Investigation of the three dimensional organisation of the genome in B cell development

Abstract

In order to detect and eliminate a myriad of different antigens, the adaptive immune systems of jawed vertebrates have evolved to generate diverse repertoires of antigen receptors (AgRs). AgR diversity is primarily generated by a form of somatic recombination known as V(D)J recombination, where different variable (V), diversity (D) and joining (J) gene segments are cut-and-pasted together. V(D)J recombination of the immunoglobulin (Ig) loci, critical to the generation of diverse antibody repertoires, occurs during B lymphocyte development in the bone marrow.

The three dimensional conformations of the multimegabase murine immunoglobulin heavy chain (Igh) and immunoglobulin kappa chain (Igκ) loci must not only facilitate recombination between gene segments separated by up to 3Mb, but diverse recombination events. To understand how this is achieved a capture Hi-C (CHi-C) approach was developed to investigate interactions within the Ig loci at high resolution. Hi-C libraries were enriched for the Ig loci and other genes of interest, using a biotinylated RNA bait system.

In this thesis, I used the CHi-C assay to generate a high-resolution, unbiased view of murine Igh locus conformation in pro-B cells, resolving conflicting models in the field. I observed that the Igh locus folds into subdomains and two regulatory elements that bind CTCF, the 3’ CTCF Binding Elements (3’CBEs) and the Intergenic Control Region 1 (IGCR1), mediate contacts with the entire VH region. In collaborative studies the CHi-C data was used for polymer modelling to generate possible single conformations of the locus, giving further insights into the mechanisms of recombination.

I revealed the first high-resolution, unbiased spatial conformation model of the murine Igκ locus in both pro-B and pre-B cells. I confirmed that the locus folds into discrete subdomains at the pro-B cell stage, but undergoes reorganisation at the pre-B cell stage. I then examined the roles of regulatory elements in the locus, including using a machine learning approach to probe the mechanisms underpinning their interactions with the Vκ region. Furthermore, I have proposed that the conformation of the Igκ locus, combined with published data on the binding of transcription factors and the distribution of active histone modifications and non-coding transcription, suggests the existence of a, so far, undescribed regulatory element in the Igκ locus.

Moreover, the CHi-C assay used enabled the interrogation of interchromosomal contacts of baited regions and revealed that the Ig loci mediate numerous interchromosomal interactions during B cell development. I have shown that the interchromosomal interactions of the Ig loci are developmental stage specific. Additionally, I have demonstrated that, predominantly at the pre-B cell stage, the Ig loci and key lymphocyte-specific transcription factors share interaction partners. I characterised these interaction partners, and showed that many are implicated in immune system processes, indicative that these interaction networks have a regulatory function.

Together these findings give new insights into the three dimensional organisation of the genome in B cell development.