Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element.
Bottom Line: The second element is a chromatin barrier that protects the tether from hyperactive RC chromatin.When the second element is removed, active RC chromatin spreads upstream, forcing the tether to serve as a new barrier.Acquisition of barrier function by the CTCF element disrupts contacts between distal Vβ gene segments and significantly alters Tcrb repertoires.
Affiliation: Department of Pathology and Immunology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110.Show MeSH
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Mentions: In addition to Eβ, transcription and rearrangement of the RC is controlled by two promoters, termed PDβ1 and PDβ2, situated within their respective DβJβ clusters (Fig. 1 A; Sikes et al., 1998, 2002). Activation of the Dβ1Jβ, but not Dβ2Jβ, cluster is crippled in thymocytes harboring a 3.5-kb deletion spanning PDβ1 (ΔPDβ1 allele; Fig. 1 A; Whitehurst et al., 1999). To test whether activities associated with the promoter region contribute to folding of Tcrb into its active conformation, we performed 3C analyses on DN thymocytes from ΔPDβ1/Rag1−/− mice. Because ΔPDβ1 removes one relevant restriction site near Dβ1, we focused RC interactome experiments on Dβ2 and Eβ. As shown in Fig. 3 A (top), Dβ2 interactions with the most proximal portion of the Trbv cluster are unaffected by the ΔPDβ1 mutation (Trbv16-30). However, we observe a significant reduction in Dβ2 cross-linking with distal portions of the Trbv array (Trbv1-14). Precisely the same bifurcation in long-range interactions is observed when Eβ is used as the 3C viewpoint (Fig. 3 B). The ΔPDβ1 mutation also reduced CTCF levels at sites in the distal Trbv array (Fig. 3 C), which may be a consequence of disrupting their association with CTCF-rich elements near the RC (see Discussion). However, RAD21 binding and germline Trbv transcription throughout Tcrb are unaffected in ΔPDβ1 thymocytes (Fig. 3, D and E).
Affiliation: Department of Pathology and Immunology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110.