The Mismatch Repair Pathway Functions Normally at a non-AID Target in Germinal Center B cells

Deficiency in Msh2, a component of the mismatch repair (MMR) system, leads to a ~10-fold increase in the mutation frequency in most tissues. By contrast, Msh2-deficiency in germinal center (GC) B cells decreases the mutation frequency at the IgH V-region, as a dU:dG mismatch produced by AID initiates modifications by MMR resulting in mutations at nearby A:T basepairs. This raises the possibility that GC B cells express a factor that converts MMR into a globally mutagenic pathway. To test this notion, we investigated whether MMR corrects mutations in GC B cells at a gene not mutated by AID. We found that GC B cells accumulate 5-times more mutations than follicular B cells. Notably, the mutation frequency was ~10 times higher in Msh2-/- compared to wildtype GC B cells. These results show that in GC B cells MMR functions normally at an AID-insensitive gene.MAS

Elevated incidence of polyp formation in APC(Min/⁺)Msh2⁻/⁻ mice is independent of nitric oxide-induced DNA mutations.

Gut microbiota has been linked to a number of human diseases including colon cancer. However, the mechanism through which gut bacteria influence colon cancer development and progression remains unclear. Perturbation of the homeostasis between the host immune system and microbiota leads to inflammation and activation of macrophages which produce large amounts of nitric oxide that acts as a genotoxic effector molecule to suppress bacterial growth. However, nitric oxide also has genotoxic effects to host cells by producing mutations that can predispose to colon cancer development. The major DNA lesions caused by nitric oxide are 8oxoG and deamination of deoxycytosine bases. Cellular glycosylases that belong to the base excision repair pathway have been demonstrated to repair these mutations. Recent evidence suggests that the mismatch repair pathway (MMR) might also repair nitric oxide-induced DNA damage. Since deficiency in MMR predisposes to colon cancer, we hypothesized that MMR-deficient colon epithelial cells are incapable of repairing nitric-oxide induced genetic lesions that can promote colon cancer. Indeed, we found that the MMR pathway repairs nitric oxide-induced DNA mutations in cell lines. To test whether nitric oxide promotes colon cancer, we genetically ablated the inducible nitric oxide synthase (iNOS) or inhibited iNOS activity in the APC(Min/+)Msh2(-/-) mouse model of colon cancer. However, despite the fact that nitric oxide production was strongly reduced in the colon using both approaches, colon cancer incidence was not affected. These data show that nitric oxide and iNOS do not promote colon cancer in APC(Min/+)Msh2(-/-) mice

Nitric oxide induces DNA mutations that are repaired by the MMR pathway.

<p>(A) Measurements of the amounts of nitric oxide in culture medium generated by various concentrations of SNAP or by macrophages that were stimulated by LPS and IFN-γ. (B) The mutant frequency at the <i>lac</i>I gene in Msh2^+/− (WT, n = 3) and Msh2^−/− (n = 3) SNAP-treated or non-treated macrophages was measured by the Big Blue mutagenesis screen. Bone marrow-derived macrophages were cultured in presence of 150 µM SNAP for 20 hrs. Genomic DNA was isolated and the mutant frequency at <i>lac</i>I gene was calculated. (C) Mutation spectrum in SNAP-treated macrophages as determined by sequencing analysis. The C:G → T:A transitions are shown. The tranversion mutations include C:G → A:T, C:G → G:C, T:A → G:C and T:A → A:T substitutions. The insertions and deletions are presents as other mutations. (D) The mutant frequency at the <i>hprt</i> gene in Msh2^+/− (WT, n = 8) and Msh2^−/− (n = 8) SNAP-treated or non-treated Pre-B cells. (E) The mutant frequency at the <i>hprt</i> gene in SW680 (n = 8) and DLD-1 (n = 8) SNAP-treated or non-treated human colorectal cancer cell lines.</p

Characteristics of the mutations within the <i>lacI</i> gene in Msh2 WT and Msh2^−/− macrophages that were SNAP-treated or untreated.

<p>Characteristics of the mutations within the <i>lacI</i> gene in Msh2 WT and Msh2^−/− macrophages that were SNAP-treated or untreated.</p

iNOS-deficiency does not impact polyp number in APC^Min/+MSH2

<p>^−/−<b>mice.</b> Polyp count in the small intestine (A) or in the colon (B) of APC^Min/+MSH2^+/− (A^+/−M^+/−) mice and APC^Min/+MSH2^−/− (A^+/−M^−/−) mice that were iNOS proficient (N^+/−) or iNOS deficient (N^−/−).</p

Three Distinct Patterns of Histone H3Y41 Phosphorylation Mark Active Genes

The JAK2 tyrosine kinase is a critical mediator of cytokine-induced signaling. It plays a role in the nucleus, where it regulates transcription by phosphorylating histone H3 at tyrosine 41 (H3Y41ph). We used chromatin immunoprecipitation coupled to massively parallel DNA sequencing (ChIP-seq) to define the genome-wide pattern of H3Y41ph in human erythroid leukemia cells. Our results indicate that H3Y41ph is located at three distinct sites: (1) at a subset of active promoters, where it overlaps with H3K4me3, (2) at distal cis-regulatory elements, where it coincides with the binding of STAT5, and (3) throughout the transcribed regions of active, tissue-specific hematopoietic genes. Together, these data extend our understanding of this conserved and essential signaling pathway and provide insight into the mechanisms by which extracellular stimuli may lead to the coordinated regulation of transcription