SummaryChromatin structure affects the accessibility of DNA to transcription, repair, and replication. Changes in chromatin structure occur during development, but less is known about changes during aging. We examined the state of chromatin structure and its effect on gene expression during aging in Drosophila at the whole genome and cellular level using whole-genome tiling microarrays of activation and repressive chromatin marks, whole-genome transcriptional microarrays and single-cell immunohistochemistry. We found dramatic reorganization of chromosomal regions with age. Mapping of H3K9me3 and HP1 signals to fly chromosomes reveals in young flies the expected high enrichment in the pericentric regions, the 4th chromosome, and islands of facultative heterochromatin dispersed throughout the genome. With age, there is a striking reduction in this enrichment resulting in a nearly equivalent level of H3K9me3 and HP1 in the pericentric regions, the 4th chromosome, facultative heterochromatin, and euchromatin. These extensive changes in repressive chromatin marks are associated with alterations in age-related gene expression. Large-scale changes in repressive marks with age are further substantiated by single-cell immunohistochemistry that shows changes in nuclear distribution of H3K9me3 and HP1 marks with age. Such epigenetic changes are expected to directly or indirectly impinge upon important cellular functions such as gene expression, DNA repair, and DNA replication. The combination of genome-wide approaches such as whole-genome chromatin immunoprecipitation and transcriptional studies in conjunction with single-cell immunohistochemistry as shown here provide a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans.

Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies against desmoglein 3 (Dsg3). Here, we clone anti-Dsg3 antibodies from four PV patients and identify pathogenic VH1-46 autoantibodies from all four patients. Unexpectedly, VH1-46 autoantibodies had relatively few replacement mutations. We reverted antibody somatic mutations to their germline sequences to determine the requirement of mutations for autoreactivity. Three of five VH1-46 germline-reverted antibodies maintain Dsg3 binding, compared to zero of five non-VH1-46 germline-reverted antibodies. Site-directed mutagenesis of VH1-46 antibodies demonstrate that acidic amino acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding. Our data suggest that VH1-46 autoantibody gene usage is commonly found in PV because VH1-46 antibodies require few to no mutations to acquire Dsg3 autoreactivity, which may favor their early selection. Common VH gene usage indicates common humoral immune responses, even among unrelated patients.

Summary In autoantibody-mediated diseases such as pemphigus serum antibodies lead to disease. Genetic analysis of B cells has allowed characterization of antibody repertoires in such diseases but would be complemented by proteomic analysis of serum autoantibodies. Here we show using proteomic analysis that the serum autoantibody repertoire in pemphigus is much more polyclonal than found by genetic studies of B cells. In addition many B cells encode pemphigus autoantibodies that are not secreted into the serum. Heavy chain variable gene usage of serum autoantibodies is not shared among patients, implying targeting of the coded proteins will not be a useful therapeutic strategy. Analysis of autoantibodies over years in individual patients indicates that many antibody clones persist but the proportion of each changes. These studies indicate a dynamic and diverse autoantibody response not revealed by genetic studies, and explain why similar overall autoantibody titers may give variable disease activity.

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Shared VH1-46 gene usage has been described in B cells reacting to desmoglein 3 (Dsg3) in the autoimmune disease pemphigus vulgaris (PV), as well as B cells responding to rotavirus capsid protein VP6. In both diseases, VH1-46 B cells bearing few to no somatic mutations can recognize the disease antigen. This intriguing connection between an autoimmune response to self-antigen and an immune response to foreign antigen prompted us to investigate whether VH1-46 B cells may be predisposed to Dsg3-VP6 cross-reactivity. Focused testing of VH1-46 mAbs previously isolated from PV and rotavirus-exposed individuals indicates that cross-reactivity is rare, found in only one of seven VH1-46 IgG clonotypes. High-throughput screening of IgG B cell repertoires from 2 PV patients identified no additional cross-reactive clonotypes. Screening of IgM B cell repertoires from one non-PV and 3 PV patients identified specific cross-reactive Abs in one PV patient, but notably all 6 cross-reactive clonotypes utilized VH1-46. Site directed mutagenesis studies indicate that amino acid residues predisposing VH1-46 Abs to Dsg3 reactivity reside in CDR2. However, somatic mutations only rarely promote Dsg3-VP6 cross-reactivity; most mutations abolish VP6 and/or Dsg3 reactivity. Nevertheless, functional testing identified two cross-reactive VH1-46 Abs that both disrupt keratinocyte adhesion and inhibit rotavirus replication, indicating the potential for VH1-46 Abs to have both pathologic autoimmune and protective immune functions. Taken together, these studies suggest that certain VH1-46 B cell populations may be predisposed to Dsg3-VP6 cross-reactivity, but multiple mechanisms prevent the onset of autoimmunity after rotavirus exposure.