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. 2021 Jan-Jun:296:100413.
doi: 10.1016/j.jbc.2021.100413. Epub 2021 Feb 11.

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

Guangsong Su  1 , Wenbin Wang  1 , Jun Chen  1 , Man Liu  1 , Jian Zheng  1 , Dianhao Guo  1 , Jinfang Bi  1 , Zhongfang Zhao  1 , Jiandang Shi  1 , Lei Zhang  2 , Wange Lu  3
Affiliations

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

Guangsong Su et al. J Biol Chem. 2021 Jan-Jun.

Erratum in

Abstract

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: CTCF-binding element; HoxA; differentiation; embryonic stem cells; enhancer; enhancer–enhancer interaction complex; long-range chromatin interaction.

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Conflict of interest statement

Conflict of interests The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Identification 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 Fig. S1.
Figure 2
Figure 2
Gene 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). D–K, 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.
Figure 3
Figure 3
CBE+47deletion potentiates RA-induced HoxA expression and perturbs ESC differentiation.A–C, 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). D–F, 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 A–F, 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 Figs. S2 and S3.
Figure 4
Figure 4
Transcriptome 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 Figs. S4–S7.
Figure 5
Figure 5
CBE+47is 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. B–D, 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.
Figure 6
Figure 6
Deletions of multiple enhancers synergize to alter RA-induced HoxA gene expression.A–E, 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 A–C 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 Figs. S8 and S9.
Figure 7
Figure 7
Schematic showing how CBE+47regulates 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.
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