Uttiya Basu

The AID antibody diversification enzyme is regulated by protein kinase A phosphorylation

Antibodies, which are produced by B-lineage cells, consist of immunoglobulin heavy (IgH) and light (IgL) chains that have amino-terminal variable regions and carboxy-terminal constant regions. In response to antigens, B cells undergo two types of genomic alterations to increase antibody diversity. Affinity for antigen can be increased by introduction of point mutations into IgH and IgL variable regions by somatic hypermutation. In addition, antibody effector functions can be altered by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). Somatic hypermutation and CSR both require the B-cell-specific activation-induced cytidine deaminase protein (AID), which initiates these reactions through its single-stranded (ss)DNA-specific cytidine deaminase activity. In biochemical assays, replication protein A (RPA), a ssDNA-binding protein, associates with phosphorylated AID from activated B cells and enhances AID activity on transcribed double-stranded (ds)DNA containing somatic hypermutation or CSR target sequences. This AID–RPA association, which requires phosphorylation, may provide a mechanism for allowing AID to access dsDNA targets in activated B cells. Here we show that AID from B cells is phosphorylated on a consensus protein kinase A (PKA) site and that PKA is the physiological AID kinase. Thus, AID from non-lymphoid cells can be functionally phosphorylated by recombinant PKA to allow interaction with RPA and promote deamination of transcribed dsDNA substrates. Moreover, mutation of the major PKA phosphorylation site of AID preserves ssDNA deamination activity, but markedly reduces RPA-dependent dsDNA deamination activity and severely impairs the ability of AID to effect CSR in vivo. We conclude that PKA has a critical role in post-translational regulation of AID activity in B cells.

The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells

The transcription factor BATF controls the differentiation of interleukin 17 (IL-17)-producing helper T cells (TH17 cells) by regulating expression of the transcription factor RORγt itself and RORγt target genes such as Il17. Here we report the mechanism by which BATF controls in vivo class-switch recombination (CSR). In T cells, BATF directly controlled expression of the transcription factors Bcl-6 and c-Maf, both of which are needed for development of follicular helper T cells (TFH cells). Restoring TFH cell activity to Batf−/− T cells in vivo required coexpression of Bcl-6 and c-Maf. In B cells, BATF directly controlled the expression of both activation-induced cytidine deaminase (AID) and of germline transcripts of the intervening heavy-chain region and constant heavy-chain region (IH-CH). Thus, BATF functions at multiple hierarchical levels in two cell types to globally regulate switched antibody responses in vivo.

The RNA Exosome Targets the AID Cytidine Deaminase to Both Strands of Transcribed Duplex DNA Substrates

Activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) heavy-chain (IgH) class switch recombination (CSR) and Ig variable region somatic hypermutation (SHM) in B lymphocytes by deaminating cytidines on template and nontemplate strands of transcribed DNA substrates. However, the mechanism of AID access to the template DNA strand, particularly when hybridized to a nascent RNA transcript, has been an enigma. We now implicate the RNA exosome, a cellular RNA-processing/degradation complex, in targeting AID to both DNA strands. In B lineage cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions in an AID-dependent fashion, and is required for optimal CSR. Moreover, both the cellular RNA exosome complex and a recombinant RNA exosome core complex impart robust AID- and transcription-dependent DNA deamination of both strands of transcribed SHM substrates in vitro. Our findings reveal a role for noncoding RNA surveillance machinery in generating antibody diversity.

Integrity of the AID serine-38 phosphorylation site is critical for class switch recombination and somatic hypermutation in mice.

Activation-induced cytidine deaminase (AID) is a single-stranded (ss) DNA-specific cytidine deaminase that initiates Ig heavy chain (IgH) class switch recombination (CSR) and Ig somatic hypermutation (SHM) by deaminating cytidines within, respectively, IgH switch (S) regions and Ig variable region (V) exons. AID that is phosphorylated on serine residue 38 interacts with replication protein A (RPA), a ssDNA binding protein, to promote deamination of transcribed double-stranded DNA in vitro, which, along with other evidence, suggests that AID may similarly gain access to transcribed S regions and V exons in vivo. However, the physiological role of AID phosphorylation at serine residue 38 (S38), and even the requirement for the S38 residue, with respect to CSR or SHM has been debated. To address this issue, we used gene targeting to generate an endogenous mouse AID locus that produces AID in which S38 is substituted with alanine (AIDS38A), a mutant form of AID that retains similar catalytic activity on ssDNA as WT AID (AIDWT). B cells homozygous for the AIDS38A mutation show substantially impaired CSR and SHM, correlating with inability of AIDS38A to interact with endogenous RPA. Moreover, mice haploinsufficient for AIDS38A have even more severely impaired CSR when compared with mice haploinsufficient for AIDWT, with CSR levels reduced to nearly background levels. These results unequivocally demonstrate that integrity of the AID S38 phosphorylation site is required for normal CSR and SHM in mice and strongly support a role for AID phosphorylation at S38 and RPA interaction in regulating CSR and SHM.

Noncoding RNA transcription targets AID to divergently transcribed loci in B cells

The vast majority of the mammalian genome has the potential to express noncoding RNA (ncRNA). The 11-subunit RNA exosome complex is the main source of cellular 3′–5′ exoribonucleolytic activity and potentially regulates the mammalian noncoding transcriptome. Here we generated a mouse model in which the essential subunit Exosc3 of the RNA exosome complex can be conditionally deleted. Exosc3-deficient B cells lack the ability to undergo normal levels of class switch recombination and somatic hypermutation, two mutagenic DNA processes used to generate antibody diversity via the B-cell mutator protein activation-induced cytidine deaminase (AID). The transcriptome of Exosc3-deficient B cells has revealed the presence of many novel RNA exosome substrate ncRNAs. RNA exosome substrate RNAs include xTSS-RNAs, transcription start site (TSS)-associated antisense transcripts that can exceed 500 base pairs in length and are transcribed divergently from cognate coding gene transcripts. xTSS-RNAs are most strongly expressed at genes that accumulate AID-mediated somatic mutations and/or are frequent translocation partners of DNA double-strand breaks generated at Igh in B cells. Strikingly, translocations near TSSs or within gene bodies occur over regions of RNA exosome substrate ncRNA expression. These RNA exosome-regulated, antisense-transcribed regions of the B-cell genome recruit AID and accumulate single-strand DNA structures containing RNA–DNA hybrids. We propose that RNA exosome regulation of ncRNA recruits AID to single-strand DNA-forming sites of antisense and divergent transcription in the B-cell genome, thereby creating a link between ncRNA transcription and overall maintenance of B-cell genomic integrity.

Regulation of AID, the B-cell genome mutator

The mechanisms by which B cells somatically engineer their genomes to generate the vast diversity of antibodies required to challenge the nearly infinite number of antigens that immune systems encounter are of tremendous clinical and academic interest. The DNA cytidine deaminase activation-induced deaminase (AID) catalyzes two of these mechanisms: class switch recombination (CSR) and somatic hypermutation (SHM). Recent discoveries indicate a significant promiscuous targeting of this B-cell mutator enzyme genome-wide. Here we discuss the various regulatory elements that control AID activity and prevent AID from inducing genomic instability and thereby initiating oncogenesis.

Mutations, kataegis and translocations in B cells: understanding AID promiscuous activity

As B cells engage in the immune response, they express activation-induced cytidine deaminase (AID) to initiate the hypermutation and recombination of immunoglobulin genes, which are crucial processes for the efficient recognition and disposal of pathogens. However, AID must be tightly controlled in B cells to minimize off-target mutations, which can drive chromosomal translocations and the development of B cell malignancies, such as lymphomas. Recent genomic and biochemical analyses have begun to unravel the mechanisms of how AID-mediated deamination is targeted outside immunoglobulin genes. Here, we discuss the transcriptional and topological features that are emerging as key drivers of AID promiscuous activity.

Evolution of Phosphorylation-Dependent Regulation of Activation Induced Cytidine Deaminase

Interaction of activation-induced cytidine deaminase (AID) with replication protein A (RPA) has been proposed to promote AID access to transcribed double-stranded (ds) DNA during immunoglobulin light chain and heavy chain class switch recombination (CSR). Mouse AID (mAID) interaction with RPA and transcription-dependent dsDNA deamination in vitro requires protein kinase A (PKA) phosphorylation at serine 38 (S38), and normal mAID CSR activity depends on S38. However, zebrafish AID (zAID) catalyzes robust CSR in mouse cells despite lacking an S38-equivalent PKA site. Here, we show that aspartate 44 (D44) in zAID provides similar in vitro and in vivo functionality as mAID S38 phosphorylation. Moreover, introduction of a PKA site into a zAID D44 mutant made it PKA dependent for in vitro activities and restored normal CSR activity. Based on these findings, we generated mAID mutants that similarly function independently of S38 phosphorylation. Comparison of bony fish versus amphibian and mammalian AIDs suggests evolutionary divergence from constitutive to PKA-regulated RPA/AID interaction.

The Saccharomyces cerevisiae 60 S Ribosome Biogenesis Factor Tif6p Is Regulated by Hrr25p-mediated Phosphorylation

The biosynthesis of 60 S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eIF6. This protein is necessary for the formation of 60 S ribosomal subunits because it is essential for the processing of 35 S pre-rRNA to the mature 25 S and 5.8 S rRNAs. In the present work, using molecular genetic and biochemical analyses, we show that Hrr25p, an isoform of yeast casein kinase I, phosphorylates Tif6p both in vitro and in vivo. Tryptic phosphopeptide mapping of in vitro phosphorylated Tif6p by Hrr25p and 32P-labeled Tif6p isolated from yeast cells followed by mass spectrometric analysis revealed that phosphorylation occurred on a single tryptic peptide at Ser-174. Sucrose gradient fractionation and coimmunoprecipitation experiments demonstrate that a small but significant fraction of Hrr25p is bound to 66 S preribosomal particles that also contain bound Tif6p. Depletion of Hrr25p from a conditional yeast mutant that fails to phosphorylate Tif6p was unable to process pre-rRNAs efficiently, resulting in significant reduction in the formation of 25 S rRNA. These results along with our previous observations that phosphorylatable Ser-174 is required for yeast cell growth and viability, suggest that Hrr25p-mediated phosphorylation of Tif6p plays a critical role in the biogenesis of 60 S ribosomal subunits in yeast cells.

Transcriptomics Identify CD9 as a Marker of Murine IL-10-Competent Regulatory B Cells

Regulatory B cells (Breg) have immune suppressive functions in various autoimmune/inflammation models and diseases and are found to be enriched in diverse B cell subsets. The lack of a unique marker or set of markers efficiently identifying Breg cells impedes detailed investigation into their origin, development, and immunological roles. Here, we perform transcriptome analysis of IL-10-expressing B cells to identify key regulators for Breg biogenesis and function and identify CD9, a tetraspanin-family transmembrane protein, as a key surface marker for most mouse IL-10(+) B cells and their progenitors. CD9 plays a role in the suppressive function of IL-10(+) B cells in ex vivo T cell proliferation assays through a mechanism that is dependent upon B/T cell interactions. CD9(+) B cells also demonstrate inhibition of Th1-mediated contact hypersensitivity in an in vivo model system. Taken together, our findings implicate CD9 in the immunosuppressive activity of regulatory B cells.