Dynamic Changes in Binding of Immunoglobulin Heavy Chain 3′ Regulatory Region to Protein Factors during Class Switching

Abstract

The 3 regulatory region (3 RR) of the Igh locus works at long distances on variable region (V H ) and switch region (I) region promoters to initiate germ line (non-coding) transcription (GT) and promote class switch recombination (CSR). The 3 RR contains multiple elements, including enhancers (hs3a, hs1.2, hs3b, and hs4) and a proposed insulator region containing CTCF (CCCTC-binding factor) binding sites, i.e. hs5/6/7 and the downstream region ("38"). Notably, deletion of each individual enhancer (hs3a-hs4) has no significant phenotypic consequence, suggesting that the 3 RR has considerable structural flexibility in its function. To better understand how the 3 RR functions, we identified transcription factor binding sites and used chromatin immunoprecipitation (ChIP) assays to monitor their occupancy in splenic B cells that initiate GT and undergo CSR (LPS؎IL4), are deficient in GT and CSR (p50 ؊/؊ ), or do not undergo CSR despite efficient GT (anti-IgM؉IL4). Like 3 RR enhancers, hs5-7 and the 38 region were observed to contain multiple Pax5 binding sites (in addition to multiple CTCF sites). We found that the Pax5 binding profile to the 3 RR dynamically changed during CSR independent of the specific isotype to which switching was induced, and binding focused on hs1.2, hs4, and hs7. CTCF-associated and CTCF-independent cohesin interactions were also identified. Our observations are consistent with a scaffold model in which a platform of active protein complexes capable of facilitating GT and CSR can be formed by varying constellations of 3 RR elements.The immunoglobulin heavy chain (Igh) 3 locus is subject to multiple processes that markedly increase the number of different antibody gene coding sequences (reviewed in Ref. 1). These include the random DNA recombination of V (variable), D (diversity) and J (joining) sequences to form multiple variable region coding segments and class switch recombination (CSR) that juxtaposes assembled variable region genes to different constant region (C H ) genes to provide distinct biological functions associated with specific antigen-binding specificities. In addition, somatic hypermutation of variable region genes augments genetic diversity. These processes are regulated by cisacting elements, including locally acting promoters, and by long distance regulators, such as the intronic enhancer (E) and the 3Ј regulatory region (3Ј RR). Knockout mouse models identified E as a major regulator of VDJ recombination (2-5) and showed that the 3Ј RR influenced germ line (non-coding) transcription (GT) of individual C H genes (6 -11). GT is initiated from a promoter upstream of each C H gene and extends through switch and C H sequences, generating a single-stranded DNA substrate for activation-induced cytidine deaminase (AID), a protein critical for CSR (12). Long-range physical interactions between an element of the 3Ј RR and I region promoters that drive GT have been shown in cells stimulated by LPS to undergo CSR by use of a chromosome conformation capture (3C) assay (13). Studies hav...

Binding of estrogen receptors to switch sites and regulatory elements in the immunoglobulin heavy chain locus of activated B cells suggests a direct influence of estrogen on antibody expression

“…When the 3′RR region is absent in mice, V-D-J joining capacity is retained, but mice express low levels of IgM. Mice lacking 3′RR are also deficient in SHM and CSR [23,38–40]. The 3′RR not only influences CSR outcome in normal animals, but a rearrangement involving the 3′RR can serve as a mechanism for B cell suicide [41–44].…”

“…AID, RNA polymerase, BATF, cohesin, Med 1, Med 12, histone-modifying enzymes, and the RNA exosome) [18,19,23,31,40,46–59]. ER should perhaps be studied in conjunction with other members of the nuclear hormone receptor superfamily, as receptors can be promiscuous and cross-regulatory [26,60].…”

Interaction between the immunoglobulin heavy chain 3′ regulatory region and the IgH transcription unit during B cell differentiation

“…3’ RR-DFL16.1 interactions associated with locus contraction and potentially with VDJ joining are detected in pro-B cells, while 3’ RR-J H interactions associated with heavy chain transcription are detected in the 63-12 Abelson-virus transformed pro-B cell line, and in pre-B and plasma cells. Interactions with I/switch regions are important for class switch recombination (Chatterjee et al, 2011; Wuerffel et al, 2007; Yan et al, 2011). The identification of proteins that facilitate these interactions is in progress.…”

“…CTCF binding, as measured by PD (pulldown)/input, was reduced by lentiviral shRNA, as shown in the gray bars. Note that the predominant CTCF binding sites are in hs5-7 (Chatterjee et al, 2011). The 5’ end of the c-myc gene was used as a positive control.…”

“…Additional 3’ RR hs sites contain binding sites for Pax5 and CTCF and display their accompanying insulator activity (hs5-7 and “38”) (Fig. 1A) (Chatterjee et al, 2011; Garrett et al, 2005). Functionally, the 3’ RR is important for immunoglobulin heavy chain gene expression in plasma cells and for class switching in activated B cells, as revealed by mouse knockout models (Pinaud et al, 2001; Vincent-Fabert et al, 2010).…”