The transcription factor E2A can bind to and cleave single-stranded immunoglobulin heavy chain locus DNA.

Class switch recombination (CSR) changes the constant region of the immunoglobulin heavy chain (IgH), and somatic hypermutation (SH) introduces point mutations in the variable regions of the antibody genes. Both these processes that optimize antibody responses of B lymphocytes are initiated by the enzyme Activation Induced cytidine Deaminase (AID). Here we have searched for CSR or SH coupled activities of the transcription factor E2A, since E2A is in a complex with AID and the transcription factors PAX5, ETS1 and IRF4 on key sequences of the Igh locus in B lymphocytes activated to CSR and SH. We report that E2A in contrast to other described transcription factors binds sequence specifically also to single-stranded DNA. The binding of E2A to single-stranded DNA has a strong sequence preference for one strand of a site in the intronic enhancer of the Igh locus. Furthermore, E2A was also found to cleave single-stranded DNA. The sequence profile of substrates cleaved by E2A is coupled to the sequences of substrates and products of AID, suggesting that E2A has a role not only in targeting of AID to switch regions and SH parts of antibody genes but also in cleavage of DNA at these sites.

ETS1 and PAX5 transcription factors recruit AID to Igh DNA.

B lymphocytes optimize antibody responses by class switch recombination (CSR), which changes the expressed constant region exon of the immunoglobulin heavy chain (IgH), and by somatic hypermutation (SH) that introduces point mutations in the variable regions of the antibody genes. Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates both these antibody diversification processes by deaminating cytosine to uracil. Here we asked the question if transcription factors can mediate the specific targeting of the antibody diversification by recruiting AID. We have recently reported that AID is together with the transcription factors E2A, PAX5 and IRF4 in a complex on key sequences of the Igh locus. Here we report that also ETS1 is together with AID in this complex on key sequences of the Igh locus in splenic B cells of mice. Furthermore, we show that both ETS1 and PAX5 can directly recruit AID to DNA sequences from the Igh locus with the specific binding site for the transcription factor. Taken together, our findings support the notion of a targeting mechanism for the selective diversification of antibody genes with limited genome wide mutagenesis by recruitment of AID by PAX5 and ETS1 in a transcription factor complex.

Regulation of the DNA Repair Complex during Somatic Hypermutation and Class-Switch Recombination.

B lymphocytes optimize Ab responses by somatic hypermutation (SH), which introduces point mutations in the variable regions of the Ab genes and by class-switch recombination (CSR), which changes the expressed C region exon of the IgH. These Ab diversification processes are initiated by the deaminating enzyme activation-induced cytidine deaminase followed by many DNA repair enzymes, ultimately leading to deletions and a high mutation rate in the Ab genes, whereas DNA lesions made by activation-induced cytidine deaminase are repaired with low error rate on most other genes. This indicates an advanced regulation of DNA repair. In this study, we show that initiation of Ab diversification in B lymphocytes of mouse spleen leads to formation of a complex between many proteins in DNA repair. We show also that BCR activation, which signals the end of successful SH, reduces interactions between some proteins in the complex and increases other interactions in the complex with varying kinetics. Furthermore, we show increased localization of SH- and CSR-coupled proteins on switch regions of the Igh locus upon initiation of SH/CSR and differential changes in the localization upon BCR signaling, which terminates SH. These findings provide early evidence for a DNA repair complex or complexes that may be of functional significance for carrying out essential roles in SH and/or CSR in B cells.

Regulated localization of an AID complex with E2A, PAX5 and IRF4 at the Igh locus.

Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates somatic hypermutation (SH) and class switch recombination (CSR) by deaminating cytosine to uracil. The targeting of AID and therefore SH and CSR to Ig genes is a central process of the immune system, but the trans-acting factors mediating the specific targeting have remained elusive. Here we show that defective calmodulin inhibition of the transcription factor E2A after activation of the B cell receptor (BCR) leads to reduced BCR, IL4 plus CD40 ligand stimulated CSR to IgE and instead CSR to other Ig classes. AID that initiates CSR is shown to be in a complex with the transcription factors E2A, PAX5 and IRF4 on key sequences of the Igh locus. Calmodulin shows proximity with each of them after BCR stimulation. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks these reductions. AID binds directly to the bHLH domain of E2A and to the PD domain of PAX5. E2A, AID, PAX5 and IRF4 are components of a CSR complex that is redistributed on the Igh locus by BCR signaling through calmodulin binding.

Allelic exclusion of IgH through inhibition of E2A in a VDJ recombination complex.

A key feature of the immune system is the paradigm that one lymphocyte has only one Ag specificity that can be selected for or against. This requires that only one of the alleles of genes for AgR chains is made functional. However, the molecular mechanism of this allelic exclusion has been an enigma. In this study, we show that B lymphocytes with E2A that cannot be inhibited by calmodulin are dramatically defective in allelic exclusion of the IgH locus. Furthermore, we provide data supporting that E2A, PAX5, and the RAGs are in a VDJ recombination complex bound to key sequences on the Igh gene. We show that pre-BCR activation releases the VDJ recombination complex through calmodulin binding to E2A. We also show that pre-BCR signaling downregulates several components of the recombination machinery, including RAG1, RAG2, and PAX5, through calmodulin inhibition of E2A.

CaMKII targets Bcl10 in T-cell receptor induced activation of NF-κB.

Recognition of antigen by T- or B-cell receptors leads to formation of an immunological synapse and initiation of signalling events that collaborate to determine the nature of the adaptive immune response. Activation of NF-κB transcription factors has a key role in regulation of numerous genes with important functions in immune responses and inflammation and is of great importance for lymphocyte activation and differentiation. The activation of NF-κB depends on changes in intracellular Ca(2+) levels, and both calmodulin (CaM) and a CaM-dependent kinase, CaMKII, help regulate NF-κB activation after T-cell receptor (TCR) stimulation, but the mechanisms are not well characterized. Here we have analyzed the functional role of CaMKII in the signalling pathway from the TCR to activation of IKK, the kinase that phosphorylates the NF-κB inhibitor IκB. We show that CaMKII is recruited to the immunological synapse where it interacts with and phosphorylates the signalling adaptor protein Bcl10. Furthermore, phosphorylation of the CARD domain of Bcl10 by CaMKII regulates the interactions within the important Carma1, Bcl10, Malt1 signalling complex and the essential signal induced ubiquitinations of Bcl10 and IKKγ. We propose a novel mechanism whereby Ca(2+) signals can be integrated at the immunological synapse through CaMKII-dependent phosphorylation of Bcl10.

Interaction of calmodulin with Bcl10 modulates NF-kappaB activation.

Calcium signals resulting from antigen receptor activation are important in determining the responses of a T or B lymphocyte to an antigen. Calmodulin (CaM), a multi-functional sensor of intracellular calcium (Ca(2+)) signals in cells, is required in the pathway from the T cell receptor (TCR) to activation of the key transcription factor NF-kappaB. Here we searched for a partner in direct interaction with CaM in the pathway, and found that CaM interacts specifically with the signaling adaptor Bcl10. The binding is Ca(2+) dependent and of high affinity, with a K(d) of approximately 160 nM. Proximity of CaM and Bcl10 in vivo is induced by increases in the intracellular Ca(2+) level. The interaction is localized to the CARD domain of Bcl10, which interacts with the CARD domain of the upstream signaling partner Carma1. Binding of CaM to Bcl10 is shown to inhibit the ability of Bcl10 to interact with Carma1, an interaction that is required for signaling from the TCR to NF-kappaB. Furthermore, a mutant of Bcl10 with reduced binding to CaM shows increased activation of an NF-kappaB reporter, which is further enhanced by activating stimuli. We propose a novel mechanism whereby the Ca(2+) sensor CaM regulates T cell responses to antigens by binding to Bcl10, thereby modulating its interaction with Carma1 and subsequent activation of NF-kappaB.

The RUNX1 Runt domain at 1.25A resolution: a structural switch and specifically bound chloride ions modulate DNA binding.

The evolutionarily conserved Runt homology domain is characteristic of the RUNX family of heterodimeric eukaryotic transcription factors, including RUNX1, RUNX2 and RUNX3. The genes for RUNX1, also termed acute myeloid leukemia protein 1, AML1, and its dimerization partner core-binding factor beta, CBFbeta, are essential for hematopoietic development and are together the most common targets for gene rearrangements in acute human leukemias. Here, we describe the crystal structure of the uncomplexed RUNX1 Runt domain at 1.25A resolution and compare its conformation to previously published structures in complex with DNA, CBFbeta or both. We find that complex formation induces significant structural rearrangements in this immunoglobulin (Ig)-like DNA-binding domain. Most pronounced is the movement of loop L11, which changes from a closed conformation in the free Runt structure to an open conformation in the CBFbeta-bound and DNA-bound forms. This transition, which we refer to as the S-switch, and accompanying structural movements that affect other parts of the Runt domain are crucial for sustained DNA binding. The closed to open transition can be induced by CBFbeta alone; suggesting that one role of CBFbeta is to trigger the S-switch and to stabilize the Runt domain in a conformation enhanced for DNA binding.A feature of the Runt domain hitherto unobserved in any Ig-like DNA-binding domain is the presence of two specifically bound chloride ions. One chloride ion is coordinated by amino acid residues that make direct DNA contact. In a series of electrophoretic mobility-shift analyses, we demonstrate a chloride ion concentration-dependent stimulation of the DNA-binding activity of Runt in the physiological range. A comparable DNA-binding stimulation was observed for negatively charged amino acid residues. This suggests a regulatory mechanism of RUNX proteins through acidic amino acid residues provided by activation domains during cooperative interaction with other transcription factors.