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Background: Human endosomal Toll-like receptors TLR7 and TLR8 recognize self and non-self RNA ligands, and are important mediators of innate immunity and autoimmune pathogenesis. TLR7 and TLR8 are, respectively, encoded by adjacent X-linked genes. We previously established that TLR7 evades X chromosome inactivation (XCI) in female immune cells. Whether TLR8 also evades XCI, however, has not yet been explored. Method: In the current study, we used RNA fluorescence in situ hybridization (RNA FISH) to directly visualize, on a single-cell basis, primary transcripts of TLR7 and TLR8 relative to X chromosome territories in CD14+ monocytes and CD4+ T lymphocytes from women, Klinefelter syndrome (KS) men, and euploid men. To assign X chromosome territories in cells lacking robust expression of a XIST compartment, we designed probes specific for X-linked genes that do not escape XCI and therefore robustly label the active X chromosome. We also assessed whether XCI escape of TLR8 was associated with sexual dimorphism in TLR8 protein expression by western blot and flow cytometry. Results: Using RNA FISH, we show that TLR8, like TLR7, evades XCI in immune cells, and that cells harboring simultaneously TLR7 and TLR8 transcript foci are more frequent in women and KS men than in euploid men, resulting in a sevenfold difference in frequency. This transcriptional bias was again observable when comparing the single X of XY males with the active X of cells from females or KS males. Interestingly, TLR8 protein expression was significantly higher in female mononuclear blood cells, including all monocyte subsets, than in male cells. Conclusions: TLR8, mirroring TLR7, escapes XCI in human monocytes and CD4+ T cells. Co-dependent transcription from the active X chromosome and escape from XCI could both contribute to higher TLR8 protein abundance in female cells, which may have implications for the response to viruses and bacteria, and the risk of developing inflammatory and autoimmune diseases. Highlights: TLR8, like TLR7, escapes X chromosome inactivation in immune cells from women and 47,XXY men with Klinefelter syndrome. The frequency of double-positive cells for TLR7 and TLR8 primary transcripts is sevenfold higher in women than in men. TLR7 and TLR8 form a co-regulated gene cluster on the human X chromosome, with sex-specific, divergent transcriptional patterns observable in monocytes and CD4+ T lymphocytes. Co-dependent transcription of the TLR7 and TLR8 genes on the active X was observed in women and in men with Klinefelter syndrome, contrasting with mutually exclusive transcription in euploid men. Blood mononuclear cells, including monocyte subsets, expressed higher levels of TLR8 protein in females than in euploid males. Plain Language summary: Human endosomal Toll-like receptors TLR7 and TLR8, encoded by two adjacent X-linked genes, recognize self and non-self RNA ligands, and are important mediators of innate immunity and autoimmune pathogenesis. We previously reported that TLR7 evades X chromosome inactivation (XCI) in female immune cells, correlating with enhanced functional properties in B cells harboring biallelic expression of this gene. Here, we conducted a comprehensive single-cell resolution analysis of the transcriptional regulation of both TLR7 and TLR8, in CD14+ monocytes and CD4+ T lymphocytes. We unequivocally demonstrated that TLR8, like TLR7, escapes XCI in immune cells from female and Klinefelter syndrome males. When we analyzed TLR7 and TLR8 transcripts together, cells from women and KS men exhibited higher frequencies of cells co-transcribing the two genes. Surprisingly, these differences were attributable not only to the ability of TLR7 and TLR8 to be expressed on the Xi, but also to the joint transcriptional behavior of the TLR7–TLR8 gene pair on the active X chromosome specifically. This contrasted with a striking pattern of mutually exclusive transcription on the single X of euploid men. Corroborating our RNA FISH results, we found higher TLR8 protein expression in female than in male leukocytes, including all monocyte subpopulations. In summary, our data suggest that sex-biased co-regulation of the Toll-like receptor locus and XCI escape of TLR8 contribute to the sexual dimorphism in TLR8 expression, which may have important consequences for the functional make-up of monocyte and T cell populations. [ABSTRACT FROM AUTHOR]

Misrepair of DNA double-strand breaks produced by the V(D)J recombinase (the RAG1/RAG2 proteins) at immunoglobulin (Ig) and T cell receptor (Tcr) loci has been implicated in pathogenesis of lymphoid malignancies in humans (1) and in mice (2-7). Defects in DNA damage response factors such as ataxia telangiectasia mutated (ATM) protein and combined deficiencies in classical non-homologous end joining and p53 predispose to RAG-initiated genomic rearrangements and lymphomagenesis (2-11). Although we showed previously that RAG1/RAG2 shepherd the broken DNA ends to classical nonhomologous end joining for proper repair (12,13), roles for the RAG proteins in preserving genomic stability remain poorly defined. Here we show that the RAG2 carboxy (C) terminus, although dispensable for recombination (14,15), is critical for maintaining genomic stability. Thymocytes from 'core' Rag2 homozygotes ([Rag2.sup.c/c] mice) show dramatic disruption of Tcrα/δ locus integrity. Furthermore, all [Rag2.sup.c/c] [p53.sup.-/-] mice, unlike [Rag1.sup.c/c] [p53.sup.-/-] and [p53.sup.-/-] animals, rapidly develop thymic lymphomas bearing complex chromosomal translocations, amplifications and deletions involving the Tcrα/δ and Igh loci. We also find these features in lymphomas from [Atm.sup.-/-] mice. We show that, like ATM-deficiency (3), core RAG2 severely destabilizes the RAG post-cleavage complex. These results reveal a novel genome guardian role for RAG2 and suggest that similar 'end release/end persistence' mechanisms underlie genomic instability and lymphomagenesis in [Rag2.sup.c/c] [p53.sup.-/-] and [Atm.sup.-/-] mice., RAG mutations can cause specific defects in the joining stage of V(D)J recombination (12,13,16). The 'dispensable' RAG2 C terminus (murine amino acids 1-383) is of particular interest: loss of the [...]

Examination of the three-dimensional organization of X-linked genes reveals that when transcribed, they are always located at the periphery of, or outside, the Xist RNA domain, in contact with the transcription machinery. Upon silencing, gene shifts to a more internal location, within the Xist RNA compartment devoid of transcription factors and the appearance of this compartment is not dependent on the A-repeats of the Xist transcript, which are essential for gene silencing.

Given their essential role in adaptive immunity, antigen receptor loci have been the focus of analysis for many years and are among a handful of the most well-studied genes in the genome. Their investigation led initially to a detailed knowledge of linear structure and characterization of regulatory elements that confer control of their rearrangement and expression. However, advances in DNA FISH and imaging combined with new molecular approaches that interrogate chromosome conformation have led to a growing appreciation that linear structure is only one aspect of gene regulation and in more recent years, the focus has switched to analyzing the impact of locus conformation and nuclear organization on control of recombination. Despite decades of work and intense effort from numerous labs, we are still left with an incomplete picture of how the assembly of antigen receptor loci is regulated. This chapter summarizes our advances to date and points to areas that need further investigation. [ABSTRACT FROM AUTHOR]

A correction to the article "RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobin loci," that was published online on May 17, 2009 is presented.

A correction to the article "RAG-1 and ATM Coordinate Monoallelic Recombination and Nuclear Positioning of Immunoglobulin Loci" that was published online in the May 17, 2009 issue is presented.