Cis-Regulatory Elements and Epigenetic Changes Control Genomic Rearrangements of the IgH Locus

Thomas Perlot*,†, Frederick W. Alt*    * The Howard Hughes Medical Institute, The Children's Hospital, Immune Disease Institute, and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
† University of Vienna, Dr-Karl-Lueger-Ring1, Vienna, Austria

1 Introduction

An individual clone of mature B-cells expresses immunoglobulin (Ig) molecules as an antigen receptor. The typical subunit of an Ig molecule consists of two identical heavy chains (HC) and two identical light chains (LC). The N-terminal region of these chains contains the highly variable antigen binding site; whereas the C-terminal part is called constant region (C region). The C region of the IgH chain (CH) determines the effector functions of antibodies, which are the secreted form of Ig molecules.

Immunoglobulin (Ig) and T-cell receptor (TCR) variable region exons are assembled from large arrays of V (variable), D (diversity), and J (joining) gene segments during the development, respectively, of B and T lymphocytes. Once a functional immunoglobulin chain is expressed, allelic exclusion operates through a feedback mechanism to prevent further rearrangements of Ig heavy (IgH) and Ig light (IgL) chain genes. V(D)J recombination is mediated by a common recombinase complex that includes the recombination-activating gene products RAG1 and RAG2, which harbor endonuclease activity that introduces DNA double-strand breaks (DSBs) at V, D, and J segments. The V(D)J reaction is completed by the ubiquitously expressed nonhomologous end-joining (NHEJ) factors that join the broken V, D, and J segments together. Still, Ig loci are only fully assembled in B lineage cells and TCR loci are only assembled in T lineage cells. Within a lineage, different loci are rearranged in a specific order. For example, IgH locus variable region exons are assembled before those of Ig light chains (IgL), and within the IgH locus D to JH recombination precedes VH to DJH recombination. Given such locus-specific regulation and a common V(D)J recombinase, accessibility of the different loci to the common V(D)J recombinase must underlie the cell-type and stage-dependent assembly of the different IgH and TCR gene families (Jung et al., 2006).

Activation of mature B-cells can alter their IgH loci through a separate form of genomic rearrangement which is termed IgH class switch recombination (CSR). CSR allows B-cells to express IgH chains with different constant regions which can change the effector functions of antibodies without altering variable region specificity. CSR is initiated by activation-induced cytosine deaminase (AID), the activity of which ultimately leads to DSBs in regions upstream of CH genes which are then joined by NHEJ or other end-joining pathways to complete the CSR reaction (Chaudhuri et al., 2007).

Ig and TCR loci contain a number of cis-regulatory elements which regulate V(D)J rearrangements, IgH CSR, and Ig gene expression at various levels. In this chapter, we will focus on the impact of cis-regulatory elements on genetic and epigenetic regulation of recombination events within the IgH locus.

2 The Immunoglobulin Heavy Chain Locus

The murine IgH locus is a complex genomic region, spanning about 3 Mb close to the telomere of the long arm on chromosome 12. The IgH locus comprises arrays of V, D, and J segments upstream of several constant region exons (Fig. 1.1A). Different mouse strains carry varying numbers of VH and D elements. Some 150 VH segments are distributed over ∼2.5 Mb in the 5′ part of the IgH locus and are classified in 16 VH gene families defined by sequence similarities (Johnston et al., 2006). These VH gene families are partially interspersed with one another but, depending on position, can be divided into proximal (3′ part of the VH cluster, close to IgH–D region, for example, VH7183), intermediate (e.g., VHS107), and distal (5′ part of the VH cluster, distant from IgH–D region, for example, VHJ558) families. 3′ of the VH elements, separated by ∼90 kb, lie 10–15 D segments (Retter et al., 2007; Ye, 2004) followed by 4 JH elements. Because of the uniform transcriptional orientation of V, D, and J segments, V(D)J recombination events at the IgH locus result in deletion of the intervening sequence. The 3′ part of the IgH locus contains a series of sets of different constant (C) region exons Cμ, Cδ, Cγ3, Cγ1, Cγ2b, Cγ2a, Cɛ, Cα, which will be referred to as “CH genes” (Fig. 1.1B).

A large number of cis-regulatory elements were identified throughout the IgH locus. The intronic enhancer, Eμ, is located in the intron between JH4 and the CH exons (Fig. 1.1); the 3′ IgH regulatory region (IgH 3′RR) consists of several DNase hypersensitive sites and is located at the very 3′ end of the IgH locus (Fig. 1.1). Transcriptional promoters are present upstream of every VH segment (Fig. 1.2B), upstream of DH segments (Fig. 1.2A and C), and upstream of CH genes (Fig. 1.2A). In addition, antisense transcripts from less well-defined promoters were described in the VH, DH, JH regions, and upstream of CH genes. Section 7 of this chapter contains a detailed discussion of these IgH cis-regulatory elements.