Loop extrusion promotes an alternate pathway for isotype switching.

Class-switch recombination (CSR) involves replacement of the Cµ constant region with another downstream CH region. CSR is initiated by activation-induced cytidine deaminase (AID)-mediated DNA breaks that are targeted to transcriptionally active switch (S) regions. S region promoters (Prs) direct synapsis by associating with the Eµ and 3'Eα enhancers that jointly anchor a chromatin loop. We report that asymmetric loop extrusion allows 3'Eα to track along the locus and form Pr-Pr-E interactions that mediate CSR between downstream S regions, followed by switching to donor Sµ. This alternative pathway bypasses sequential switching and creates immunoglobulin (Ig)E+ B cells in the absence of IgG1 expression. Based on the analysis of diagnostic CSR products in B cell subsets, we identify a BCR-negative cell intermediate that is pivotal to efficient CSR.

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

The activation-induced cytidine deaminase (AID) initiates somatic hypermutation, class-switch recombination, and gene conversion of immunoglobulin genes. In vitro, AID has been shown to target single-stranded DNA, relaxed double-stranded DNA, when transcribed, or supercoiled DNA. To simulate the in vivo situation more closely, we have introduced two copies of a nucleosome positioning sequence, MP2, into a supercoiled AID target plasmid to determine where around the positioned nucleosomes (in the vicinity of an ampicillin resistance gene) cytidine deaminations occur in the absence or presence of transcription. We found that without transcription nucleosomes prevented cytidine deamination by AID. However, with transcription AID readily accessed DNA in nucleosomes on both DNA strands. The experiments also showed that AID targeting any DNA molecule was the limiting step, and they support the conclusion that once targeted to DNA, AID acts processively in naked DNA and DNA organized within transcribed nucleosomes.

Expression of AID transgene is regulated in activated B cells but not in resting B cells and kidney.

Activation-induced DNA cytidine deaminase (AID) is required for somatic hypermutation (SHM) and efficient class switch recombination (CSR) of immunoglobulin (Ig) genes. We created AID-transgenic mice that express AID ubiquitously under the control of a beta-actin promoter. When crossed with AID-/- mice, the AID-transgenic,AID-/- mice carried out SHM and CSR, showing that the AID transgenes were functional. However, the frequencies of SHM in V- and switch-regions, and CSR were reduced compared to those in a wild type AID background. Several criteria suggested that the inefficiency of SHM was due to reduced AID activity, rather than lack of recruiting error-prone DNA repair. High levels of AID mRNA were produced in resting B cells and kidney, cells that do not express AID in wild type mice. Compared with these cells, activated B cells expressed about an order of magnitude less AID mRNA suggesting that there may be a post-transcriptional mechanism that regulates AID mRNA levels in professional AID producers but not other cells. The AID protein expressed in resting B cells and kidney was phosphorylated at serine-38. Despite this modification, known to enhance AID activity, resting B cells did not undergo SHM. Apparently, the large amounts of AID in resting B cells are not targeted to Ig genes in vivo, in contrast to findings in vitro.

Activation-induced cytidine deaminase acts on double-strand breaks in vitro.

Activation-induced cytidine deaminase (AID) is likely responsible for DNA cytidine deamination, although it may also act as an RNA deaminase. It functions on single-stranded DNA, the non-template strand in double-stranded DNA during transcription, or both strands in supercoiled DNA. To ask whether AID is able to deaminate cytidine at DNA breaks, plasmids, containing a SnaBI site (TAC downward arrowGTA) that forms blunt ends after digestion with SnaBI, were generated. If AID deaminates cytidine at the upstream blunt end, the ATG start codon in either of two drug resistance genes will be regenerated after ligation and replication in UDG-null E. coli cells. This study shows that AID targets cytidine at the break. The extent of deamination activity beyond the break is correlated with the base composition in the break region. If the break region is A, T-rich, C > T transitions are extensive. However, when the break region is not A, T-rich, mutations are mainly restricted to the break, similar to findings in vivo. The results indicate that AID has activity on double strand breaks (DSBs). Based on previous and current findings, a somatic hypermutation (SHM) model is proposed, in which collision between the transcription apparatus and the replication fork generates DSBs. After AID acts on break ends, the error-prone DNA repair machinery fixes and creates mutations.

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.

[Study of gastric function after esophagectomy and cardiectomy with vagus nerve preserved and reconstruction of gastric funds in patients with esophageal and cardiac cancer].

OBJECTIVE: To study the gastric function after esophagectomy and cardiectomy with vagus nerve preserved and reconstruction of gastric funds (VPRG)in patients with esophageal cancer (EC) and cardiac cancer (CC). METHODS: Sixty-eight patients with early or middle staged EC or CC received esophagectomy and cardiectomy with vagus nerve preserved and reconstruction of gastric funds (VPRG),while other 68 patients esophagectomy and cardiectomy with vagus nerve severed and no reconstruction of gastric funds (VSNG) as control. The symptoms,the pressure of the residual esophagus and thoracic stomach, 24-hour pH monitoring, mean basic gastric acid output, gastric emptying time of the intrathoracic stomach,fasting serum gastrin level, fibreoptic endoscopic results were compared before and after operation between the two groups. RESULTS: The patients with VPRG had less symptoms after operation than those with VSNG such as anorexia, belch, reflux, heartburn, nausea, diarrhea, postcibal satiety (P< 0.01). In VPRG group,compared with the results before operation,there were no significant differences in 24-hour pH monitoring,the mean basic gastric acid output, the fasting serum gastrin level,the gastric emptying time of intrathoracic stomach one month and one year after operation (both P > 0.05). The pressure of the residual esophagus above the anastomosis in VPRG group was significantly higher than that in VSNG group (both P< 0.05). Fibreoptic endoscopic examination revealed higher incidences of postoperative atrophic gastritis and reflux esophagitis in VPRG group one month and one year after operation than those in VSNG group (P< 0.01). CONCLUSION: Preservation of the vagus nerve and reconstruction of gastric funds after esophagectomy and cardiectomy for esophageal and cardiac cancer can prevent digestive disorder and improve the life quality of the patients.

Targeting of the activation-induced cytosine deaminase is strongly influenced by the sequence and structure of the targeted DNA.

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 approximately 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.

Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled.

The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination of Ig genes. It has been shown that in vitro, AID protein deaminates C in single-stranded DNA or the coding-strand DNA that is being transcribed but not in double-stranded DNA. However, in vivo, both DNA strands are mutated equally during SHM. We show that AID efficiently deaminates C on both DNA strands of a supercoiled plasmid, acting preferentially on SHM hotspot motifs. However, this DNA is not targeted by AID when it is relaxed after treatment with topoisomerase I, and thus, supercoiling plays a crucial role for AID targeting to this DNA. Most of the mutations are in negatively supercoiled regions, suggesting a mechanism of AID targeting in vivo. During transcription the DNA sequences upstream of the elongating RNA polymerase are negatively supercoiled, and this transient change in DNA topology may allow AID to access both DNA strands.

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.