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A role of the CTCF binding site at enhancer Eα in the dynamic chromatin organization of the Tcra-Tcrd locus - PubMed

  • ️Wed Jan 01 2020

A role of the CTCF binding site at enhancer Eα in the dynamic chromatin organization of the Tcra-Tcrd locus

Hao Zhao et al. Nucleic Acids Res. 2020.

Abstract

The regulation of T cell receptor Tcra gene rearrangement has been extensively studied. The enhancer Eα plays an essential role in Tcra rearrangement by establishing a recombination centre in the Jα array and a chromatin hub for interactions between Vα and Jα genes. But the mechanism of the Eα and its downstream CTCF binding site (here named EACBE) in dynamic chromatin regulation is unknown. The Hi-C data showed that the EACBE is located at the sub-TAD boundary which separates the Tcra-Tcrd locus and the downstream region including the Dad1 gene. The EACBE is required for long-distance regulation of the Eα on the proximal Vα genes, and its deletion impaired the Tcra rearrangement. We also noticed that the EACBE and Eα regulate the genes in the downstream sub-TAD via asymmetric chromatin extrusion. This study provides a new insight into the role of CTCF binding sites at TAD boundaries in gene regulation.

© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.
Figure 1.

EACBE is located at the sub-TAD boundary. (A) Heatmap of 10 and 5 kb binned Hi-C data at the Tcra–Tcrd locus in DP thymocytes obtained from anti-CD3 injected Rag1−/− mice. (B) CTCF ChIP-seq signal at the Tcra–Tcrd locus in DN and DP thymocytes of Rag2−/– mice (up). 4C-seq profiles of interactions from the viewpoints of the left and the right of EACBE in DP thymocytes generated from anti-CD3 injected Rag2−/− mice (down). The data are presented as reads per million mapped reads (RPM) within the Tcra–Tcrd locus. The green line and yellow line represent EACBE-left and EACBE-right viewpoint respectively. The 4C data are representative of two independent experiments.

Figure 2.
Figure 2.

EACBE deletion doesn’t influence Eα activity and T cell development. (A) Schematic of the EACBE deletion. (B) CTCF and Cohesin occupancy on the Eα and negative control (50 kb downstream) were assessed by ChIP-qPCR. Values of bound/input were relative to the Actb gene promoter (normalized to 1). Data are representative of two independent experiments. The data represent mean ± SD of three PCR results of one experiment. **P <0.01 by two side Student's t test. (C) ChIP-qPCR of histone H3K4me3 and H3K27ac modification at Eα. Values of bound/input were relative to the Actb gene promoter (normalized to 1) in each sample. The data represent mean ± SD of three experiments. (D) Flow cytometric analysis of thymocyte subsets in 6 week-old wild type and EACBE−/− mice. Data are representative of three independent experiments. (E) Cell numbers of thymocytes, spleen and lymph node (LN) in 6 week-old EACBE wild type and knockout mice (n = 6).

Figure 3.
Figure 3.

EACBE deletion impaired Tcra rearrangement. (A) Schematic of the Tcra-Tcrd locus. (B) Relative Jα usage and (C) Heatmap of Vα−Jα combination determined by high-throughput sequencing of Tcra transcripts amplified by 5′RACE of wild type and EACBE−/− mice respectively. The relative Jα usage was calculated by dividing the number of the clonotypes containing the Jα gene by the total clonotype number. The data plotted as mean ± SD of two experiments. The signal in the heatmap is the number of the Vα−Jα clonotypes divided by the total clonotype number. Data are representative of two independent experiments. ** P<0.0005, by two side multiple Student's T test. (D) Jα usage was assessed by using qPCR with primers specific for Vα8 (Trav12), Vα2 (Trav14), or Vα10 (Trav13) families in conjunction with different Jα primers. Data are representative of three independent experiments. The data represent mean ± SD of three PCR results of one experiment. (E) Double strand breaks in two-fold serially diluted genomic DNA from wild type and EACBE−/− thymocytes detected by ligation-mediated PCR and visualized by Southern blotting with Jα-specific probes. Data are representative of three independent experiments. (F) Vα–Jα combination quantified by using qPCR with Vα and Jα gene-specific primers in sorted CD71+ DP thymocytes. The qPCR values were determined by standard curve of thymus genomic DNAs. The data plotted as mean ± SD of three experiments, each with one mouse per genotype, with values for EACBE-deficient (KO) thymocytes normalized to those for wild type (WT) littermates. * P< 0.05, ** P< 0.01, **** P< 0.0001 by two side Student's t test.

Figure 4.
Figure 4.

EACBE deletion reduced the accessibility of the proximal Vα genes. (A) H3K4me3 and H3K27ac ChIP-seq on the Tcra-Tcrd locus in Rag2−/− (WT) and Rag2−/− × EACBE−/− (KO) DP thymocytes from anti-CD3 injected mice. Data are representative of two independent experiments. (B) and (C) Histone H3K4me3 and H3K27ac modification were analysed by ChIP qPCR. Values of bound/input were relative to the Actb gene promoter. (D) Relative transcription of the unrearranged Vα and Jα region was analysed using reverse-transcription qPCR. The expressions are normalized to the Actb gene and then to WT. The data represent the mean ± SD of three experiments. * P < 0.05 by two side multiple Student's t test.

Figure 5.
Figure 5.

EACBE deletion impaired the interactions between the proximal Vα and the 5′ Jα genes. (A) Heatmap and subtraction heatmap of 10 kb binned Hi-C data of DP thymocytes generated from anti-CD3 injected EACBE+/+ × Rag1−/- and EACBE−/− × Rag1−/- mice. (B) 4C signal normalized using 4C-ker program from Eα (EACBE left), TEAp and Trav17 viewpoint in CD3-stimulated-DP thymocytes of WT and EACBE−/− mice at Rag2−/− background. It was analyzed with two independent replicates. Filled circles highlight significant differences. (C) Insulation profiles of the Tcra-Tcrd locus. (D) Cohesin occupancy on the Eα was assessed by using ChIP-qPCR. Values of bound/input were normalized to the Actb promoter CBE. Data are mean ± SD of three PCR replicates of one of two independent experiments. *** P< 0.005 by two side Student's t test.

Figure 6.
Figure 6.

EACBE regulates the genes in the downstream region. (A) CTCF, Rad21 and Nipbl ChIP-seq signals are plotted for the downstream region (B) 4C signal normalized by 4C-ker program from Eα downstream viewpoints in DP thymocytes generated from anti-CD3 injected WT and EACBE−/− mice at Rag2−/− background. It was analyzed with two independent replicates. Filled circles highlight significant differences. (C) H3K4me3 and H3K27ac ChIP-seq signals. The ChIP-seq data are representative of two independent experiments. (D) and (E) ChIP-qPCR of H3K4me3 and H3K27ac on EACBE downstream region in DP thymocytes. The ChIP-qPCR data represent mean ± SD of three experiments, with normalization to values for the Actb promoter. (F) Relative transcription of the genes in the downstream region. The expressions are normalized to the Actb gene and then to WT. The data represent mean ± SD of three experiments for WT and EACBE−/−. * P< 0.05, ** P< 0.01, **** P< 0.001 by two side multiple Student's t test.

Figure 7.
Figure 7.

The Eα regulates expression of the genes at the downstream region. (A) Schematic of the Eα and LCR deletion. (B) The LCR deletion region includes two ATAC peaks, one of which is overlapped with Nibpl peak. Red line represents the LCR deletion region. (C and D) Relative transcription of the genes in the downstream region in DP thymocytes from Eα-deleted and LCR-deleted mice. The expressions are normalized to the Actb gene and then to WT. The data represent mean ± SD of four (Eα-deleted mice) or three (LCR-deleted mice) experiments. * P< 0.05, *** P< 0.005 by two side multiple Student's t test.

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