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

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.


Fig. S3 EACBE deletion didn't influence with thymocyte development and TCR expression on surface. A) Percentages of thymocyte subsets and B) DN subsets from 6-
week-old wild type and EACBE -/mice were analyzed by using flow cytometry. Data represent mean ± SD of three independent experiments. C) Flow cytometry plot and D) Cell numbers of CD4 + and CD8 + lymphocytes in spleen of 6-week-old wild type and EACBE -/mice were analyzed by using flow cytometry. The flow cytometry dot plots show gating of CD4 + and CD8 + cells in CD3 + population. E) Flow cytometry showed the TCRβ and CD3 on cell surface. Data are two independent experiments.

Fig. S4 Activation of EACBE-deleted T cells.
A) Flow cytometry of CD4 + and CD8 + T cell from spleen or lymph node of wild type and EACBE -/mice before and after 24-hour platebound CD3/CD28 stimulate. Cell proportion of activated CD4 + and CD8 + T cells from B) spleen and C) peripheral lymph node after 24-hour stimulate. Data are representative of seven (spleen) or four (PLN, peripheral lymph node) independent experiments (one mouse per experiment). * P < 0.05 by two-side Student's T test.

Fig. S5
The repertoire of Tcrb and Tcrd in EACBE -/thymocytes. Relative clonotype numbers of A) Jβ, B) Vβ genes and C) heatmap of Vβ−Jβ rearrangements determined by deep sequencing of Tcrb transcripts amplified by 5'RACE of wild type and EACBE -/mice respectively. Data are representative of two independent experiments. D) Vδ usage determined by high-throughput sequencing of Tcrb transcripts amplified by 5'RACE of wild type and EACBE -/mice respectively. Data are mean ± SD of two experiments. * P < 0.05, ** P < 0.01 by two side Student's T test.

Fig. S6 EACBE deletion didn't influence thymocyte survival.
A) Flow cytometry plot of apoptosis assay of thymocytes cultured on 0, 6 hours, 24 hours, and 48 hours in medium with 10% FBS. Data are representative of three independent experiments. B) Apoptosis cell percentage and C) survival cell percentage of thymocytes after 0-hour, 6-hour, 24-hour-s, and 48-hour cultures. The data represent mean of three experiments with normalization to the 0hour.

Fig. S8 EACBE mediates interactions of the Eα with the genes in the downstream sub-TAD.
4C data normalized using 4C-ker program from EACBE right and INT 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. Table S1 the primer sequences used in the paper.

Primers
Sequence