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

Msh2 ATPase Activity Is Essential for Somatic Hypermutation at A-T Basepairs and for Efficient Class Switch Recombination

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytidine deaminase–mediated cytidine deamination of immunoglobulin genes. MutS homologue (Msh) 2−/− mice have reduced A-T mutations and CSR. This suggests that Msh2 may play a role in repairing activation-induced cytidine deaminase–generated G-U mismatches. However, because Msh2 not only initiates mismatch repair but also has other functions, such as signaling for apoptosis, it is not known which activity of Msh2 is responsible for the effects observed, and consequently, many models have been proposed. To further dissect the role of Msh2 in SHM and CSR, mice with a “knockin” mutation in the Msh2 gene that inactivates the adenosine triphosphatase domain were examined. This mutation (i.e., Msh2G674A), which does not affect apoptosis signaling, allows mismatches to be recognized but prevents Msh2 from initiating mismatch repair. Here, we show that, similar to Msh2−/− mice, SHM in Msh2G674A mice is biased toward G-C mutations. However, CSR is partially reduced, and switch junctions are more similar to those of postmeiotic segregation 2−/− mice than to Msh2−/− mice. These results indicate that Msh2 adenosine triphosphatase activity is required for A-T mutations, and suggest that Msh2 has more than one role in CSR

A Randomized Study Evaluating Oral Fusidic Acid (CEM-102) in Combination With Oral Rifampin Compared With Standard-of-Care Antibiotics for Treatment of Prosthetic Joint Infections: A Newly Identified Drug–Drug Interaction

BACKGROUND: Fusidic acid (FA) has been used for decades for bone infection, including prosthetic joint infection (PJI), often in combination with rifampin (RIF). An FA/RIF pharmacokinetic interaction has not previously been described. METHODS: In a phase 2 open-label randomized study, we evaluated oral FA/RIF vs standard-of-care (SOC) intravenous antibiotics for treatment of hip or knee PJI. Outcome assessment occurred at reimplantation (week 12) for subjects with 2-stage exchange, and after 3 or 6 months of treatment for subjects with hip or knee debride and retain strategies, respectively. RESULTS: Fourteen subjects were randomized 1:1 to FA/RIF or SOC. Pharmacokinetic profiles were obtained for 6 subjects randomized to FA/RIF. FA concentrations were lower than anticipated in all subjects during the first week of therapy, and at weeks 4 and 6, blood levels continued to decline. By week 6, FA exposures were 40%-45% lower than expected. CONCLUSIONS: The sponsor elected to terminate this study due to a clearly illustrated drug-drug interaction between FA and RIF, which lowered FA levels to a degree that could influence subject outcomes. Optimization of FA exposure if used in combination with RIF should be a topic of future research. CLINICAL TRIALS REGISTRATION: NCT01756924

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

Differential chromatin accessibility in peripheral blood mononuclear cells underlies COVID-19 disease severity prior to seroconversion

Abstract SARS-CoV-2 infection triggers profound and variable immune responses in human hosts. Chromatin remodeling has been observed in individuals severely ill or convalescing with COVID-19, but chromatin remodeling early in disease prior to anti-spike protein IgG seroconversion has not been defined. We performed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and RNA-seq on peripheral blood mononuclear cells (PBMCs) from outpatients with mild or moderate symptom severity at different stages of clinical illness. Early in the disease course prior to IgG seroconversion, modifications in chromatin accessibility associated with mild or moderate symptoms were already robust and included severity-associated changes in accessibility of genes in interleukin signaling, regulation of cell differentiation and cell morphology. Furthermore, single-cell analyses revealed evolution of the chromatin accessibility landscape and transcription factor motif accessibility for individual PBMC cell types over time. The most extensive remodeling occurred in CD14+ monocytes, where sub-populations with distinct chromatin accessibility profiles were observed prior to seroconversion. Mild symptom severity was marked by upregulation of classical antiviral pathways, including those regulating IRF1 and IRF7, whereas in moderate disease, these classical antiviral signals diminished, suggesting dysregulated and less effective responses. Together, these observations offer novel insight into the epigenome of early mild SARS-CoV-2 infection and suggest that detection of chromatin remodeling in early disease may offer promise for a new class of diagnostic tools for COVID-19