Inducible CTCF insulator delays the IgH 3′ regulatory region-mediated activation of germline promoters and alters class switching

Abstract

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to switch from IgM expression to the expression of downstream isotypes. CSR is preceded by inducible germline (GL) transcription of the constant genes and is controlled by the 3′ regulatory region (3′RR) in a stimulus-dependent manner. Why the 3′RR-mediated upregulation of GL transcription is delayed to the mature B-cell stage is presently unknown. Here we show that mice devoid of an inducible CTCF binding element, located in the α constant gene, display a marked isotype-specific increase of GL transcription in developing and resting splenic B cells and altered CSR in activated B cells. Moreover, insertion of a GL promoter downstream of the CTCF insulator led to premature activation of the ectopic promoter. This study provides functional evidence that the 3′RR has a developmentally controlled potential to constitutively activate GL promoters but that this activity is delayed, at least in part, by the CTCF insulator, which borders a transcriptionally active domain established by the 3′RR in developing B cells. E xpression of complex loci is developmentally programmed or induced by specific stimuli and is often controlled by distant regulatory elements within relatively large chromatin domains. Transcriptional and architectural factors play an important role in the establishment and maintenance of these domains and facilitate long-range interactions between regulatory elements and target promoters (1, 2). The Ig heavy chain (IgH) locus is expressed in a lineage-and developmental stage-dependent manner. Various cis-acting elements including promoters, enhancers, and insulators control IgH locus expression and are engaged in multiple long-range interactions (3, 4).Factors such as YY1, PAX5, IKAROS, CTCF, and Cohesin play important roles in various aspects of long-range events at the IgH locus, including V(D)J recombination, CSR, and promoter/enhancer and enhancer/enhancer interactions (3-6). Multiple CTCF binding elements (CBEs) were reported along the IgH locus. The majority of these CBEs lie within the variable domain (7), and two CBEs were identified within the V H -D intergenic region (7-9). At the 3′ end of the locus, ∼10 CBEs were identified downstream of the 3′RR and are thought to delineate the 3′ border of the IgH locus (10). More recently, a discrete CBE was identified within the α constant gene (11), but its role in vivo is presently unknown.Upon antigen challenge, mature B cells can undergo CSR that allows B cells to change the heavy-chain constant domain of an IgM to IgG, IgE, or IgA. CSR to a particular isotype is induced by specific external stimuli, including antigens, mitogens, cytokines, and intercellular interactions. CSR is mediated by highly repetitive sequences called switch (S) sequences located upstream of the constant exons and is preceded by germline (GL) transcription of the S sequences that originates from GL promoters, named I promoters (12).The 3′RR is composed of four enhance...

Two modes ofcis-activation of switch transcription by theIgHsuperenhancer

“…Purified resting or LPS-activated splenic B cells were singlecell-sorted and assayed for monoallelic transcription of Sγ3 and Sγ2b, as described above. Interestingly, and in agreement with the high levels of Sγ3 transcripts detected in resting B cell populations (15), already at 0 h, over 40% of alleles expressed Sγ3, while only 6.5% transcribed Sγ2b and 4% coexpressed Sγ3 and Sγ2b (Fig. 3 B-D).…”

“…To test this, we used a mouse genetic setting that magnifies initial monoallelic activation of the Iγ3 promoter. We have recently shown that deletion of the inducible insulator 5′hs1RI in Cα leads to premature activation of the Iγ3 and Iγ2b promoters (15). In resting (nonactivated) 5′hs1RI Δ/Δ splenic B cells, Sγ3 and Sγ2b transcript levels are unusually high compared with WT counterparts, with Sγ3 transcripts displaying the biggest difference…”

“…For clarity, only transcripts originating from allele 2 are displayed. (15). We reasoned that that might reflect an initial higher number of Sγ3-expressing alleles.…”

“…To explore whether the 3′RR displays a bidirectional activity, we used a mouse line in which the Iα promoter has been duplicated downstream of the 3′RR (2Iα mice) (15,19). We derived a heterozygous mouse line, here called 2Iα/Bl6 mice, carrying a WT allele and a mutant 2Iα allele, of C57BL/6J and 129Sv1 origin, respectively (SI Appendix, Fig.…”

“…S7). In addition, the 2Iα allele transcribes human β-globin transcripts that can be detected with specific primers (15,19) (SI Appendix, Fig. S10A).…”