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

• Published in: Proceedings of the National Academy of Sciences - PNAS 2011-06, Vol.108 (23), p.9566-9571
• Main Authors: Degner, Stephanie C., Verma-Gaur, Jiyoti, Wong, Timothy P., Bossen, Claudia, Iverson, G. Michael, Torkamani, Ali, Vettermann, Christian, Lin, Yin C., Ju, Zhongliang, Schulz, Danae, Murre, Caroline S., Birshtein, Barbara K., Schork, Nicholas J., Schlissel, Mark S., Riblet, Roy, Murre, Cornelis, Feeney, Ann J.
• Format: Article
• Language: English
• Subjects: Animals; Antibodies; Antisense; B lymphocytes; Binding sites; Binding Sites - genetics; Biological Sciences; Blotting, Western; CCCTC-Binding Factor; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Cell Line; Cell lines; Cells; Cells, Cultured; Chromatin; Chromatin Immunoprecipitation; Chromosomal Proteins, Non-Histone - genetics; Chromosomal Proteins, Non-Histone - metabolism; Chromosomes; cohesin; Cohesins; Compaction; Conformation; DNA, Antisense - genetics; DNA-Binding Proteins; Enhancer Elements, Genetic - genetics; Enhancers; Genes; Genetic loci; Genomics; Heavy chains; Immunoglobulin Heavy Chains - genetics; Immunoglobulin Heavy Chains - metabolism; Immunoglobulins; Mice; Mice, Inbred C57BL; Mice, Knockout; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Phosphoproteins - genetics; Phosphoproteins - metabolism; Precursor Cells, B-Lymphoid - metabolism; Protein Binding; Proteins; Regulatory sequences; Repressor Proteins - genetics; Repressor Proteins - metabolism; Retroviridae; Retrovirus; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Antisense - genetics; Rosette; Signal transduction; Stem cells; Transcription; Transcription, Genetic; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1019391108

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.