Cooperative interactions enable singular olfactory receptor expression in mouse olfactory neurons.

The monogenic and monoallelic expression of only one out of >1000 mouse olfactory receptor (ORs) genes requires the formation of large heterochromatic chromatin domains that sequester the OR gene clusters. Within these domains, intergenic transcriptional enhancers evade heterochromatic silencing and converge into interchromosomal hubs that assemble over the transcriptionally active OR. The significance of this nuclear organization in OR choice remains elusive. Here, we show that transcription factors Lhx2 and Ebf specify OR enhancers by binding in a functionally cooperative fashion to stereotypically spaced motifs that defy heterochromatin. Specific displacement of Lhx2 and Ebf from OR enhancers resulted in pervasive, long-range, and trans downregulation of OR transcription, whereas pre-assembly of a multi-enhancer hub increased the frequency of OR choice in cis. Our data provide genetic support for the requirement and sufficiency of interchromosomal interactions in singular OR choice and generate general regulatory principles for stochastic, mutually exclusive gene expression programs.

Mapping an epitope in EBNA-1 that is recognized by monoclonal antibodies to EBNA-1 that cross-react with dsDNA.

INTRODUCTION: The Epstein Barr Virus (EBV) has been associated with the autoimmune disease, Systemic Lupus Erythematosus (SLE). EBV nuclear antigen-I (EBNA-1) is the major nuclear protein of EBV. We previously generated an IgG monoclonal antibody (MAb) to EBNA-1, 3D4, and demonstrated that it cross-reacts with double stranded DNA (dsDNA) and binds the 148 amino acid viral binding site (VBS) in the carboxyl region of EBNA-1. The aim of the present study was to characterize another antibody to EBNA-1 that cross-reacts with dsDNA, compare its immunoglobulin genes to 3D4, and finely map the epitope in EBNA-1 that is recognized by these cross-reactive antibodies. METHODS: We generated an IgM MAb to EBNA-1, 16D2, from EBNA-1 injected mice and demonstrated by ELISA that it cross-reacts with dsDNA and binds the 148 amino acid VBS. We sequenced the variable heavy and light chain genes of 3D4 and 16D2 and compared V gene usage. To more finely map the epitope in EBNA-1 recognized by these MAbs, we examined their binding by ELISA to 15 overlapping peptides spanning the 148 amino acid domain. RESULTS: Sequence analysis revealed that 3D4 and 16D2 utilize different VH and VL genes but identical JH and Jk regions with minimal junctional diversity. This accounts for similarities in their CDR3 regions and may explain their similar dual binding specificity. Epitope mapping revealed 3D4 and 16D2 bind the same peptide in the VBS. Based on the crystal structure of EBNA-1, we observed that this peptide resides at the base of an exposed proline rich loop in EBNA-1. CONCLUSION: We have demonstrated that two MAbs that bind EBNA-1 and cross-react with dsDNA, recognize the same peptide in the VBS. This peptide may serve as a mimetope for dsDNA and may be of diagnostic and therapeutic value in SLE.

Antibodies elicited in response to EBNA-1 may cross-react with dsDNA.

BACKGROUND: Several genetic and environmental factors have been linked to Systemic Lupus Erythematosus (SLE). One environmental trigger that has a strong association with SLE is the Epstein Barr Virus (EBV). Our laboratory previously demonstrated that BALB/c mice expressing the complete EBNA-1 protein can develop antibodies to double stranded DNA (dsDNA). The present study was undertaken to understand why anti-dsDNA antibodies arise during the immune response to EBNA-1. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we demonstrated that mouse antibodies elicited in response to EBNA-1 cross-react with dsDNA. First, we showed that adsorption of sera reactive with EBNA-1 and dsDNA, on dsDNA cellulose columns, diminished reactivity with EBNA-1. Next, we generated monoclonal antibodies (MAbs) to EBNA-1 and showed, by several methods, that they also reacted with dsDNA. Examination of two cross-reactive MAbs--3D4, generated in this laboratory, and 0211, a commercial MAb--revealed that 3D4 recognizes the carboxyl region of EBNA-1, while 0211 recognizes both the amino and carboxyl regions. In addition, 0211 binds moderately well to the ribonucleoprotein, Sm, which has been reported by others to elicit a cross-reactive response with EBNA-1, while 3D4 binds only weakly to Sm. This suggests that the epitope in the carboxyl region may be more important for cross-reactivity with dsDNA while the epitope in the amino region may be more important for cross-reactivity with Sm. CONCLUSIONS/SIGNIFICANCE: In conclusion, our results demonstrate that antibodies to the EBNA-1 protein cross-react with dsDNA. This study is significant because it demonstrates a direct link between the viral antigen and the development of anti-dsDNA antibodies, which are the hallmark of SLE. Furthermore, it illustrates the crucial need to identify the epitopes in EBNA-1 responsible for this cross-reactivity so that therapeutic strategies can be designed to mask these regions from the immune system following EBV exposure.

BAFF overexpression promotes anti-dsDNA B-cell maturation and antibody secretion.

Overexpression of BAFF is believed to play an important role in systemic lupus erythematosus and elevated levels of serum BAFF have been found in lupus patients. Excess BAFF also leads to overproduction of anti-dsDNA antibodies and a lupus-like syndrome in mice. In the present study, we use mice transgenic for the R4A-Cmu (IgM) heavy chain of an anti-dsDNA antibody, to study the effects of BAFF overexpression on anti-dsDNA B-cell regulation. We observe that overexpression of BAFF promotes anti-dsDNA B-cell maturation and secretion of antibody and enriches for transgenic anti-dsDNA B cells in the marginal zone and follicular splenic compartments. In addition, our data suggests that BAFF rescues a subset of anti-dsDNA B cells from a regulatory checkpoint in the transitional stage of development.