE+47 region. Gray shadowing indicates HoxA locus. In (A), blue dotted box area shows the TAD boundary region. Other results relevant to these findings are shown in .
      Here, we focused on a significant CBE closest to the HoxA 3'-end, ∼47 kb from HoxA and located in the second intron of Halr1; we designate that element CBE+47 (Fig. 1B and Fig. S1). We observed significant binding of MED1, MED12, and YY1 upstream of CBE+47 (Fig. 1B) as well as enrichment for the epigenetic modification-related markers H3K27ac and H3K4me1/2/3 (Fig. 1C). We also observed significant chromatin accessibility (based on DNase and ATAC-seq analysis) and transcriptional activity marked by PolⅡenrichment in these regions (Fig. 1D). These data indicate that CBE+47 is located between HoxA and potential regulatory elements. Others had shown that CBEs regulate target gene expression by organizing chromatin interactions between a functional element and a target gene (
      • Hark A.T.
      • Schoenherr C.J.
      • Katz D.J.
      • Ingram R.S.
      • Levorse J.M.
      • Tilghman S.M.
      CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus.
      ,
      • Kurukuti S.
      • Tiwari V.K.
      • Tavoosidana G.
      • Pugacheva E.
      • Murrell A.
      • Zhao Z.
      • Lobanenkov V.
      • Reik W.
      • Ohlsson R.
      CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2.
      ). Therefore, we postulated that CBE+47 may play a regulatory role.

      Gene expression analysis in cultured WT and CBE+47-deleted cells grown in self-renewal conditions

      To assess CBE+47 function, we employed CRISPR-Cas9 knockout methodology (
      • Su G.
      • Guo D.
      • Chen J.
      • Liu M.
      • Zheng J.
      • Wang W.
      • Zhao X.
      • Yin Q.
      • Zhang L.
      • Zhao Z.
      • Shi J.
      • Lu W.
      A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.
      ). Specifically, we designed two sgRNAs, upstream and downstream of CBE+47 (Fig. 2A) and after Cas9 cleavage and DNA recombination, obtained two homozygous CBE+47 knockout lines (CBE+47-KO). PCR of genomic DNA using specific primers and Sanger sequencing confirmed CBE+47 deletion (Fig. 2B). AP staining of CBE+47 knockout lines revealed no significant differences in cell morphology relative to WT cells in self-renewing culture conditions (Fig. 2C). Moreover, expression of Halr1 and Sakp2 mRNAs was comparable in WT and KO lines, based on qRT-PCR analysis (Fig. 2D). However, most of the 3'-end genes of HoxA, including Hoxa2-a6, were significantly upregulated, while the TAD boundary gene Hoxa9 was significantly downregulated in CBE+47-KO relative to WT cells (Fig. 2E). These data show that CBE+47 deletion significantly changes HoxA expression and indicate that even in an undifferentiated ESC state, CBE+47 is required to maintain proper HoxA expression.
      Figure thumbnail gr2
      Figure 2Gene expression analysis in WT and CBE+47-deleted cells grown in self-renewal culture conditions. A, schematic showing CRISPR/Cas9-mediated deletion of CBE+47 (blue shadowing) using two sgRNAs. Indicated primers shown in blue were used to distinguish CBE+47-KO (CBE+47 knockout) from wild-type (WT) clones. B, validation of knockout lines by genomic DNA PCR and sequencing. Left, images of agarose gel showing PCR analysis of representative clones. Right, DNA sequencing of CBE+47-KO cell clones #1 and #2 using the indicated primer. C, appearance of WT and CBE+47-KO cells from lines 1 and #2 stained with alkaline phosphatase (AP). DK, data derived from qRT-PCR analysis of WT and CBE+47-KO cells showing indicated transcripts in undifferentiated ESC. Expression levels in WT ESC were set to 1 and shown as the blue dotted line. Data are represented as means ± SD. Significance was based on Student’s t-test (two-tailed; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). In all analyses, n = 4 (two CBE+47-KO lines (#1 and #2) and two biological replicates per line). In (B): M, DNA marker. In (C): scale bar, 50 μm.
      Given the critical function of HoxA in ESC differentiation (
      • Martinez-Ceballos E.
      • Chambon P.
      • Gudas L.J.
      Differences in gene expression between wild type and Hoxa1 knockout embryonic stem cells after retinoic acid treatment or leukemia inhibitory factor (LIF) removal.
      ,
      • Yin Y.
      • Yan P.
      • Lu J.
      • Song G.
      • Zhu Y.
      • Li Z.
      • Zhao Y.
      • Shen B.
      • Huang X.
      • Zhu H.
      • Orkin S.H.
      • Shen X.
      Opposing roles for the lncRNA haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation.
      ,
      • De Kumar B.
      • Parker H.J.
      • Parrish M.E.
      • Lange J.J.
      • Slaughter B.D.
      • Unruh J.R.
      • Paulson A.
      • Krumlauf R.
      Dynamic regulation of Nanog and stem cell-signaling pathways by Hoxa1 during early neuro-ectodermal differentiation of ES cells.
      ) and the observation that CBE+47 deletion significantly increased expression of a subset of HoxA genes, we asked whether ESC pluripotency was perturbed by CBE+47 deletion by evaluating expression of pluripotency- and differentiation-related genes by qRT-PCR (Fig. 2, FK). We observed no significant differences in expression of pluripotency-regulated genes in CBE+47-KO versus WT cells, except for slight upregulation of Sox2. However, mesoderm and trophectoderm genes were significantly upregulated in CBE+47-KO cells. Expression of other germ layer genes such as Otx2 (epiblast), Sox17 (endoderm), and Sox11 (ectoderm) also significantly increased in CBE+47-KO cells. These results indicate that CBE+47 deletion does not regulate ESC pluripotency but may alter its differentiation capacity.

      CBE+47 deletion significantly potentiates RA-induced HoxA expression and promotes abnormal differentiation of early ESC

      During early RA-induced differentiation of ESC, HoxA genes become significantly activated (
      • De Kumar B.
      • Parrish M.E.
      • Slaughter B.D.
      • Unruh J.R.
      • Gogol M.
      • Seidel C.
      • Paulson A.
      • Li H.
      • Gaudenz K.
      • Peak A.
      • McDowell W.
      • Fleharty B.
      • Ahn Y.
      • Lin C.
      • Smith E.
      • et al.
      Analysis of dynamic changes in retinoid-induced transcription and epigenetic profiles of murine Hox clusters in ES cells.
      ,
      • Yin Y.
      • Yan P.
      • Lu J.
      • Song G.
      • Zhu Y.
      • Li Z.
      • Zhao Y.
      • Shen B.
      • Huang X.
      • Zhu H.
      • Orkin S.H.
      • Shen X.
      Opposing roles for the lncRNA haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation.
      ) (Fig. S2). Therefore, we asked whether CBE+47 regulates those activities. To do so, we treated CBE+47-KO and WT ESC with RA for 12, 24, and 48 h (Fig. S3A) and then compared HoxA expression levels by qRT-PCR. HoxA 3'-end genes (Hoxa2-a6) showed significantly higher expression in CBE+47-KO cells, as did the TAD boundary genes Hoxa7 and Hoxa9. However, expression of HoxA 5'-end genes (Hoxa10, Hoxa11, and Hoxa13) was comparable in both genotypes (Fig. 3, AC). Also, antisense long noncoding RNAs (such as Hoxaas3) at the HoxA locus were significantly upregulated in CBE+47-KO relative to WT cells (Fig. S3, BD). These data suggest overall that CBE+47 functions to restrict RA-induced overexpression of HoxA.
      Figure thumbnail gr3
      Figure 3CBE+47 deletion potentiates RA-induced HoxA expression and perturbs ESC differentiation. AC, qRT-PCR of WT and CBE+47-KO cells showing HoxA gene expression in ESC after RA treatment for 12 h (A), 24 h (B), and 48 h (C). DF, qRT-PCR of WT and CBE+47-KO ESC showing transcripts of indicated genes in ESC after RA treatment for 12 h (D), 24 h (E), and 48 h (F). In AF, expression levels in WT ESC were set to 1 and shown as the blue dotted line. Data are represented as means ± SD. Significance is based on Student’s t-test (two-tailed; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). In all analyses, n = 4 (two CBE+47-KO lines (#1 and #2) and two biological replicates per line). Other results relevant to this figure are shown in and .
      To assess whether CBE+47 knockout alters ESC differentiation, we used qRT-PCR to analyze potential changes in pluripotency- or differentiation-related master control genes in WT and CBE+47-KO ESC under RA induction conditions. Expression of pluripotency genes (Nanog and Klf4) did not change significantly at 12 h after ESC differentiation but significantly decreased in CBE+47-KO relative to WT cells by 24 and 48 h. The epiblast master control gene Otx2, endoderm master control gene Gata4, and ectoderm master control gene Pax6 showed significantly higher expression levels in CBE+47-KO versus WT cells, while the mesoderm master gene T and the trophectoderm master gene Hand1 were significantly downregulated (Fig. 3, D and E). These findings suggest overall that CBE+47 is essential to maintain RA-induced HoxA expression and proper early ESC differentiation.

      Transcriptome analysis of WT and CBE+47kb-deleted cells during RA-induced early ESC differentiation

      To further assess effects of CBE+47 loss on early (24 h) RA-induced ESC differentiation, we performed transcriptome analysis using RNA-seq after RA-induced differentiation of WT and CBE+47-KO cells. Relative to WT cells, statistical analysis of CBE+47-KO cells revealed 869 genes upregulated and 360 downregulated (fold-change ≥ 2, p < 0.05) (Fig. 4A). Among them, the most significantly upregulated genes were at the 3’-end of HoxA (Fig. 4B). Because these genes located in the same TAD with CBE+47, we hypothesized that CBE+47 deletion significantly promotes gene expression restricted to that TAD. These findings reveal that CBE+47 serves as a cis-regulatory element playing a local regulatory role (Fig. S4). Moreover, relative to WT cells, CBE+47 KO cells expressed significantly higher levels of HoxB/C/D cluster genes (Fig. S5A). Genes relevant to RA signaling, such as Crabp2, Cyp26a1, and Stra6, were also significantly overexpressed in CBE+47 KO cells, as were the differentiation-related genes Cbx4 and Foxa1 (Fig. S5B). By contrast, pluripotency genes such as Myc, Lin28a, and Etv4 were significantly suppressed in CBE+47 KO relative to WT cells (Fig. S5C). These data suggest that in normal cells CBE+47 functions to enable proper differentiation.
      Figure thumbnail gr4
      Figure 4Transcriptome analysis in WT and CBE+47-deleted cells following RA-induced early ESC differentiation. A, heatmap depicting gene expression changes in WT and CBE+47-KO cells treated 24 h with RA (Fold-change ≥ 2; p < 0.05, as determined by DESeq2). B, RNA-seq results shown heatmap of HoxA. C and D, GO-BP analyses indicating differentially expressed genes. E, GSEA plots for the top three KEGG signaling pathways showing log2 fold change for the entire transcriptome. NES, Normalized Enrichment Score. Other results related to this figure are shown in .
      To assess mechanisms underlying these activities, we performed bioinformatic analysis of up- and downregulated genes in CBE+47 KO cells (Fig. S6). GO analysis indicated that biological processes (BP) relevant to upregulated genes include gland development and positive regulation of nervous system development (Fig. 4C). GO-BPs of downregulated genes mainly involved embryonic placenta development, positive regulation of vasoconstriction, and epithelial cell differentiation (Fig. 4D). KEGG pathway enrichment analysis revealed upregulated genes to be enriched in pathways including cGMP-PKG signaling, neuroactive ligand–receptor interaction, axon guidance, stem cell pluripotency, Hippo signaling, cAMP signaling, Wnt and Hedgehog signaling (Fig. S7A). Enrichment results relevant to downregulated genes revealed pathways related to proteoglycans in cancer, transcriptional dysregulation in cancer, arachidonic acid metabolism, HTLV-I infection, Hippo signaling, and thyroid hormone signaling (Fig. S7A). These findings, which were further validated by Gene Set Enrichment Analysis (GSEA) (Fig. 4E and Fig. S7B), suggest that CBE+47 deletion promotes abnormal gene expression and perturbs proper RA-induced early ESC differentiation.

      CBE+47 is required for proper chromatin interactions between adjacent enhancers and HoxA

      Previous studies report that CTCF regulates target gene expression by controlling chromatin interaction between enhancers and target genes and that this activity occurs within a TAD (
      • Zhao H.
      • Li Z.
      • Zhu Y.
      • Bian S.
      • Zhang Y.
      • Qin L.
      • Naik A.K.
      • He J.
      • Zhang Z.
      • Krangel M.S.
      • Hao B.
      A role of the CTCF binding site at enhancer Eα in the dynamic chromatin organization of the Tcra–Tcrd locus.
      ). Studies by us and others have identified three enhancers (E1, E2, and E3) at the HoxA 3'-end (Fig. 5A) required for HoxA expression following RA-induced ESC differentiation (
      • Su G.
      • Guo D.
      • Chen J.
      • Liu M.
      • Zheng J.
      • Wang W.
      • Zhao X.
      • Yin Q.
      • Zhang L.
      • Zhao Z.
      • Shi J.
      • Lu W.
      A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.
      ,
      • Yin Y.
      • Yan P.
      • Lu J.
      • Song G.
      • Zhu Y.
      • Li Z.
      • Zhao Y.
      • Shen B.
      • Huang X.
      • Zhu H.
      • Orkin S.H.
      • Shen X.
      Opposing roles for the lncRNA haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation.
      ,
      • Cao K.
      • Collings C.K.
      • Marshall S.A.
      • Morgan M.A.
      • Rendleman E.J.
      • Wang L.
      • Sze C.C.
      • Sun T.
      • Bartom E.T.
      • Shilatifard A.
      SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.
      ,
      • Liu G.Y.
      • Zhao G.N.
      • Chen X.F.
      • Hao D.L.
      • Zhao X.
      • Lv X.
      • Liu D.P.
      The long noncoding RNA Gm15055 represses Hoxa gene expression by recruiting PRC2 to the gene cluster.
      ). Among them, E1 and E3 reportedly mediate significant long-range interactions with HoxA chromatin. Remarkably, CBE+47 is located between those three enhancers and HoxA (Fig. 5A). Therefore, we speculate that the CBE+47 may be involved in organizing the interactions between the three enhancers and HoxA chromatin.
      Figure thumbnail gr5
      Figure 5CBE+47 is required for proper chromatin interactions between HoxA and adjacent enhancers. A, IGV (Integrative Genomics Viewer) screenshots showing gene tracks of H3K27ac (+RA, 24 h) and CTCF ChIP-seq signal occupancy at Skap2 and HoxAloci in WT ESC. BD, chromatin interaction profiles using indicated enhancers as anchors in WT or CBE+47-KO cells after 24 h of RA treatment. Numbers on Y-axis denote interaction frequency. Light green, light purple, light red, and light gray shadows mark E1, E2, E3, and HoxA regions, respectively. E, quantitative results showing interaction differences seen in WT and CBE+47-KO cells.
      Next we used Capture-C methodology, which is widely used to define chromatin interactions between enhancers and target genes (
      • Su G.
      • Guo D.
      • Chen J.
      • Liu M.
      • Zheng J.
      • Wang W.
      • Zhao X.
      • Yin Q.
      • Zhang L.
      • Zhao Z.
      • Shi J.
      • Lu W.
      A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.
      ,
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      • Roberts N.
      • McGowan S.
      • Hay D.
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      • Lynch M.
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      • Gibbons R.
      • Higgs D.R.
      Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment.
      ,
      • Davies J.O.
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      • McGowan S.J.
      • Roberts N.A.
      • Taylor S.
      • Higgs D.R.
      • Hughes J.R.
      Multiplexed analysis of chromosome conformation at vastly improved sensitivity.
      ,
      • Liang Y.C.
      • Wu P.
      • Lin G.W.
      • Chen C.K.
      • Yeh C.Y.
      • Tsai S.
      • Yan J.
      • Jiang T.X.
      • Lai Y.C.
      • Huang D.
      • Cai M.
      • Choi R.
      • Widelitz R.B.
      • Lu W.
      • Chuong C.M.
      Folding keratin gene clusters during skin regional specification.
      ,
      • Zhang L.
      • He A.
      • Chen B.
      • Bi J.
      • Chen J.
      • Guo D.
      • Qian Y.
      • Wang W.
      • Shi T.
      • Zhao Z.
      • Shi J.
      • An W.
      • Attenello F.
      • Lu W.
      A HOTAIR regulatory element modulates glioma cell sensitivity to temozolomide through long-range regulation of multiple target genes.
      ,
      • Qian Y.
      • Zhang L.
      • Cai M.
      • Li H.
      • Xu H.
      • Yang H.
      • Zhao Z.
      • Rhie S.K.
      • Farnham P.J.
      • Shi J.
      • Lu W.
      The prostate cancer risk variant rs55958994 regulates multiple gene expression through extreme long-range chromatin interaction to control tumor progression.
      ), to define interactions between E1, E2, and E3 and HoxA chromatin in WT and CBE+47-KO cells. For this analysis we used cells induced 24 h with RA, with enhancers E1, E2, and E3 as respective anchor regions (Fig. 5, BD). In WT cells, we observed significant interactions between all three enhancers and HoxA chromatin, with E1 interacting most strongly (Fig. 5B). However, in CBE+47 KO cells, interactions between the E1 and HoxA chromatin were significantly reduced relative to WT cells. However, E2 and E3 interaction with HoxA chromatin increased significantly in CBE+47 KO relative to WT cells, and the intensity of chromatin interactions shifted from the 3'-end to the 5'-end of HoxA (Fig. 5, C and D). In addition, the results also show that the interaction between enhancers decreases significantly except for the slight increase of E1 and E2 (Fig. 5E). These results indicate overall that CBE+47 is essential to maintain these proper chromatin interactions during RA-induced early ESC differentiation.

      Multiple enhancer deletions show synergistic effect on RA-induced HoxA expression

      Next, we used CRISPR-Cas9 to delete enhancers individually or in groups to establish multiple homozygous enhancer knockout lines (namely, ΔE1, ΔE2, ΔE3, ΔE1/2, ΔE1/2/3) (Fig. S8). We then treated each line 24 h with RA and performed qRT-PCR to determine HoxA expression (Fig. 6, AE). ΔE1 and ΔE2 cells showed similar phenotypes in terms of HoxA regulation, that is, both 3’-end (Hoxa1) and central (Hoxa5-a10) genes were significantly suppressed relative to WT cells (Fig. 6, A and B). Also relative to WT cells, in ΔE3 cells, Hoxa1-a7 were significantly inhibited, indicating that these enhancers have specific and unique regulatory effects (Fig. 6C). In ΔE1/2 and ΔE1/2/3 cells, HoxA expression was significantly reduced compared with ΔE1, ΔE2, or ΔE3 (Fig. 6, D and E and Fig. S9, A and B). In addition, considering the closest distance between E1 and CBE+47, we also compared the expression differences of HoxA cluster gene between them. The results showed that expressions of HoxA 3'-end gene ware significantly different (Fig. S9C), which indicated that the regulation of HoxA by CBE+47 and E1 was opposite. Over all, these findings indicate that interactions among E1, E2, and E3 enhancers with HoxA chromatin synergize to promote RA-induced HoxA activation.
      Figure thumbnail gr6
      Figure 6Deletions of multiple enhancers synergize to alter RA-induced HoxA gene expression. AE, transcripts of HoxA genes were measured by qRT–PCR and normalized to WT levels in ΔE1 (A), ΔE2 (B), ΔE3 (C), ΔE1/2 (D), andΔE1/2/3 (E) cells following 24 h of RA treatment. Expression levels in WT ESC were set to 1 and shown as the blue dotted line. Data are represented as means ± SD. Significance is based on Student’s t-test (two-tailed; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.) In AC and E, n = 4, including two enhancer knockout lines and two biological replicates per line. In D, n = 2, including one enhancer knockout line and two biological replicates per line. Other results relevant to these are shown in and .

      Discussion

      Precise expression of HoxA genes is critical for ESC differentiation. Although others have shown that CBEs play an important role in ESC differentiation by regulating high-order chromatin structure (
      • Narendra V.
      • Rocha P.P.
      • An D.
      • Raviram R.
      • Skok J.A.
      • Mazzoni E.O.
      • Reinberg D.
      CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation.
      ,
      • Beagan J.A.
      • Duong M.T.
      • Titus K.R.
      • Zhou L.
      • Cao Z.
      • Ma J.
      • Lachanski C.V.
      • Gillis D.R.
      • Phillips-Cremins J.E.
      YY1 and CTCF orchestrate a 3D chromatin looping switch during early neural lineage commitment.
      ), identification of a functional CBE and mechanisms regulating HoxA expression remain largely unknown. Here, we identified a new functional CBE+47 and propose a novel model based on our results. Specifically, in WT cells (Fig. 7, left), three enhancers near CBE+47 act as an EEIC to interact with the 3'-end of HoxA chromatin to maintain normal HoxA expression and promote correct early differentiation of ESC induced by RA. In CBE+47-KO cells (Fig. 7, right), enhancer interactions decrease, resulting in a relatively loose EEIC. Furthermore, EEIC interactions with central HoxA chromatin regions increase following CBE+47 loss, promoting HoxA overexpression. These results indicate that a functional CBE+47 maintains normal HoxA expression during ESC differentiation by organizing precise interactions between adjacent enhancers and HoxA chromatin. Our findings highlight indispensable fine-regulatory roles of CBEs as functional elements in high-order chromatin structure and reveal a direct regulatory effect of the CBE+47 on RA-induced HoxA expression and early ESC differentiation.
      Figure thumbnail gr7
      Figure 7Schematic showing how CBE+47 regulates RA-induced HoxA expression and early ESC differentiation by orchestrating long-range chromatin interactions between HoxA and adjacent enhancers. Left, model showing CBE+47 activity in WT cells. Three enhancers near CBE+47 act as an EEIC and interact with the 3'-end of HoxA chromatin to maintain normal HoxA normal expression, allowing proper ESC differentiation after RA treatment. Right, in CBE+47-KO cells interactions between enhancers decrease, resulting in a relatively loose EEIC and allowing increased interactions between that EEIC and the central region of HoxA chromatin. As a result, HoxA is overexpressed and early ESC differentiation proceeds abnormally.
      Previously, Ferraiuolo et al. (
      • Ferraiuolo M.A.
      • Rousseau M.
      • Miyamoto C.
      • Shenker S.
      • Wang X.Q.
      • Nadler M.
      • Blanchette M.
      • Dostie J.
      The three-dimensional architecture of Hox cluster silencing.
      ) showed that HoxA exhibits a special chromatin structure required for its expression and that CTCF regulates formation of that structure and thus gene expression. Recent studies focusing on CBEs within the HoxA locus, including the TAD boundary, have found that CBEs organize HoxA chromatin structure, maintain normal HoxA expression, and regulate ESC differentiation (
      • Narendra V.
      • Rocha P.P.
      • An D.
      • Raviram R.
      • Skok J.A.
      • Mazzoni E.O.
      • Reinberg D.
      CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation.
      ). Narendra et al. (
      • Narendra V.
      • Rocha P.P.
      • An D.
      • Raviram R.
      • Skok J.A.
      • Mazzoni E.O.
      • Reinberg D.
      CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation.
      ) showed that CBE5/6 knockout significantly promoted HoxA expression, altered HoxA chromatin structure, and enhanced ESC neural differentiation. Studies in other cells, such as AML cells, also reveal that CBEs located at the HoxA locus significantly regulate HoxA expression and affect tumorigenesis (
      • Luo H.
      • Wang F.
      • Zha J.
      • Li H.
      • Yan B.
      • Du Q.
      • Yang F.
      • Sobh A.
      • Vulpe C.
      • Drusbosky L.
      • Cogle C.
      • Chepelev I.
      • Xu B.
      • Nimer S.D.
      • Licht J.
      • et al.
      CTCF boundary remodels chromatin domain and drives aberrant HOX gene transcription in acute myeloid leukemia.
      ,
      • Ghasemi R.
      • Struthers H.
      • Wilson E.R.
      • Spencer D.H.
      Contribution of CTCF binding to transcriptional activity at the HOXA locus in NPM1-mutant AML cells.
      ). Here, we find that besides several CBEs in the HoxA locus, there are several CBEs downstream of HoxA within the same TAD (Fig. 1B). Among them, CBE+47 KO significantly enhanced RA-induced HoxA expression and promoted early ESC differentiation, strongly suggesting that CBE+47 is a functional CBE. However, other CBEs have not yet been evaluated and their potential activity requires future analysis. Recently, others developed a CRISPR-Cas9-based genetic screen method to detect functional CBEs in breast cancer and AML (
      • Luo H.
      • Sobh A.
      • Vulpe C.D.
      • Brewer E.
      • Dovat S.
      • Qiu Y.
      • Huang S.
      HOX loci focused CRISPR/sgRNA library screening identifying critical CTCF boundaries.
      ,
      • Korkmaz G.
      • Manber Z.
      • Lopes R.
      • Prekovic S.
      • Schuurman K.
      • Kim Y.
      • Teunissen H.
      • Flach K.
      • Wit E.
      • Galli G.G.
      • Zwart W.
      • Elkon R.
      • Agami R.
      A CRISPR-Cas9 screen identifies essential CTCF anchor sites for estrogen receptor-driven breast cancer cell proliferation.
      ). Comparable approaches may be taken in the future to screen for functional CBEs that regulate ESC differentiation.
      Our transcriptome analysis showed that after CBE+47 deletion, genes in the same TAD with CBE+47 are significantly upregulated relative to genes in nearby TADs (Fig. S4), suggesting that CBE+47 is a cis-regulatory element that plays a local regulatory role. We also observed upregulation of RA signaling-related genes (Crabp2, Cyp26a1, Stra6) when CBE+47 was deleted, while pluripotency-related genes (Myc and Lin28a) were significantly downregulated (Fig. S5), supporting the idea that CBE+47 loss promotes RA-induced early ESC differentiation. Recent studies show that Lin28a inhibits HoxA expression to maintain limb development (
      • Sato T.
      • Kataoka K.
      • Ito Y.
      • Yokoyama S.
      • Inui M.
      • Mori M.
      • Takahashi S.
      • Akita K.
      • Takada S.
      • Ueno-Kudoh H.
      • Asahara H.
      Lin28a/let-7 pathway modulates the Hox code via polycomb regulation during axial patterning in vertebrates.
      ), while De Kumar et al. (
      • De Kumar B.
      • Parker H.J.
      • Parrish M.E.
      • Lange J.J.
      • Slaughter B.D.
      • Unruh J.R.
      • Paulson A.
      • Krumlauf R.
      Dynamic regulation of Nanog and stem cell-signaling pathways by Hoxa1 during early neuro-ectodermal differentiation of ES cells.
      ) found that HOXA1 binds to the Myc gene near enhancers, suggesting positive feedback between HoxA and Myc or Lin28a in early ESC differentiation induced by RA. These possibilities require further analysis.
      RA significantly induced HoxA expression during early ESC differentiation (Fig. S2). Others have shown that at least three enhancers (E1, E2, and E3) are needed for HoxA expression in this context (
      • Su G.
      • Guo D.
      • Chen J.
      • Liu M.
      • Zheng J.
      • Wang W.
      • Zhao X.
      • Yin Q.
      • Zhang L.
      • Zhao Z.
      • Shi J.
      • Lu W.
      A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.
      ,
      • Yin Y.
      • Yan P.
      • Lu J.
      • Song G.
      • Zhu Y.
      • Li Z.
      • Zhao Y.
      • Shen B.
      • Huang X.
      • Zhu H.
      • Orkin S.H.
      • Shen X.
      Opposing roles for the lncRNA haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation.
      ,
      • Cao K.
      • Collings C.K.
      • Marshall S.A.
      • Morgan M.A.
      • Rendleman E.J.
      • Wang L.
      • Sze C.C.
      • Sun T.
      • Bartom E.T.
      • Shilatifard A.
      SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.
      ,
      • Liu G.Y.
      • Zhao G.N.
      • Chen X.F.
      • Hao D.L.
      • Zhao X.
      • Lv X.
      • Liu D.P.
      The long noncoding RNA Gm15055 represses Hoxa gene expression by recruiting PRC2 to the gene cluster.
      ). CBE+47 lies between these three enhancers and HoxA (Fig. 5A). As an insulator, CBE may block interaction between enhancers and target genes, exerting an inhibitory effect (
      • Kim T.H.
      • Abdullaev Z.K.
      • Smith A.D.
      • Ching K.A.
      • Loukinov D.I.
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      • Ren B.
      Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome.
      ,
      • Gaszner M.
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      Insulators: Exploiting transcriptional and epigenetic mechanisms.
      ,
      • Dowen J.M.
      • Fan Z.P.
      • Hnisz D.
      • Ren G.
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      • Zhang L.N.
      • Weintraub A.S.
      • Schujiers J.
      • Lee T.I.
      • Zhao K.
      • Young R.A.
      Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes.
      ). Our analysis first reveals that the three enhancers interact significantly as an EEIC. Second, we find that the EEIC interacts significantly with HoxA. Third, the interaction of these enhancers with HoxA also has its own pattern. When CBE+47 is deleted, enhancer interactions significantly decrease, resulting in formation of a relatively loose EEIC. We conclude that CBE+47 is required to organize enhancer interaction. Following CBE+47 loss, interaction of E1 becomes more concentrated in the middle of the HoxA locus, and interaction of E2 and E3 with HoxA moves from the 3'-end to middle regions. Furthermore, CBE+47 deletion significantly promotes higher expression of Hoxa2-a9, which corresponds to changes in chromatin interactions between enhancers and HoxA. Thus CBE+47 not only organizes enhancer interactions but orchestrates interactions between enhancers and HoxA, ensuring correct HoxA expression. Although the CBE+47 organizes high-order chromatin structures and previous studies show that the binding direction of CTCF alters formation of a CTCF-mediated chromatin loop (
      • de Wit E.
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      • Krijger P.H.
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      CTCF binding polarity determines chromatin looping.
      ), other CBEs may pair with CBE+47 to form chromatin loops, a possibility that requires further analysis.
      Multiple enhancers reportedly maintain RA-induced HoxA expression through long-range chromatin interactions with HoxA (
      • Su G.
      • Guo D.
      • Chen J.
      • Liu M.
      • Zheng J.
      • Wang W.
      • Zhao X.
      • Yin Q.
      • Zhang L.
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      • Lu W.
      A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.
      ,
      • Yin Y.
      • Yan P.
      • Lu J.
      • Song G.
      • Zhu Y.
      • Li Z.
      • Zhao Y.
      • Shen B.
      • Huang X.
      • Zhu H.
      • Orkin S.H.
      • Shen X.
      Opposing roles for the lncRNA haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation.
      ,
      • Cao K.
      • Collings C.K.
      • Marshall S.A.
      • Morgan M.A.
      • Rendleman E.J.
      • Wang L.
      • Sze C.C.
      • Sun T.
      • Bartom E.T.
      • Shilatifard A.
      SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.
      ,
      • Liu G.Y.
      • Zhao G.N.
      • Chen X.F.
      • Hao D.L.
      • Zhao X.
      • Lv X.
      • Liu D.P.
      The long noncoding RNA Gm15055 represses Hoxa gene expression by recruiting PRC2 to the gene cluster.
      ). Here, we show that knockout of multiple enhancers synergizes to alter HoxA expression (Fig. 6), a finding partially consistent with a previous report (
      • Cao K.
      • Collings C.K.
      • Marshall S.A.
      • Morgan M.A.
      • Rendleman E.J.
      • Wang L.
      • Sze C.C.
      • Sun T.
      • Bartom E.T.
      • Shilatifard A.
      SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.
      ). Although E1/E2/E3 enhancers are known to have regulatory effects, we also observed other enhancers located in the Skap2 region in the same TAD with HoxA (Fig. 5A), although their functional effects are unknown. Furthermore, although CBE+47 organizes interactions between E1, E2, and E3 enhancers, we did not observe significant CTCF-binding peaks in the E2 region, suggesting that other factors may regulate enhancer interactions. A recent study using the ChIA-PET method (Chromatin Interaction Analysis by Paired-End Tag sequencing) from Wang et al. (
      • Wang P.
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      • Ruan Y.
      Chromatin topology reorganization and transcription repression by PML-RARalpha in acute promyeloid leukemia.
      ) shows that RARα (Retinoic Acid Receptor alpha) mediates chromatin interactions in AML cells. Activation of target genes by RA signaling requires RAR/RXR (retinoic acid receptor/retinoid X receptor) signaling (
      • Valcarcel R.
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      • Jimenez C.G.
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      Retinoid-dependent in vitro transcription mediated by the RXR/RAR heterodimer.
      ,
      • Rhinn M.
      • Dolle P.
      Retinoic acid signalling during development.
      ). Remarkably, we also found significant RAR/RXR binding peaks in E1, E2, and HoxA regions over the course of RA-induced ESC differentiation (data not shown) (
      • Simandi Z.
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      • Wright L.C.
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      • Nagy L.
      OCT4 acts as an integrator of pluripotency and signal-induced differentiation.
      ), suggesting that RAR/RXR regulates these interactions, a possibility that needs further verification. We also found that chromatin interactions between a single enhancer and HoxA are different. Thus, mechanisms underlying maintenance of these different interactions require further analysis.
      In summary, we identified a new functional CBE+47 that regulates RA-induced HoxA expression and early ESC differentiation via orchestrating long-range chromatin interactions between its adjacent enhancers and HoxA. These findings further our understanding of intrinsic mechanisms governing RA-induced early ESC differentiation and highlight specific regulatory roles of CBE in high-order chromatin structure.

      Experimental procedures

      Embryonic stem cell culture

      Mouse ESC E14 were cultured as previously described with some modifications (
      • Su G.
      • Guo D.
      • Chen J.
      • Liu M.
      • Zheng J.
      • Wang W.
      • Zhao X.
      • Yin Q.
      • Zhang L.
      • Zhao Z.
      • Shi J.
      • Lu W.
      A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.
      ). ESC were grown in culture dishes coated with 0.1% gelatin (Sigma, Lot # SLBQ9498V) in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Lot # 8119213) supplemented with 15% fetal bovine serum (FBS, AusGeneX, Lot # FBS00717-1), 1x nonessential amino acids (100x, Gibco, Lot # 2027433), 1x L-Glutamate (100x, Gibco, Lot # 2085472), 1x Penicillin-streptomycin (100x, Gibco, Lot # 2029632), 50 μM β-mercaptoethanol (Sigma, Cas # 60-24-2), 10 ng/ml LIF (ESGRO, Lot # ESG1107), 1 μM PD0325901 (a MEK inhibitor, MedChemExpress, Lot # HY-10254), and 3 μM CHIR99021 (a GSK inhibitor, MedChemExpress, Lot # HY-10182). The medium was replaced every 1 to 2 days. All cells were maintained at 37 °C in a 5% CO2 incubator.

      Retinoic acid (RA)-induced early ESC differentiation

      Cells were gently washed with 1x phosphate buffer saline solution (PBS), dissociated, and plated at an appropriate density on gelatin-coated plates in LIF/2i withdrawal medium supplemented with 2 μm RA (Solarbio, Lot # 1108F031). The culture medium was replaced at the given point in time.

      RNA extraction, reverse transcription, and quantitative real-time PCR (qRT-PCR)

      Total RNA was extracted from differentiated or undifferentiated ESC using TRIzol Reagent (Life Technologies, Lot # 213504). cDNA synthesis was performed using a PrimerScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Lot # AJ51485A) according to the manufacturer’s instructions. PCR reactions were performed using HieffTMqPCR SYBR Green Master Mix (YEASEN, Lot # H28360) and a BioRad CFX Connect Real-Time system. PCR cycling conditions were as previously reported: 95 °C for 5 min, 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s. We then constructed a melting curve of amplified DNA (
      • Su G.
      • Guo D.
      • Chen J.
      • Liu M.
      • Zheng J.
      • Wang W.
      • Zhao X.
      • Yin Q.
      • Zhang L.
      • Zhao Z.
      • Shi J.
      • Lu W.
      A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.
      ). Target gene values were normalized to Gapdh expression and the experimental control using ΔΔCt methods (
      • Schmittgen T.D.
      • Livak K.J.
      Analyzing real-time PCR data by the comparative C(T) method.
      ). Primer sequences used in this study are shown in Table S1.

      CRISPR/Cas9-mediated CBE+47 and enhancers deletion in ESC

      The CRISPR/Cas9 system was used following published protocols (
      • Su G.
      • Guo D.
      • Chen J.
      • Liu M.
      • Zheng J.
      • Wang W.
      • Zhao X.
      • Yin Q.
      • Zhang L.
      • Zhao Z.
      • Shi J.
      • Lu W.
      A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.
      ,
      • Cong L.
      • Ran F.A.
      • Cox D.
      • Lin S.
      • Barretto R.
      • Habib N.
      • Hsu P.D.
      • Wu X.
      • Jiang W.
      • Marraffini L.A.
      • Zhang F.
      Multiplex genome engineering using CRISPR/Cas systems.
      ,
      • Engreitz J.M.
      • Haines J.E.
      • Perez E.M.
      • Munson G.
      • Chen J.
      • Kane M.
      • McDonel P.E.
      • Guttman M.
      • Lander E.S.
      Local regulation of gene expression by lncRNA promoters, transcription and splicing.
      ). Briefly, target-specific guide RNAs (sgRNAs) were designed using an online tool (http://chopchop.cbu.uib.no/). sgRNAs of the appropriate site and score were selected. sgRNA sequences are shown in Table S2. For CBE+47 and enhancer knockout, sgRNAs were cloned into a Cas9-puro vector using the Bsmb1 site. ESC were transfected with two sgRNA plasmids using Lipofectamine 3000 (Life Technologies, Lot # 2125386), and 24 h later, cells were treated with 5 μM puromycin (MCE, Lot # 64358) for 24 h and then cultured in medium without puromycin for another 5 ∼ 7 days. Individual colonies were picked and validated by genomic DNA PCR and subsequent Sanger DNA sequencing. PCR primers used for genotyping are listed in Table S3.

      Alkaline phosphatase (AP) staining of WT and CBE+47-deleted cells

      ESC were plated at low density in 12-well plates coated with gelatin for 4 days and then washed twice with PBS and incubated with reagents from the AP Staining kit (Cat # AP100R-1, System Biosciences) following the manufacturer’s instructions. Digital images were taken using an Olympus Inverted Fluorescence Microscope.

      Enhancer capture-C and data analysis

      Capture-C probes were designed using an online tool within three enhancer regions (http://apps.molbiol.ox.ac.uk/CaptureC/cgi-bin/CapSequm.cgi): the E1 bait (Mouse, mm10, chr6: 52012160–52015360), the E2 bait (Mouse, mm10, chr6:52075358–52076639), and the E3 bait (Mouse, mm10, chr6: 52101884–52104217). Probe sequences are listed in Table S4.
      Capture-C libraries were prepared as previously described with minor modifications (
      • Su G.
      • Guo D.
      • Chen J.
      • Liu M.
      • Zheng J.
      • Wang W.
      • Zhao X.
      • Yin Q.
      • Zhang L.
      • Zhao Z.
      • Shi J.
      • Lu W.
      A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.
      ,
      • Hughes J.R.
      • Roberts N.
      • McGowan S.
      • Hay D.
      • Giannoulatou E.
      • Lynch M.
      • De Gobbi M.
      • Taylor S.
      • Gibbons R.
      • Higgs D.R.
      Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment.
      ,
      • Davies J.O.
      • Telenius J.M.
      • McGowan S.J.
      • Roberts N.A.
      • Taylor S.
      • Higgs D.R.
      • Hughes J.R.
      Multiplexed analysis of chromosome conformation at vastly improved sensitivity.
      ). Briefly, RA-induced differentiated ESC (WT and CBE+47-KO) were fixed with 1% (vol/vol) formaldehyde for 10 min at room temperature, quenched with 125 mM glycine in PBS, and then lysed in cold lysis buffer [10 mM Tris-HCl, pH7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 0.2% NP-40, 1× complete protease inhibitor cocktail (Roche, Lot # 3024150)]. Chromatin was digested with DpnII (New England Biolabs, Lot # 10014860) at 37 °C overnight. Fragments were then diluted and ligated with T4 DNA ligase (Takara, Lot # 1211707) at 16 °C overnight. Cross-linking was reversed by overnight incubation at 60 °C with proteinase K (Bioline, Lot # BIO-37037). Then 3C libraries were purified by phenol-chloroform followed by chloroform extraction and ethanol-precipitated at −80 °C overnight. Sequencing libraries were prepared from 10 μg of the 3C library by sonication to an average size of 200 ∼ 300 bp and indexed using NEBnext reagents (New England Biolabs, Lot # 0031607), according to the manufacturer's protocol. Enrichment of 2 μg of an indexed library incubated with 3 μM of a pool of biotinylated oligonucleotides was performed using the SeqCap EZ Hybridization reagent kit (Roche/NimbleGen, Lot # 05634261001), following the manufacturer's instructions. Two rounds of capture employing 48∼72 and 24 h hybridizations, respectively, were used. Correct library size was confirmed by agarose gel electrophoresis, and DNA concentration was determined using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Lot # 0000248352). All sequencing was performed on Hi-Seq 2500 platforms using paired 150 bp protocols (Illumina).
      Capture-C data were analyzed using previously described methods (
      • Su G.
      • Guo D.
      • Chen J.
      • Liu M.
      • Zheng J.
      • Wang W.
      • Zhao X.
      • Yin Q.
      • Zhang L.
      • Zhao Z.
      • Shi J.
      • Lu W.
      A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.
      ,
      • Hughes J.R.
      • Roberts N.
      • McGowan S.
      • Hay D.
      • Giannoulatou E.
      • Lynch M.
      • De Gobbi M.
      • Taylor S.
      • Gibbons R.
      • Higgs D.R.
      Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment.
      ,
      • Davies J.O.
      • Telenius J.M.
      • McGowan S.J.
      • Roberts N.A.
      • Taylor S.
      • Higgs D.R.
      • Hughes J.R.
      Multiplexed analysis of chromosome conformation at vastly improved sensitivity.
      ). Briefly, clean paired-end reads were reconstructed into single reads using FLASH (
      • Magoc T.
      • Salzberg S.L.
      FLASH: Fast length adjustment of short reads to improve genome assemblies.
      ). After in silico DpnII digestion using the DpnII2E.pl script, reads were mapped back to the mm10 mouse genome using Bowtie1. Finally, chimeric reads containing captured reads and Capture-Reporter reads were analyzed using CCanalyser3.pl. Results were visualized using the Integrated Genome Browser (IGV) (
      • Freese N.H.
      • Norris D.C.
      • Loraine A.E.
      Integrated genome browser: Visual analytics platform for genomics.
      ) and online tool (https://epgg-test.wustl.edu/browser/) (
      • Zhou X.
      • Lowdon R.F.
      • Li D.
      • Lawson H.A.
      • Madden P.A.
      • Costello J.F.
      • Wang T.
      Exploring long-range genome interactions using the WashU epigenome browser.
      ).

      RNA-seq and bioinformatics analysis

      ESC were lysed with Trizol reagent (Life Technologies, Lot # 265709) and RNA was extracted based on the manufacturer's instructions. RNA was then sequenced by a company (Novogene). Clean reads were mapped to the Ensemble mm10 mouse genome using Hisat2 with default parameters. Gene reads were counted by Htseq (
      • Anders S.
      • Pyl P.T.
      • Huber W.
      HTSeq-a Python framework to work with high-throughput sequencing data.
      ). Fold changes were computed as a log2 ratio of normalized reads per gene using the DEseq2 R package (
      • Love M.I.
      • Huber W.
      • Anders S.
      Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.
      ). Gene expression with∣log2 (fold change)∣≥ 1 (p < 0.05) was considered significantly altered. Heatmaps were drawn using the heatmap.2 function. Two biological replicates were analyzed for each experimental condition.

      Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses

      GO and KEGG pathway analyses were performed using the following online tools: Metascape (http://metascape.org/gp/index.html#/main/step1) (
      • Zhou Y.
      • Zhou B.
      • Pache L.
      • Chang M.
      • Khodabakhshi A.H.
      • Tanaseichuk O.
      • Benner C.
      • Chanda S.K.
      Metascape provides a biologist-oriented resource for the analysis of systems-level datasets.
      ) and DAVID Functional Annotation Bioinformatics Microarray Analysis tool (http://david.abcc.ncifcrf.gov/) (
      • Huang D.W.
      • Sherman B.T.
      • Lempicki R.A.
      Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
      ).

      Gene set enrichment analysis (GSEA)

      GSEA was carried out using the online tool (https://www.gsea-msigdb.org/gsea/index.jsp), as previously reported (
      • Subramanian A.
      • Tamayo P.
      • Mootha V.K.
      • Mukherjee S.
      • Ebert B.L.
      • Gillette M.A.
      • Paulovich A.
      • Pomeroy S.L.
      • Golub T.R.
      • Lander E.S.
      • Mesirov J.P.
      Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles.
      ,
      • Mootha V.K.
      • Lindgren C.M.
      • Eriksson K.F.
      • Subramanian A.
      • Sihag S.
      • Lehar J.
      • Puigserver P.
      • Carlsson E.
      • Ridderstråle M.
      • Laurila E.
      • Houstis N.
      • Daly M.J.
      • Patterson N.
      • Mesirov J.P.
      • Golub T.R.
      • et al.
      PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes.
      ). KEGG pathway-related gene sets were obtained from the KEGG database (https://www.genome.jp/kegg/) (
      • Kanehisa M.
      • Sato Y.
      • Kawashima M.
      • Furumichi M.
      • Tanabe M.
      KEGG as a reference resource for gene and protein annotation.
      ).

      Statistical analyses

      Data were analyzed by Student’s t-test (two-tailed) unless otherwise specified. Statistically significant p-values are indicated in Figures as follows: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

      Data availability

      Data supporting this study are available from the corresponding author upon request. Raw data reported here (DNA-seq and RNA-seq) have been deposited in the NCBI Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE154495.
      Published data sets were downloaded using online tools (http://cistrome.org/db/#/ and http://promoter.bx.psu.edu/hi-c/) and analyzed in this study (
      • Wang Y.
      • Song F.
      • Zhang B.
      • Zhang L.
      • Xu J.
      • Kuang D.
      • Li D.
      • Choudhary M.N.K.
      • Li Y.
      • Hu M.
      • Hardison R.
      • Wang T.
      • Yue F.
      The 3D genome browser: A web-based browser for visualizing 3D genome organization and long-range chromatin interactions.
      ,
      • Zheng R.
      • Wan C.
      • Mei S.
      • Qin Q.
      • Wu Q.
      • Sun H.
      • Chen C.H.
      • Brown M.
      • Zhang X.
      • Meyer C.A.
      • Liu X.S.
      Cistrome data browser: Expanded datasets and new tools for gene regulatory analysis.
      ). These include: GSM2588420 for H3K27ac ChIP-seq analyses and GSE124306 for E3 enhancer Capture-C in mouse ESC (WT) 24 h after RA induction (
      • Su G.
      • Guo D.
      • Chen J.
      • Liu M.
      • Zheng J.
      • Wang W.
      • Zhao X.
      • Yin Q.
      • Zhang L.
      • Zhao Z.
      • Shi J.
      • Lu W.
      A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.
      ,
      • Cao K.
      • Collings C.K.
      • Marshall S.A.
      • Morgan M.A.
      • Rendleman E.J.
      • Wang L.
      • Sze C.C.
      • Sun T.
      • Bartom E.T.
      • Shilatifard A.
      SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.
      ); GSE96107 for Hi-C (
      • Bonev B.
      • Mendelson Cohen N.
      • Szabo Q.
      • Fritsch L.
      • Papadopoulos G.L.
      • Lubling Y.
      • Xu X.
      • Lv X.
      • Hugnot J.P.
      • Tanay A.
      • Cavalli G.
      Multiscale 3D genome rewiring during mouse neural development.
      ); GSM859491 for H3K27ac (
      • Xiao S.
      • Xie D.
      • Cao X.
      • Yu P.
      • Xing X.
      • Chen C.C.
      • Musselman M.
      • Xie M.
      • West F.D.
      • Lewin H.A.
      • Wang T.
      • Zhong S.
      Comparative epigenomic annotation of regulatory DNA.
      ); GSM2630487 for H3K4me1 (
      • Cao K.
      • Collings C.K.
      • Morgan M.A.
      • Marshall S.A.
      • Rendleman E.J.
      • Ozark P.A.
      • Smith E.R.
      • Shilatifard A.
      An Mll4/COMPASS-Lsd1 epigenetic axis governs enhancer function and pluripotency transition in embryonic stem cells.
      ); GSM881353 for H3K4me2 (
      • Xiao S.
      • Xie D.
      • Cao X.
      • Yu P.
      • Xing X.
      • Chen C.C.
      • Musselman M.
      • Xie M.
      • West F.D.
      • Lewin H.A.
      • Wang T.
      • Zhong S.
      Comparative epigenomic annotation of regulatory DNA.
      ); GSM1258237 for H3K4me3 (
      • Denissov S.
      • Hofemeister H.
      • Marks H.
      • Kranz A.
      • Ciotta G.
      • Singh S.
      • Anastassiadis K.
      • Stunnenberg H.G.
      • Stewart A.F.
      Mll2 is required for H3K4 trimethylation on bivalent promoters in embryonic stem cells, whereas Mll1 is redundant.
      ); GSM699165 for CTCF (
      • Handoko L.
      • Xu H.
      • Li G.
      • Ngan C.Y.
      • Chew E.
      • Schnapp M.
      • Lee C.W.
      • Ye C.
      • Ping J.L.
      • Mulawadi F.
      • Wong E.
      • Sheng J.
      • Zhang Y.
      • Poh T.
      • Chan C.S.
      • et al.
      CTCF-mediated functional chromatin interactome in pluripotent cells.
      ); GSM2645432 for YY1 (
      • Weintraub A.S.
      • Li C.H.
      • Zamudio A.V.
      • Sigova A.A.
      • Hannett N.M.
      • Day D.S.
      • Abraham B.J.
      • Cohen M.A.
      • Nabet B.
      • Buckley D.L.
      • Guo Y.E.
      • Hnisz D.
      • Jaenisch R.
      • Bradner J.E.
      • Gray N.S.
      • et al.
      YY1 is a structural regulator of enhancer-promoter loops.
      ); GSM1439567 for Med1 (
      • Buganim Y.
      • Markoulaki S.
      • van Wietmarschen N.
      • Hoke H.
      • Wu T.
      • Ganz K.
      • Akhtar-Zaidi B.
      • He Y.
      • Abraham B.J.
      • Porubsky D.
      • Kulenkampff E.
      • Faddah D.A.
      • Shi L.
      • Gao Q.
      • Sarkar S.
      • et al.
      The developmental potential of iPSCs is greatly influenced by reprogramming factor selection.
      ); GSM560345 for Med12 (
      • Kagey M.H.
      • Newman J.J.
      • Bilodeau S.
      • Zhan Y.
      • Orlando D.A.
      • van Berkum N.L.
      • Ebmeier C.C.
      • Goossens J.
      • Rahl P.B.
      • Levine S.S.
      • Taatjes D.J.
      • Dekker J.
      • Young R.A.
      Mediator and cohesin connect gene expression and chromatin architecture.
      ); GSM1276711 for RNA PolⅡ (
      • Denissov S.
      • Hofemeister H.
      • Marks H.
      • Kranz A.
      • Ciotta G.
      • Singh S.
      • Anastassiadis K.
      • Stunnenberg H.G.
      • Stewart A.F.
      Mll2 is required for H3K4 trimethylation on bivalent promoters in embryonic stem cells, whereas Mll1 is redundant.
      ); GSM1014154 for DNase (
      • Vierstra J.
      • Rynes E.
      • Sandstrom R.
      • Zhang M.
      • Canfield T.
      • Hansen R.S.
      • Stehling-Sun S.
      • Sabo P.J.
      • Byron R.
      • Humbert R.
      • Thurman R.E.
      • Johnson A.K.
      • Vong S.
      • Lee K.
      • Bates D.
      • et al.
      Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution.
      ); and GSM2651154 for ATAC-seq data (
      • Tastemel M.
      • Gogate A.A.
      • Malladi V.S.
      • Nguyen K.
      • Mitchell C.
      • Banaszynski L.A.
      • Bai X.
      Transcription pausing regulates mouse embryonic stem cell differentiation.
      ) in mouse undifferentiated ESC.

      Supporting information

      This article contains supporting information.

      Conflict of interests

      The authors declare that they have no conflict of interest.

      Acknowledgments

      We thank all members of our laboratory for many helpful discussions. We also thank Dr Elise Lamar for editing our article. This work was supported by the National Key R&D Program of China (NO. 2017YFA0102600 ), Chinese National Natural Science Foundation of China ( NSFC31530027 ).

      Author contributions

      W. L., G. S., and L. Z. conceived and supervised the study and designed the experiments. G. S., J. C., M. L., J. Z., J. B., Z. Z., and J. S. performed the experiments and analyzed the data. J. C., D. G., and W. W. analyzed the sequencing data. G. S. and W. W. performed bioinformatic analysis. G. S., W. L., and L. Z. wrote the article. All the authors have read and approved the final article.

      Supporting information

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