Detection of chromatin-associated single-stranded DNA in regions targeted for somatic hypermutation

After encounter with antigen, the antibody repertoire is shaped by somatic hypermutation (SHM), which leads to an increase in the affinity of antibodies for the antigen, and class-switch recombination (CSR), which results in a change in the effector function of antibodies. Both SHM and CSR are initiated by activation-induced cytidine deaminase (AID), which deaminates deoxycytidine to deoxyuridine in single-stranded DNA (ssDNA). The precise mechanism responsible for the formation of ssDNA in V regions undergoing SHM has yet to be experimentally established. In this study, we searched for ssDNA in mutating V regions in which DNA–protein complexes were preserved in the context of chromatin in human B cell lines and in primary mouse B cells. We found that V regions that undergo SHM were enriched in short patches of ssDNA, rather than R loops, on both the coding and noncoding strands. Detection of these patches depended on the presence of DNA-associated proteins and required active transcription. Consistent with this, we found that both DNA strands in the V region were transcribed. We conclude that regions of DNA that are targets of SHM assemble protein–DNA complexes in which ssDNA is exposed, making it accessible to AID

A Weakened Transcriptional Enhancer Yields Variegated Gene Expression

Identical genes in the same cellular environment are sometimes expressed differently. In some cases, including the immunoglobulin heavy chain (IgH) locus, this type of differential gene expression has been related to the absence of a transcriptional enhancer. To gain additional information on the role of the IgH enhancer, we examined expression driven by enhancers that were merely weakened, rather than fully deleted, using both mutations and insulators to impair enhancer activity. For this purpose we used a LoxP/Cre system to place a reporter gene at the same genomic site of a stable cell line. Whereas expression of the reporter gene was uniformly high in the presence of the normal, uninsulated enhancer and undetectable in its absence, weakened enhancers yielded variegated expression of the reporter gene; i.e., the average level of expression of the same gene differed in different clones, and expression varied significantly among cells within individual clones. These results indicate that the weakened enhancer allows the reporter gene to exist in at least two states. Subtle aspects of the variegation suggest that the IgH enhancer decreases the average duration (half-life) of the silent state. This analysis has also tested the conventional wisdom that enhancer activity is independent of distance and orientation. Thus, our analysis of mutant (truncated) forms of the IgH enhancer revealed that the 250 bp core enhancer was active in its normal position, ∼1.4 kb 3′ of the promoter, but inactive ∼6 kb 3′, indicating that the activity of the core enhancer was distance-dependent. A longer segment – the core enhancer plus ∼1 kb of 3′ flanking material, including the 3′ matrix attachment region – was active, and the activity of this longer segment was orientation-dependent. Our data suggest that this 3′ flank includes binding sites for at least two activators

Examination of Msh6- and Msh3-deficient Mice in Class Switching Reveals Overlapping and Distinct Roles of MutS Homologues in Antibody Diversification

Somatic hypermutation and class switch recombination (CSR) contribute to the somatic diversification of antibodies. It has been shown that MutS homologue (Msh)6 (in conjunction with Msh2) but not Msh3 is involved in generating A/T base substitutions in somatic hypermutation. However, their roles in CSR have not yet been reported. Here we show that Msh6−/− mice have a decrease in CSR, whereas Msh3−/− mice do not. When switch regions were analyzed for mutations, deficiency in Msh6 was associated with an increase in transition mutations at G/C basepairs, mutations at RGYW/WRCY hotspots, and a small increase in the targeting of G/C bases. In addition, Msh6−/− mice exhibited an increase in the targeting of recombination sites to GAGCT/GGGGT consensus repeats and hotspots in Sγ3 but not in Sμ. In contrast to Msh2−/− mice, deficiency in Msh6 surprisingly did not change the characteristics of Sμ-Sγ3 switch junctions. However, Msh6−/− mice exhibited a change in the positioning of Sμ and Sγ3 junctions. Although none of these changes were seen in Msh3−/− mice, they had a higher percentage of large inserts in their switch junctions. Together, our data suggest that MutS homologues Msh2, Msh3, and Msh6 play overlapping and distinct roles during antibody diversification processes

Regulation of Glutamine Carrier Proteins by RNF5 Determines Breast Cancer Response to ER Stress-Inducing Chemotherapies

SummaryMany tumor cells are fueled by altered metabolism and increased glutamine (Gln) dependence. We identify regulation of the L-glutamine carrier proteins SLC1A5 and SLC38A2 (SLC1A5/38A2) by the ubiquitin ligase RNF5. Paclitaxel-induced ER stress to breast cancer (BCa) cells promotes RNF5 association, ubiquitination, and degradation of SLC1A5/38A2. This decreases Gln uptake, levels of TCA cycle components, mTOR signaling, and proliferation while increasing autophagy and cell death. Rnf5-deficient MMTV-PyMT mammary tumors were less differentiated and showed elevated SLC1A5 expression. Whereas RNF5 depletion in MDA-MB-231 cells promoted tumorigenesis and abolished paclitaxel responsiveness, SLC1A5/38A2 knockdown elicited opposing effects. Inverse RNF5hi/SLC1A5/38A2lo expression was associated with positive prognosis in BCa. Thus, RNF5 control of Gln uptake underlies BCa response to chemotherapies

Creating a positive casual academic identity through change and loss

Neoliberalism has significantly impacted higher education institutes across the globe by increasing the number of casual and non-continuing academic positions. Insecure employments conditions have not only affected the well-being of contingent staff, but it has also weakened the democratic, intellectual and moral standing of academic institutions. This chapter provides one practitioner’s account of the challenges of casual work, but rather than dwelling on the negativities, it outlines the potential richness of an identity based on insecurity and uncertainty. This exploration draws on the literature of retired academics and identity theory to illustrate the potential generative spaces within an undefined and incoherent identity

Detection and Characterization of Oncogene Mutations in Preneoplastic and Early Neoplastic Lesions

While it has been nearly 30 years since its discovery, the ras family of genes has not yet lost its impact on basic and clinical oncology. These genes remain central to the field of molecular oncology as tools for investigating carcinogenesis and oncogenic signaling, as powerful biomarkers for the identification of those who have or are at high risk of developing cancer, and as oncogene targets for the design and development of new chemotherapeutic drugs. Mutational activation of the K-RAS proto-oncogene is an early event in the development and progression of the colorectal, pancreatic, and lung cancers that are the major causes of cancer death in the world. The presence of point mutational "hot spots" at sites necessary for the activation of this proto-oncogene has led to the development of a number of highly sensitive PCR-based methods that are feasible for the early detection of K-RAS oncogene mutations in the clinical setting. In light of these facts, mutation at the K-RAS oncogene has the potential to serve as a useful biomarker in the early diagnosis and risk assessment of cancers with oncogenic ras signaling. This chapter describes a highly sensitive method for detecting mutant K-RAS, enriched PCR, and its application to early detection of alterations in this oncogene in preneoplastic and early neoplastic lesions of the colon and rectum

Association between Age and the 7 Repeat Allele of the Dopamine D4 Receptor Gene

Longevity is in part (25%) inherited, and genetic studies aim to uncover allelic variants that play an important role in prolonging life span. Results to date confirm only a few gene variants associated with longevity, while others show inconsistent results. However, GWAS studies concentrate on single nucleotide polymorphisms, and there are only a handful of studies investigating variable number of tandem repeat variations related to longevity. Recently, Grady and colleagues (2013) reported a remarkable (66%) accumulation of those carrying the 7 repeat allele of the dopamine D4 receptor gene in a large population of 90-109 years old Californian centenarians, as compared to an ancestry-matched young population. In the present study we demonstrate the same association using continuous age groups in an 18-97 years old Caucasian sample (N = 1801, p = 0.007). We found a continuous pattern of increase from 18-75, however frequency of allele 7 carriers decreased in our oldest age groups. Possible role of gene-environment interaction effects driven by historical events are discussed. In accordance with previous findings, we observed association preferentially in females (p = 0.003). Our results underlie the importance of investigating non-disease related genetic variants as inherited components of longevity, and confirm, that the 7-repeat allele of the dopamine D4 receptor gene is a longevity enabling genetic factor, accumulating in the elderly female population

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 Anaphase-Promoting Complex or Cyclosome Supports Cell Survival in Response to Endoplasmic Reticulum Stress

The anaphase-promoting complex or cyclosome (APC/C) is a multi-subunit ubiquitin ligase that regulates exit from mitosis and G1 phase of the cell cycle. Although the regulation and function of APC/CCdh1 in the unperturbed cell cycle is well studied, little is known of its role in non-genotoxic stress responses. Here, we demonstrate the role of APC/CCdh1 (APC/C activated by Cdh1 protein) in cellular protection from endoplasmic reticulum (ER) stress. Activation of APC/CCdh1 under ER stress conditions is evidenced by Cdh1-dependent degradation of its substrates. Importantly, the activity of APC/CCdh1 maintains the ER stress checkpoint, as depletion of Cdh1 by RNAi impairs cell cycle arrest and accelerates cell death following ER stress. Our findings identify APC/CCdh1 as a regulator of cell cycle checkpoint and cell survival in response to proteotoxic insults