Gut microbial metabolism drives transformation of MSH2-deficient colon epithelial cells.

The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)MSH2(-/-) mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2(-/-) colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating ß-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. PAPERCLIP:

The multifaceted role of the intestinal microbiota in colon cancer.

In recent years, our understanding of the mechanisms underlying colorectal carcinogenesis has vastly expanded. Underlying inflammation within the intestine, diet, and most recently, the gut microbiota, have been demonstrated to influence the development of colorectal cancer. However, since cancer is ultimately a genetic disease, these factors are thought to create genotoxic stress within the intestinal environment to promote genetic and epigenetic alterations leading to cancer. In this review, we will focus on how gut microbes intersect with inflammation, diet, and host genetics to influence the development of colon cancer.

MicroRNAs at the epicenter of intestinal homeostasis.

Maintaining intestinal homeostasis is a key prerequisite for a healthy gut. Recent evidence points out that microRNAs (miRNAs) act at the epicenter of the signaling networks regulating this process. The fine balance in the interaction between gut microbiota, intestinal epithelial cells, and the host immune system is achieved by constant transmission of signals and their precise regulation. Gut microbes extensively communicate with the host immune system and modulate host gene expression. On the other hand, sensing of gut microbiota by the immune cells provides appropriate tolerant responses that facilitate the symbiotic relationships. While the role of many regulatory proteins, receptors and their signaling pathways in the regulation of the intestinal homeostasis is well documented, the involvement of non-coding RNA molecules in this process has just emerged. This review discusses the most recent knowledge about the contribution of miRNAs in the regulation of the intestinal homeostasis.

Gut microbial metabolism and colon cancer: can manipulations of the microbiota be useful in the management of gastrointestinal health?

The gut microbiota is an important component of the human body and its immune-modulating and metabolic activities are critical to maintain good health. Gut microbes, however, are sensitive to changes in diet, exposure to antibiotics, or infections, all of which cause transient disruptions in the microbial composition, a phenomenon known as dysbiosis. It is now recognized that microbial dysbiosis is at the root of many gastrointestinal disorders. However, the mechanisms through which bacterial dysbiosis initiates disease are not fully understood. Microbially-derived metabolites and their role in disease have also attracted significant attention. Identification of cancer-associated bacteria and understanding the contributions of microbial metabolism in health and disease are exciting but challenging areas that will allow defining microbial biomarkers for predicting gastrointestinal disorders. Understanding the complex interactions between gut microbiota, diet, host immune system and host genetics will be critical to developing more personalized therapies and approaches to treat disease.

The mitochondrial protein NLRX1 controls the balance between extrinsic and intrinsic apoptosis.

NLRX1 is a mitochondrial Nod-like receptor (NLR) protein whose function remains enigmatic. Here, we observed that NLRX1 expression was glucose-regulated and blunted by SV40 transformation. In transformed but not primary murine embryonic fibroblasts, NLRX1 expression mediated resistance to an extrinsic apoptotic signal, whereas conferring susceptibility to intrinsic apoptotic signals, such as glycolysis inhibition, increased cytosolic calcium and endoplasmic reticulum stress. In a murine model of colorectal cancer induced by azoxymethane, NLRX1-/- mice developed fewer tumors than wild type mice. In contrast, in a colitis-associated cancer model combining azoxymethane and dextran sulfate sodium, NLRX1-/- mice developed a more severe pathology likely due to the increased sensitivity to dextran sulfate sodium colitis. Together, these results identify NLRX1 as a critical mitochondrial protein implicated in the regulation of apoptosis in cancer cells. The unique capacity of NLRX1 to regulate the cellular sensitivity toward intrinsic versus extrinsic apoptotic signals suggests a critical role for this protein in numerous physiological processes and pathological conditions.

Negative supercoiling creates single-stranded patches of DNA that are substrates for AID-mediated mutagenesis.

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates-which we found to be unique to actively transcribed genes-as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID.

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 an approximately 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 base pairs. 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 that is not mutated by AID. Strikingly, we found that GC B cells accumulate 5 times more mutations at a reporter gene than during the development of the mouse. Notably, the mutation frequency at this reporter gene was approximately 10 times greater in Msh2(-/-) compared with wild-type GC B cells cells. In contrast to the V region, the increased level of mutations at A:T base pairs in GC B cells was not caused by MMR. These results show that in GC B cells, (1) MMR functions normally at an AID-insensitive gene and (2) the frequency of background mutagenesis is greater in GC B cells than in their precursor follicular B cells.

Roles of DNA sequence and sigma A factor in transcription of the vraSR operon.

Cell wall damage in Staphylococcus aureus induces a rapid genome-wide response, referred to as the cell wall stress stimulon. This response is mediated by a two-component system, the vancomycin resistance-associated sensor/regulator (VraSR). The response regulator protein VraR is a transcription factor. Here, we demonstrate that two VraR binding sites in the vraSR operon control region are involved in the regulation of the vraSR operon. The sites are centered at the -60 and -35 nucleotide positions and are referred to as R1 and R2, respectively. DNase I footprinting and lux operon reporter vector studies showed that both of these sites communicate intimately with each other to fine-tune the activity of the vraSR operon. Mutagenesis of the VraR binding sites showed that dimerization of unphosphorylated VraR at R1 is driven by a hierarchy in VraR binding and by the proximity of the two tandem VraR binding sequences at this site. On the other hand, these studies show that the lack of sequence conservation and the distance between the VraR binding sequences in R2 ensure that VraR is recruited to this site only when phosphorylated (hence, under stress conditions). Furthermore, we demonstrate that sigma A (SigA) factor is involved in the regulation of the vraSR operon. Our study shows that sigma A factor does not bind to the vraSR operon control region in the absence of VraR, suggesting that VraR may interact directly with this factor.

Loss of mismatch repair signaling impairs the WNT-bone morphogenetic protein crosstalk and the colonic homeostasis.

The fine balance between proliferation, differentiation, and apoptosis in the colonic epithelium is tightly controlled by the interplay between WNT, Notch, and bone morphogenetic protein (BMP) signaling. How these complex networks coordinate the colonic homeostasis, especially if cancer predisposing mutations such as mutations in the DNA mismatch repair (MMR) are present, is unclear. Inactivation of the MMR system has long been linked to colorectal cancer; however, little is known about its role in the regulation of the colonic homeostasis. It has been shown that loss of MMR promotes the proliferation of colon epithelial cells that renders them highly susceptible to transformation. The mechanism through which MMR mediates this effect, yet, remains to be determined. Using an MMR-deficient mouse model, we show that increased methylation of Dickkopf1 impacts its expression, and consequently, the ability to negatively regulate WNT signaling. As a result, excessive levels of active ß-catenin promote strong crypt progenitor-like phenotype and abnormal proliferation. Under these settings, the development and function of the goblet cells are affected. MMR-deficient mice have fewer goblet cells with enlarged mucin-loaded vesicles. We further show that MMR inactivation impacts the WNT-BMP signaling crosstalk.

DNA-binding activity of the vancomycin resistance associated regulator protein VraR and the role of phosphorylation in transcriptional regulation of the vraSR operon.

In Staphylococcus aureus the VraSR two-component system acts as a sentinel that can rapidly sense cell wall peptidoglycan damage and coordinate a response to enhance the resistance phenotype. VraR is a transcription factor and its cognate kinase, VraS, modulates the DNA-binding activity of VraR by regulating its phosphorylation state and hence its dimerization state. Here we provide the first report on the VraR transcriptional activity by investigating the interaction with the vraSR operon control region. We found that this region contains three VraR-binding sites, each with unique VraR-binding features. VraR binding to the most conserved site is phosphorylation independent, and dimerization is proposed to be induced through binding to DNA. By contrast, binding to the less conserved site requires phosphorylation of VraR. This site overlaps with the binding site of the sigma subunit of the RNA polymerase complex, suggesting that VraR could be interacting with the transcription machinery in the presence of the cell wall stress signal. Mutagenesis studies on the VraR binding sites suggest that there is directionality in the binding of VraR to the target DNA, probably dictated by VraR dimerization. We also constructed a P(vraSR)-fused lux operon reporter vector to investigate in vivo the significance of our in vitro studies. These studies show that upon cell wall stress, induced by oxacillin, the expression level of the lux operon goes up and it is affected by the integrity of the two identified VraR-binding sites in agreement with the in vitro studies. Further, they demonstrate that the VraR most conserved binding site is essential to the vraSR operon expression. On the other hand, they suggest that the role of the VraR less conserved site could be that of mediating high levels of vraSR operon expression during cell wall stress conditions.