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Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element.

Majumder K, Koues OI, Chan EA, Kyle KE, Horowitz JE, Yang-Iott K, Bassing CH, Taniuchi I, Krangel MS, Oltz EM - J. Exp. Med. (2014)

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.

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Affiliation: Department of Pathology and Immunology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110.

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Deletion of the 5′RC flank resolves two Trbv interaction domains. (A and B) 3C analysis of RAG-deficient thymocytes (WT, ΔPDβ1, or mEβ alleles) and pro-B cells using the Dβ2 (A, top), Trbv5 (A, bottom), and Eβ (B) viewpoints (anchors). Individual HindIII fragments are represented by alternating white and gray bars. Bold black bars indicate viewpoint locations. Schematics of Tcrb are shown on top and below primary 3C data, which are presented as mean values (±SEM) from at least three independent experiments. Thymocytes were pooled from 5–10 mice per 3C experiment. Significant differences between WT and ΔPDβ1 samples are denoted as *, P < 0.05 (Student’s t test). See Fig. 1 for details of cartoon data summaries. Here, red shading indicates that Trbv-Dβ2 cross-linking in ΔPDb1 relative to WT alleles was unchanged (darkest red) or reduced to background levels in pro-B cells (white). (C and D) ChIP-qPCR assay for CTCF (C) and RAD21 (D) binding at sites near the indicated Trbv segments. Refer to Fig. 2 C for details. Data are presented as mean percent input (±SEM) with thymocytes pooled from at least 5–10 mice per experiment. (E) Trbv germline transcription was quantified relative to Actb by qRT-PCR from at least three independent experiments (involving one to three mice per experiment). Data are presented as mean relative expression (±SEM). Statistically significant differences are denoted as *, P < 0.05 (Student’s t test). (F) 3C assays were performed with the Trbv23 viewpoint (anchor). Schematic of Tcrb is shown on top. Data are presented as mean relative cross-linking (±SEM). Statistically significant differences between WT and ΔPDβ1 are denoted as *, P < 0.05 (Student’s t test).
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fig3: Deletion of the 5′RC flank resolves two Trbv interaction domains. (A and B) 3C analysis of RAG-deficient thymocytes (WT, ΔPDβ1, or mEβ alleles) and pro-B cells using the Dβ2 (A, top), Trbv5 (A, bottom), and Eβ (B) viewpoints (anchors). Individual HindIII fragments are represented by alternating white and gray bars. Bold black bars indicate viewpoint locations. Schematics of Tcrb are shown on top and below primary 3C data, which are presented as mean values (±SEM) from at least three independent experiments. Thymocytes were pooled from 5–10 mice per 3C experiment. Significant differences between WT and ΔPDβ1 samples are denoted as *, P < 0.05 (Student’s t test). See Fig. 1 for details of cartoon data summaries. Here, red shading indicates that Trbv-Dβ2 cross-linking in ΔPDb1 relative to WT alleles was unchanged (darkest red) or reduced to background levels in pro-B cells (white). (C and D) ChIP-qPCR assay for CTCF (C) and RAD21 (D) binding at sites near the indicated Trbv segments. Refer to Fig. 2 C for details. Data are presented as mean percent input (±SEM) with thymocytes pooled from at least 5–10 mice per experiment. (E) Trbv germline transcription was quantified relative to Actb by qRT-PCR from at least three independent experiments (involving one to three mice per experiment). Data are presented as mean relative expression (±SEM). Statistically significant differences are denoted as *, P < 0.05 (Student’s t test). (F) 3C assays were performed with the Trbv23 viewpoint (anchor). Schematic of Tcrb is shown on top. Data are presented as mean relative cross-linking (±SEM). Statistically significant differences between WT and ΔPDβ1 are denoted as *, P < 0.05 (Student’s t test).

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).


Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element.

Majumder K, Koues OI, Chan EA, Kyle KE, Horowitz JE, Yang-Iott K, Bassing CH, Taniuchi I, Krangel MS, Oltz EM - J. Exp. Med. (2014)

Deletion of the 5′RC flank resolves two Trbv interaction domains. (A and B) 3C analysis of RAG-deficient thymocytes (WT, ΔPDβ1, or mEβ alleles) and pro-B cells using the Dβ2 (A, top), Trbv5 (A, bottom), and Eβ (B) viewpoints (anchors). Individual HindIII fragments are represented by alternating white and gray bars. Bold black bars indicate viewpoint locations. Schematics of Tcrb are shown on top and below primary 3C data, which are presented as mean values (±SEM) from at least three independent experiments. Thymocytes were pooled from 5–10 mice per 3C experiment. Significant differences between WT and ΔPDβ1 samples are denoted as *, P < 0.05 (Student’s t test). See Fig. 1 for details of cartoon data summaries. Here, red shading indicates that Trbv-Dβ2 cross-linking in ΔPDb1 relative to WT alleles was unchanged (darkest red) or reduced to background levels in pro-B cells (white). (C and D) ChIP-qPCR assay for CTCF (C) and RAD21 (D) binding at sites near the indicated Trbv segments. Refer to Fig. 2 C for details. Data are presented as mean percent input (±SEM) with thymocytes pooled from at least 5–10 mice per experiment. (E) Trbv germline transcription was quantified relative to Actb by qRT-PCR from at least three independent experiments (involving one to three mice per experiment). Data are presented as mean relative expression (±SEM). Statistically significant differences are denoted as *, P < 0.05 (Student’s t test). (F) 3C assays were performed with the Trbv23 viewpoint (anchor). Schematic of Tcrb is shown on top. Data are presented as mean relative cross-linking (±SEM). Statistically significant differences between WT and ΔPDβ1 are denoted as *, P < 0.05 (Student’s t test).
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fig3: Deletion of the 5′RC flank resolves two Trbv interaction domains. (A and B) 3C analysis of RAG-deficient thymocytes (WT, ΔPDβ1, or mEβ alleles) and pro-B cells using the Dβ2 (A, top), Trbv5 (A, bottom), and Eβ (B) viewpoints (anchors). Individual HindIII fragments are represented by alternating white and gray bars. Bold black bars indicate viewpoint locations. Schematics of Tcrb are shown on top and below primary 3C data, which are presented as mean values (±SEM) from at least three independent experiments. Thymocytes were pooled from 5–10 mice per 3C experiment. Significant differences between WT and ΔPDβ1 samples are denoted as *, P < 0.05 (Student’s t test). See Fig. 1 for details of cartoon data summaries. Here, red shading indicates that Trbv-Dβ2 cross-linking in ΔPDb1 relative to WT alleles was unchanged (darkest red) or reduced to background levels in pro-B cells (white). (C and D) ChIP-qPCR assay for CTCF (C) and RAD21 (D) binding at sites near the indicated Trbv segments. Refer to Fig. 2 C for details. Data are presented as mean percent input (±SEM) with thymocytes pooled from at least 5–10 mice per experiment. (E) Trbv germline transcription was quantified relative to Actb by qRT-PCR from at least three independent experiments (involving one to three mice per experiment). Data are presented as mean relative expression (±SEM). Statistically significant differences are denoted as *, P < 0.05 (Student’s t test). (F) 3C assays were performed with the Trbv23 viewpoint (anchor). Schematic of Tcrb is shown on top. Data are presented as mean relative cross-linking (±SEM). Statistically significant differences between WT and ΔPDβ1 are denoted as *, P < 0.05 (Student’s t test).
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).

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine in St. Louis, St. Louis, MO 63110.

Show MeSH
Related in: MedlinePlus