Epigenetics of V(D)J Recombination, CSR, and SHM

KEY CONCEPTS
-Noncoding RNAs are associated with V(D)J recombination, class switch recombination (CSR), and somatic hypermutation (SHM).
- miRNAs regulate activation-induced deaminase (AID) expression.
- Transcription factors and transcription target histone posttranslational modifications.

DNA recombination and/or mutagenesis in Ig and TCR loci are stringently orchestrated at multiple levels, including regulation of chromatin structure and transcriptional elongation. Both DNA and its associated histones in Ig and TCR loci chromatin are epigenetically marked during B and T cell development and differentiation. Epigenetic modifications are changes in the cell progeny that are independent from the genomic DNA sequence. They include histone posttranslational modifications, DNA methylation, and alteration of gene expression by noncoding RNAs, including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) (discussed in the chapters Chromatin, Epigenetics I, Epigenetics II, and Regulatory RNA). Epigenetic modifications act in concert with transcription factors and play critical roles in B and T cell development and differentiation. Upon antigen encounter by mature B cells in the periphery, alterations of the epigenetic landscape in these lymphocytes are induced by the same stimuli that drive the antibody response. Such alterations instruct B cells to undergo CSR and SHM, as well as differentiation to memory B cells or longlived plasma cells. Inducible histone modifications, together with DNA methylation and miRNAs, modulate the transcriptome, particularly the expression of AID. These inducible B cell–intrinsic epigenetic marks guide the maturation of antibody responses.
For the V(D)J recombination, CSR, and SHM machineries to access their respective DNA targets in the antigen receptor loci, the targeted regions need to be in an open chromatin state, which is associated with transcription and specific patterns of epigenetic modifications. The transcription is mediated by cis-activating elements, such as V and I promoters as well as iEμ and 3′Eα enhancers, and transcription factors specifically recruited by these elements. During transcription elongation, chromatin remodeling generates nucleosome-free regions by repositioning or evicting  nucleosomes or acts more subtly by transiently lifting a loop of DNA off of the nucleosome surface. Transcription elongation results in nucleosome disassembly or disassociation from DNA. DNA freed from repressive associations with nucleosomes is, therefore, amenable to react with factors of the V(D)J recombination, CSR, or SHM machinery. Accordingly, RNA polymerase II is detected at a high density in S regions that will undergo CSR, suggesting that this molecule facilitates recruitment or targeting of CSR factors.
lncRNAs generated by noncoding transcription in the IgH loci have been shown to play an important role in the targeting of the V(D)J recombination and CSR machineries. lncRNAs are evolutionarily conserved noncoding RNA molecules that are longer than 200 nucleotides and located within the intergenic stretches or overlapping antisense transcripts of coding genes . Production of lncRNA transcripts from V(D)J region DNA in Ig or TCR loci can trigger changes in chromatin structure and modulate recombination. In addition, lncRNA transcription targets AID to divergently transcribed loci in B cells. In B cells undergoing CSR, the RNA exosome, a cellular RNA-processing/degradation complex, associates with AID, accumulates on S regions in an AID-dependent fashion, and is required for optimal CSR. RNA exosome-regulated, antisensetranscribed regions of the B cell genome recruit AID and accumulate single- strand DNA structures containing RNA–DNA hybrids. The RNA exosome regulates transcription of lncRNAs that are engaged in long-range DNA interactions to regulate the function of IgH 3′ regulatory region super-enhancer and modulate CSR.

In addition, an lncRNA generated by S region transcription followed by lariat debranching can fold into G-quadruplex structures, which can be directly bound by AID and mediate targeting of AID to S region DNA. A critical role of chromatin accessibility in antibody diversification is emphasized by the fact that though all S regions contain 5′-AGCT-3′ repeats and can, therefore, potentially be targeted by 14-3-3 adaptors for the recruitment of AID to unfold CSR, only the S regions that undergo germline I_H -S-C_H transcription and enrichment of activating histone modification can
be targeted by the CSR machinery, including 14-3-3 and AID.
As a potent mutator, AID is tightly regulated to avoid damages, such as chromosomal translocations, resulting from its dysregulation in both B cells and non-B cells. The expression of Aicda is modulated by changes of Aicda epigenetic status.
Repression of Aicda expression in naïve B cells is mediated by promoter DNA hypermethylation. Upon B cell activation, Aicda DNA is demethylated and the locus becomes enriched in H3K9ac/K14ac and H3K4me3. These epigenetic changes, together with induction of Homeobox protein HoxC4, NF-κB, and other transcription factors, activate gene transcription. Transcription elongation depends on induction of H3K36me3, an intragenic mark of gene activation. miRNAs provide an additional and more important mechanism of modulation of AID expression. miR-155, miR-181b, and miR-361 modulate AID expression by binding to the evolutionarily conserved target sites in the 3′ UTR of AICDA/Aicda mRNA, thereby reducing both AICDA/Aicda mRNA and AID protein levels. These miRNAs likely repress AID in naïve B cells and in B cells that completed SHM and CSR. Histone deacetylase inhibitors (HDIs) can upregulate these miRNAs by increasing histone acetylation, and therefore expression of their host genes, and lead to downregulation of AID expression.
AID targets are predominantly associated within super-enhancers and regulatory clusters, which are enriched in chromatin modifications associated with active enhancers (such as H3K27Ac). They are also associated with marks of active transcription (such as H3K36me3), indicating that these features are universal  mediators of AID recruitment. In both human and mouse B cells, a strong overlap exists between hypermutated genes and superenhancer domains. Chromatin in the target region(s) of V(D)J recombination, CSR, and SHM is also marked by multiple activating histone modifications. One of the most important activating histone modifications, trimethylation of the Lys4 residue of H3 (H3K4me3), is a specific mark of open chromatin in the genome and is highly enriched in V(D)J gene segments and S regions that will undergo V(D)J recombination and CSR, respectively. Concomitant with enrichment of activating histone modifications in those regions, repressive histone modifications, such as H3K9me3 and H3K27me3, are decreased.
The change from a repressive to a permissive chromatin state in targeted Ig loci regions is controlled by the stage of lymphoid differentiation, tissue specificity, and allelic exclusion in a fashion virtually identical to how V(D)J recombination, CSR, and SHM per se are regulated. Transcription and change of combinatorial patterns of histone modifications in those regions are coregulated by cis-activating elements and transcription factors activated by environmental cues, such as cytokines critical for B cell development or specification of Ig isotypes. In addition, the transcription process itself plays a role in the induction (“writing”) of selective histone modifications, as suggested by profoundly decreased H3K4me3 in the TCRα locus downstream of an artificially inserted transcription termination sequence.
According to the histone code hypothesis, combinatorial patterns of histone modifications not only encrypt information on the specification of distinct chromatin states but also increase the complexity of chromatin-interacting effectors (histone code “reading”), thereby determining specific biological information outputs. In V(D)J recombination, RAG2 is a specific reader of H3K4me3, which is enriched in the recombination center, a small region containing J gene segments (and the D gene segments in some cases). This, together with strong RAG1 binding to RSSs, ensures targeting of the RAG1/RAG2 complex to the recombination center. In CSR, a combinatorial histone modification H3K9acS10ph (acetylation of Lys9 and phosphorylation of Ser10 of the same H3 tail) is read by 14-3-3 adaptors, thereby stabilizing 5′-AGCT-3′- bound 14-3-3 on the S regions that will undergo recombination.

Some histone code readers, such as RAG2, can directly mediate enzymatic reactions upon reading histone modifications. Others do not possess intrinsic enzymatic activities and, by virtue of their scaffold functions, instead transduce epigenetic information to downstream enzymatic factors. For instance, 14-3-3 adaptors read H3K9acS10ph (as well as binding to 5′-AGCT-3′ repeats) and, in turn, recruit AID to S-region DNA. Together with elements of the CSR and SHM machinery, such as Rev1 in Ung, these histone code transducers nucleate the assembly of multicomponent complexes through simultaneous interaction with multiple protein and/or nucleic acid ligands via different domains or subunits.

Another potential mechanism of accessibility control is DNA methylation, which occurs mainly at dCs of CpG sites . CpG methylation has an important function in regulating transcription and chromatin structure. It represses gene expression directly by impeding the binding of transacting factors, and indirectly by the recruitment of HDACs through methyl CpG-binding–domain (MBD) family proteins. Differences in methylation status are also correlated with antigen–receptor gene rearrangement and expression. In addition, DNA methylation around the RSS may also regulate V(D)J recombination by directly inhibiting the cleavage activity of the RAG1/RAG2 complex.
Although the density of CpG sites is much lower than overall genome-wide CpG level, increased DNA methylation at these CpG sites results in significantly reduced germline transcription and CSR. The role of DNA hypomethylation in SHM has also been suggested by the finding that only the hypomethylated allele is hypermutated in B cells carrying two nearly identical prerearranged transgenic Igκ alleles, despite comparable transcription of both alleles. DNA demethylation probably facilitates SHM
targeting by promoting H3K9ac/K14ac, H4K8ac, and H3K4me3 histone modifications that are associated with an open chromatin state and are enriched in the V(D)J region.