CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells

Abstract

Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V(H) genes to be brought in proximity with D(H)-J(H) segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ~60 sites throughout the V(H) region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5' of DFL16 and the 3' regulatory region, and also the intronic enhancer (Eμ), creating a D(H)-J(H)-Eμ-C(H) domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D(H) region and parts of the V(H) locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.

Fig. 1.

CTCF knockdown results in decreased Igh locus compaction. (A) RNA expression in CTCF knockdown and control (scramble shRNA) Rag1^−/− pro-B cells. Data are presented as mean ± SEM (n = 3). (B) Western blot of CTCF in CTCF knockdown and control pro-B cells. GAPDH served as a loading control. (C) Diagram of Igh locus indicating the position of the BAC probes. (D) Igh locus contraction as measured by 3D-FISH in CTCF knockdown and control pro-B cells. YY1^−/− pro-B and E2A^−/− pre–pro-B cells were also analyzed. The graph represents the percentage of alleles with spatial distances within three ranges: <0.3 μm, 0.3–0.5 μm, and 0.5–1.5 μm. (E) Dot plots showing distribution of spatial distances between V_HJ558 and V_H7183 probes. For CTCF knockdown, control, and YY1^−/− pro-B cells, 204, 202, and 106 alleles, respectively, were analyzed. ***P < 0.0001 in comparison to control pro-B cells. (F) CTCF ChIP in CTCF knockdown and control pro-B cells. Data are presented as mean ± SEM (n = 2).

Fig. 2.

3C shows the 3D conformation of the Igh locus in E2A^−/− pre–pro-B cells, Rag1^−/− pro-B cells, and MEFs. (A) Number of reads from the CTCF and Rad21 ChIP-seq experiments in the 3′ portion of the Igh locus from the 3′RR to the first V_H gene. A schematic map of the relevant portion of the Igh locus is shown, with locations of hs sites (red), constant regions (blue), Eμ (red line), J_H genes (purple lines), and D_H genes (black lines). (B) Relative cross-linking frequencies between CTCF/DFL anchor fragment and HindIII fragments within the Igh locus in E2A^−/− pre–pro-B cells, RAG1^−/− pro-B cells, and MEFs using a CTCF/DFL TaqMan probe (gray bar). Data are presented as mean ± SEM (n = 3). In comparison to MEFs: *P < 0.05; **P < 0.01.

Fig. 3.

CTCF and Rad21 knockdown reduces 3D interactions within the Igh locus. R2K cells were transduced with control (scramble), CTCF, or Rad21 shRNA retroviruses. Cells were treated with puromycin on days 2–4, and the R2K cells were harvested on day 5 after transduction. Expression levels of (A) CTCF and (B) Rad21 were measured by quantitative PCR assay, and results were normalized to mouse 18S RNA. Data are presented as mean ± SEM (n = 5). (C) Western blot for CTCF and Rad 21. GAPDH was the loading control. CTCF (D) and Rad21 (E) ChIP assays for CTCF and Rad21 enrichment at selected CTCF sites within the Igh locus. Data are presented as mean ± SEM (n = 3 and n = 2, respectively). (F) Relative cross-linking frequencies between CTCF/DFL and other HindIII fragments using a CTCF/DFL probe were measured in R2K cells transduced with control, CTCF, and Rad21 shRNA retroviruses. Data are presented as mean ± SEM (n = 4). In comparison to control shRNA: *P < 0.05; **P < 0.01.

Fig. 4.

CTCF knockdown affects the level of antisense transcription in the Igh locus. (A) Relative antisense transcription levels as measured by quantitative PCR (qPCR) of cultured Rag1^−/− pro-B cells transduced with control or CTCF shRNAs. The DFL(+3) primer target is located 3 kb downstream of the DFL16.1 gene. The 5′DSP primer targets are located 0.4 kb upstream of the DSP genes. The 24, 62, and 87 primers are located 24, 62, or 87 kb downstream of V_H7183.2.3 (81×). The gray bar indicates the relative location of the CTCF/DFL sites. Results are normalized to mouse 18S RNA and are presented as mean ± SEM (n = 6). (B) Relative antisense transcription levels in the V_HJ558 region as measured by qPCR of CTCF knockdown and control Rag1^−/− pro-B cells. Results are presented as mean ± SEM (n = 5). (C) Sense transcription levels from J558 region and μ° were measured by qPCR on CTCF knockdown and control Rag1^−/− pro-B cells. Results are presented as mean ± SEM (n = 4). In comparison to control pro-B cells: *P < 0.075; **P ≤ 0.05 (details in Table S7).