Hong Ming Shen

The E Box Motif CAGGTG Enhances Somatic Hypermutation without Enhancing Transcription

The frequency of somatic hypermutations of an Ig kappa transgene with an artificial test insert, RS, is at least 4-fold higher than that of three related transgenes. The four transgenes differ only in the sequence of a 96 bp insert within the variable region. RS is hypermutable over the total 625 nucleotides of the variable/joining region. The RS insert contains two CAGGTG sequences, potential binding sites for basic helix-loop-helix proteins. Changing CAGGTG to AAGGTG reduces the mutability to that of the non-RS transgenes without altering the mutation pattern. The CAGGTG motif enhances somatic hypermutation without enhancing transcription. A DNA probe containing the two CAGGTG sites, but not AAGGTG, binds E47 and gives rise to two specific EMSA bands with nuclear extracts from mutating cells. Possible actions of this enhancer of somatic hypermutation are discussed.

Cis-acting sequences that affect somatic hypermutation of Ig genes

Summary: We review our studies on the mechanism of somatic hypermutation of immunoglobulin genes. Most experiments were carried out using Ig transgenes. We showed in these experiments that all required cisacting elements are present within the 10–16 kb of a cransgene. Only the Ig variable region and its proximate flanks are mutated, not the constant region. Several Ig gene enhancers are permissive for somatic mutation. Association of the enhancer with its natural Ig promoter is not necessary. However, the mutation process seems specific for Ig genes. No mutations were found in housekeeping genes from cells with high levels of somatic hypermutation of their Ig genes. The Ig enhancers may provide the Ig gene specificity. An exception may he the BCL6 gene, which was mutated in but not hut not in mouse B cells

Somatic Hypermutation and Class Switch Recombination in Msh6−/−Ung−/− Double-Knockout Mice

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytosine deaminase (AID). The uracil, and potentially neighboring bases, are processed by error-prone base excision repair and mismatch repair. Deficiencies in Ung, Msh2, or Msh6 affect SHM and CSR. To determine whether Msh2/Msh6 complexes which recognize single-base mismatches and loops were the only mismatch-recognition complexes required for SHM and CSR, we analyzed these processes in Msh6−/−Ung−/− mice. SHM and CSR were affected in the same degree and fashion as in Msh2−/−Ung−/− mice; mutations were mostly C,G transitions and CSR was greatly reduced, making Msh2/Msh3 contributions unlikely. Inactivating Ung alone reduced mutations from A and T, suggesting that, depending on the DNA sequence, varying proportions of A,T mutations arise by error-prone long-patch base excision repair. Further, in Msh6−/−Ung−/− mice the 5′ end and the 3′ region of Ig genes was spared from mutations as in wild-type mice, confirming that AID does not act in these regions. Finally, because in the absence of both Ung and Msh6, transition mutations from C and G likely are “footprints” of AID, the data show that the activity of AID is restricted drastically in vivo compared with AID in cell-free assays.

Somatic Hypermutation of Immunoglobulin Genes is Linked to Transcription

Immunoglobulin (Ig) genes are rearranged in pre-B cells. Pre-B cells that express Ig heavy (H) and light (L) chain genes whose V(D)J recombination results in a functional reading frame mature into B cells that exit the bone marrow. The V(D)J recombination process creates a large repertoire of different variable regions from a restricted pool of germline genes. Additional variablity arises during the process of somatic hypermutation in mature B cells proliferating in germinal centers of lymphoid organs (reviewed in French et al. 1989). B cells that have mutated to express high-affinity antibodies are selected and develop into plasma cells or memory cells. B cells with mutations that decrease the affinity of the expressed Igs or that prevent Ig expression die by apoptosis. The somatic point mutations are located within the variable region and their proximate upstream and downstream flanks, but not generally within the constant region.

Targeting of the Activation-Induced Cytosine Deaminase Is Strongly Influenced by the Sequence and Structure of the Targeted DNA

ABSTRACT Activation-induced deaminase (AID) initiates immunoglobulin somatic hypermutation (SHM). Since in vitro AID was shown to deaminate cytosines on single-stranded DNA or the nontranscribed strand, it remained a puzzle how in vivo AID targets both DNA strands equally. Here we investigate the roles of transcription and DNA sequence in cytosine deamination. Strikingly different results are found with different substrates. Depending on the target sequence, the transcribed DNA strand is targeted as well as or better than the nontranscribed strand. The preferential targeting is not related to the frequency of AID hot spots. Comparison of cytosine deamination by AID and bisulfite shows different targeting patterns suggesting that AID may locally unwind the DNA. We conclude that somatic hypermutation on both DNA strands is the natural outcome of AID action on a transcribed gene; furthermore, the DNA sequence or structure and topology play major roles in targeting AID in vitro and in vivo. On the other hand, the lack of mutations in the first ∼100 nucleotides and beyond about 1 to 2 kb from the promoter of immunoglobulin genes during SHM must be due to special conditions of transcription and chromatin in vivo.

Attracting AID to targets of somatic hypermutation

The process of somatic hypermutation (SHM) of immunoglobulin (Ig) genes requires activation-induced cytidine deaminase (AID). Although mistargeting of AID is detrimental to genome integrity, the mechanism and the cis-elements responsible for targeting of AID are largely unknown. We show that three CAGGTG cis-elements in the context of Ig enhancers are sufficient to target SHM to a nearby transcribed gene. The CAGGTG motif binds E47 in nuclear extracts of the mutating cells. Replacing CAGGTG with AAGGTG in the construct without any other E47 binding site eliminates SHM. The CA versus AA effect requires AID. CAGGTG does not enhance transcription, chromatin acetylation, or overall target gene activity. The other cis-elements of Ig enhancers alone cannot attract the SHM machinery. Collectively with other recent findings, we postulate that AID targets all genes expressed in mutating B cells that are associated with CAGGTG motifs in the appropriate context. Ig genes are the most highly mutated genes, presumably because of multiple CAGGTG motifs within the Ig genes, high transcription activity, and the presence of other cooperating elements in Ig enhancers.

The inactivation of the XP-C gene does not affect somatic hypermutation or class switch recombination of immunoglobulin genes.

The mechanism of somatic hypermutation of immunoglobulin genes is not known, but appears to be linked to transcription and perhaps DNA repair. In order to determine if global DNA repair or the repair of the nontranscribed DNA strand is required for somatic mutation, we have analysed mice whose XP-C gene was inactivated by homologous recombination. Our study shows that hypermutation occurs in XP-C knockout mice with a normal frequency, suggesting that the XP-C gene product is not required for somatic hypermutation. Furthermore, we found that Ig gene switch recombination also is normal in these mice.

Somatic hypermutation: processivity of the cytosine deaminase AID and error-free repair of the resulting uracils.

The somatic hypermutation (SHM) process is initiated in activated B lymphocytes by the cytosine DNA deaminase AID creating uracils that are further treated by DNA replication and/or error-prone base excision repair (BER) and/or error-prone mismatch repair (MMR), resulting in mutations at the targeted cytosines, as well as at all four nucleotides neighboring the AID-induced uracil. In this analysis we investigate two issues that are specific to SHM, the processivity of AID in vivo in vertebrate cells versus in cell free assays, and the error-prone versus error-free repair of the AID-induced uracils. Compilation of published data shows that AID is highly processive in vitro, but shows little, although apparently real, processivity in vivo. We postulate that the combined effects of chromatin and associated transcription factors prevent AID from migrating along extensive tracks in vivo. The comparison of mutation loads in Ig genes at cytosines in wild type mice with those in mice with a combined Ung/MMR-deficiency suggests that Ig genes in wild type mice undergo error-free repair of over 47% of the uracils originally created by AID. The uracil glycosylase Ung which is involved in the error-prone repair during SHM is also involved in the error-free repair.

The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

1. 1. Shen, 2. et al . 2009. J. Exp. Med. doi: 10.1084/jem.20082678 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20082678%26rft_id%253Dinfo%253Apmid%252F19380635%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%