Objective. Inhibition of T cell DNA methylation causes autoreactivity in vitro and a lupus-like disease in vivo, suggesting that T cell DNA hypomethylation may contribute to autoimmunity. The hypomethylation effects are due, in part, to overexpression of lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18). Importantly, T cells from patients with active lupus have hypomethylated DNA and overexpress LFA-1 on an autoreactive subset, suggesting that the same mechanism could contribute to human lupus. The present study investigated the nature of the methylation change that affects LFA-1 expression in vitro and in human lupus.Methods. Bisulfite sequencing was used to determine the methylation status of the ITGAL promoter and flanking regions in T cells from lupus patients and healthy subjects, and in T cells treated with DNA methylation inhibitors. "Patch" methylation of promoter sequences in reporter constructs was used to determine the functional significance of the methylation changes.Results. Hypomethylation of specific sequences flanking the ITGAL promoter was seen in T cells from patients with active lupus and in T cells treated with 5-azacytidine and procainamide. Patch methylation of this region suppressed ITGAL promoter function.Conclusion. DNA methylation changes occur in specific sequences that regulate LFA-1 expression in lupus T cells and in the hypomethylation model, indicating that altered methylation of specific genes may play a role in the pathogenesis of lupus.The mechanisms initiating human systemic lupus erythematosus (SLE) remain unknown. The finding that exposure to certain drugs can induce a lupus-like disease has provided leads into the nature of biochemical alterations associated with lupus. The 2 drugs most frequently associated with lupus, procainamide and hydralazine, can cause a lupus-like disease through effects on T cell DNA methylation. Treating T cells with procainamide, hydralazine, or 5-azacytidine (5-azaC; the prototypic DNA methylation inhibitor) demethylates DNA, alters gene expression, and induces major histocompatibility complex-specific T cell autoreactivity (1-5). Adoptive transfer of the autoreactive cells causes a lupus-like disease in animal models (5-7). The autoimmune effects of the methylation inhibitors are due, in part, to overexpression of lymphocyte functionassociated antigen 1 (LFA-1) (CD11a/CD18), because increasing T cell LFA-1 by transfection causes an identical autoreactivity in vitro, and a similar autoimmune disease in vivo (4,8).Altered DNA methylation has also been implicated in human lupus. T cells from patients with active lupus have an ϳ17% decrease in genomic deoxymethylcytosine content and overexpress LFA-1 on an autoreactive subset (9-11). However, whether the DNA hypomethylation occurring in lupus affects transcriptionally relevant regions is not known. It is similarly unknown if the DNA hypomethylation induced by 5-azaC or procainamide affects the same sequences as those affected in SLE.DNA methylation inhibitors increase LFA-1 through thei...

Understanding the pathways that control epithelial carcinogenesis is vital to the development of effective treatments. The Polycomb group family member Bmi1 is overexpressed in numerous epithelial tumors, but its role in their development has not been established. We now show a key role for Bmi1 in lung adenocarcinoma. Whereas lung development occurs normally in Bmi1-deficient mice, loss of Bmi1 decreases the number and progression of lung tumors at a very early point in an oncogenic K-ras-initiated mouse model of lung cancer. This correlates with a defect in the ability of Bmi1-deficient putative bronchiolalveolar stem cells (BASCs) to proliferate in response to the oncogenic stimulus. Notably, in the absence of oncogenic K-ras, Bmi1-deficient BASCs show impaired proliferation and self-renewal capacity in culture and after lung injury in vivo. Abrogated lung cancer development and BASC self-renewal occur partially in a p19 ARF -dependent manner. Our data suggest that Bmi1 deficiency suppresses tumor development by limiting the expansion potential of BASCs, the apparent lung cancer cells of origin. Because Bmi1 is elevated in additional tumor types, this suggests that Bmi1 plays a key role in regulating proliferation of both stem cells and tumor cells in diverse adult epithelial tissues.Arf ͉ Ink4a ͉ non-small-cell lung cancer ͉ p16

Summary Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study, we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFβ transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFβ, and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFβ deficiency generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via Polycomb Repressive Complex 2 (PRC2) independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.