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...

The p53 tumor suppressor protein, which is commonly mutated in human cancers, has been shown to interact directly with virally encoded proteins from papillomavirus, adenovirus, and simian virus 40. The disruption of p53 function may be required for efficient replication of certain viruses and may also play a role in the development of virally induced malignancies. Infection with Epstein-Barr virus (EBV) has been associated with the development of B-cell lymphomas and nasopharyngeal carcinoma. Here we show that the EBV immediate-early protein, BZLF1 (Z), which is responsible for initiating the switch from latent to lytic infection, can interact directly in vitro and in vivo with the tumor suppressor protein, p53. This interaction requires the coiled-coil dimerization domain of the Z protein and the carboxy-terminal portion of p53.Overexpression of wild-type p53 inhibits the ability of Z to disrupt viral latency. Likewise, Z inhibits p53-dependent transactivation in lymphoid cells. The direct interaction between Z and p53 may play a role in regulating the switch from latent to lytic viral infection.The p53 tumor suppressor protein is an important negative regulator of cell proliferation (38,48,63,65) and is often mutated in human cancers (26). The p53 protein interacts with the viral E6 protein from certain cancer-associated strains of human papillomavirus (36,54,68), the Elb protein of adenovirus (53, 72), and the large T antigen of papovaviruses (35,53). The dysregulation of wild-type p53 function by viral proteins is thought to play an important role in virus-induced malignancy (37).The interaction between viral proteins and p53 may also play a role in the replication of certain viruses. The p53 protein binds to the simian virus 40 origin of replication (3) and inhibits the ability of large T antigen to mediate simian virus 40 replication in vitro (19,67). In addition, p53 colocalizes with the viral replication proteins in cells infected with herpes simplex virus (70). These data suggest that p53 could potentially regulate the replication of herpesviruses through direct interaction with viral replicative proteins.In this study, we have examined the ability of p53 to interact with the Epstein-Barr virus (EBV) protein BZLF1 (Z), which mediates lytic replication. EBV is a transforming virus in vitro, and infection with EBV has been associated with the development of both B-cell and epithelial cell malignancies in vivo (reviewed in reference 44). EBV infection of B cells in vivo is primarily latent, although productive infection can occur in immunocompromised patients. The switch from latent to productive infection is mediated by transcriptional activation of the immediate-early Z gene (6,52,62). The Z gene product (a member of the basic leucine zipper family) binds directly to APl-like motifs as a homodimer and functions as a transcriptional transactivator (5,12,14,30,34,39,49,64). A number of viral early promoters contain upstream Z-binding sites and are activated upon expression of the Z gene product (6,8,12,14...

Disruption of Epstein-Barr virus (EBV) latency is mediated through the activation of the viral immediate-early proteins, BZLF1 (Z) and BRLF1 (R).i.; (Chevallier-Greco, A., et al., (1986) EMBO J., 5, 3243-9; Countryman, and Miller, G. (1985) Proc. Natl. Acad. Sci. USA, 82, 4085-4089). We have previously demonstrated that these proteins cooperatively activate the EBV early promoter BMRF1 in lymphoid cells but not in epithelial cells. Although cooperative transactivation by these proteins has been demonstrated with a number of EBV promoters, the mechanism of this interaction is not well understood. We now show that the cooperative activation of the BMRF1 promoter by Z-plus-R requires an intact R binding site and at least one functional Z response element (ZRE). Despite the presence of an R binding site, the BMRF1 promoter is only moderately responsive to R alone in either HeLa or Jurkat cells. Efficient activation of the BMRF1 promoter by Z alone in HeLa cells requires two ZREs (located at -59 and -106), whereas two additional Z binding sites (located at -42 and -170) contribute very little to Z-induced activation. In the absence of ZREs, Z acted as a repressor of R-induced transactivation. These observations, along with observations made by other investigators (Giot, J.F. et al., (1991) Nucleic Acids Res., 19, 1251-8), suggest that Z-plus-R cooperative activation is dependent upon 1) direct binding by R and Z to responsive promoter elements and 2) contributions by cell-specific factors.

The Epstein-Barr virus (EBV) BZLF1 (Z) immediate-early transactivator initiates the switch between latent and productive infection in B cells. The Z protein, which has homology to the basic leucine zipper protein c-Fos, transactivates the promoters of several replicative cycle proteins. Transactivation efficiency of the EBV BMRF1promoter by Z is cell type dependent. In B cells, in which EBV typically exists in a latent form, Z activates the BMRF1 promoter inefficiently. We have discovered that the p65 component of the cellular factor NF-KB inhibits transactivation of several EBV promoters by Z. Furthermore, the inhibitor of NF-KB, IKBBa, can augment Z-induced transactivation in the B-cell line Raji. Using glutathione S-transferase fusion proteins and coimmunoprecipitation studies, we demonstrate a direct interaction between Z and p65. This physical interaction, which requires the dimerization domain of Z and the Rel homology domain of p65, can be demonstrated both in vitro and in vivo. Inhibition of Z transactivation function by NF-KB p65, or possibly by other Rel family proteins, may contribute to the inefficiency of Z transactivator function in B cells and may be a mechanism of maintaining B-cell-specific viral latency. (14,15,70). The immediate-early Z protein disrupts viral latency through transactivation of early EBV genes which contain upstream Z-binding sites (9,11,13,14,16,30,31,42,43,64,71 Epstein-Barr virus (EBV) is

Among the few Epstein-Barr virus (EBV) genes expressed during latency are the Epstein-Barr nuclear antigens (EBNAs), at least one of which contributes to the ability of the virus to transform B lymphocytes. We have analyzed a promoter located in the BamHI-C fragment of EBV which is responsible for the expression of EBNA-1 in some cell lines. Deletion analysis of a 1.4-kb region 5' of the RNA start site has identified a 700-bp fragment that is required for optimal promoter activity in latently infected B lymphocytes, as shown by promoter constructs linked to the chloramphenicol acetyltransferase reporter gene. This fragment is also able to enhance activity, in an orientation-independent manner, of the simian virus 40 early promoter linked to the chloramphenicol acetyltransferase gene. The enhancer element has some constitutive activity in EBV-negative lymphoid cells, which is increased in the presence of the EBNA-2 gene product. Further deletions have shown that the EBNA-2-responsive region requires a 98-bp region that contains a degenerate octamer-binding motif. In epithelial cells there was no enhancer activity regardless of the presence of EBNA-2. These results demonstrate that BamHI-C promoter activity may be dependent not on an enhancer contained in the ori-P, as was previously assumed, but rather on EBNA-2 transactivation of this more proximal enhancer located in the upstream region of the BamHI C promoter itself.