The forkhead family protein FOXP3 acts as a repressor of transcription and is both an essential and sufficient regulator of the development and function of regulatory T cells. The molecular mechanism by which FOXP3-mediated transcriptional repression occurs remains unclear. Here, we report that transcriptional repression by FOXP3 involves a histone acetyltransferase-deacetylase complex that includes histone acetyltransferase TIP60 (Tat-interactive protein, 60 kDa) and class II histone deacetylases HDAC7 and HDAC9. The N-terminal 106 -190 aa of FOXP3 are required for TIP60 -FOXP3, HDAC7-FOXP3 association, as well as for the transcriptional repression of FOXP3 via its forkhead domain. FOXP3 can be acetylated in primary human regulatory T cells, and TIP60 promotes FOXP3 acetylation in vivo. Overexpression of TIP60 but not its histone acetyltransferase-deficient mutant promotes, whereas knockdown of endogenous TIP60 relieved, FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation and can be restored by the protein deacetylation inhibitor trichostatin A, indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a previously uncharacterized complex-based mechanism by which FOXP3 actively mediates transcriptional repression.A central theme that has emerged over the last 25 years is that a process of self-regulation of the immune response occurs to limit self-reactivity. Biochemical details of how the immune system distinguishes and regulates self and non-self remain to be fully documented (1). A recently characterized CD4 ϩ CD25 ϩ regulatory T cell subset expresses the Foxp3 transcription factor. As a transcriptional repressor of cytokine gene expression (2), Foxp3 was subsequently identified as an essential and sufficient regulator of natural regulatory T cell development and function (3-5).Mammalian transcriptional repressors can execute their function by either passive or active mechanisms (6, 7). FOXP3 may, for example, function as a passive transcriptional repressor in the case of its association with 9). In this study, we explore the role of FOXP3 as an active transcriptional repressor by revealing the dynamic FOXP3 ensemble formation with a specific histone acetyltransferase (HAT) and certain class II histone deacetylases (HDACs) in expanded human CD4 ϩ CD25 ϩ regulatory T cells (10, 11).Histone acetylation and histone deacetylation affect chromatin remodeling during T cell development and differentiation (12, 13). HAT and HDAC abnormalities have been associated with leukemia (14, 15), diabetes (16) and other diseases of the immune system (17-19). The linkage of HAT and HDAC as components of a single complex permits dynamic responsiveness to extracellular stimulation (18,20). The HAT TIP60 (Tat-interactive protein, 60 kDa), originally isolated as an HIV-1 TAT-interactive protein (21), functions as eith...

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2C4 (Pertuzumab, Omnitarg) is a monoclonal antibody targeting p185 her2/neu , which is overexpressed in 30% of invasive breast cancer. 2C4 is currently in phase II clinical trials for several types of cancers. This antibody has been reported to disrupt the association between p185 her2/neu and ErbB3. In our studies of epidermal growth factor receptor (EGFR)-p185 her2/neu heterodimerization, we noted that 2C4 formed associations with the EGFR-p185 her2/neu receptor complex. Our data argue against 2C4 as a universal heterodimerization blocker for p185 her2/neu , but indicate that cocktails of monoclonal antibodies binding distinct interaction surfaces of p185 her2/neu will emerge as the most potent targeted therapy. Oncogene (2008) 27, 3870-3874; doi:10.1038/onc.2008 published online 11 February 2008 Keywords: her2; Pertuzumab; heterodimer; 4D5 p185 her2/neu (Neu, c-ErbB2), a protein product related to the oncogene neu and the second member of the ErbB family of receptor tyrosine kinases, is overexpressed in many breast and ovarian cancers (van de Vijver et al., 1987;Slamon et al., 1989), early breast tumors (Lodato et al., 1990), gastrointestinal tumors (Cohen et al., 1989, lung tumors (Kern et al., 1990) as well as tumors of the pancreas (Williams et al., 1991). Extensive studies have shown that p185 her2/neu , acting as the preferred co-receptor for other family members, plays a dominant role in mediating the malignant phenotype Kokai et al., 1989;Lodato et al., 1990). p185 her2/neu -targeted therapy was initiated more than 20 years ago when monoclonal antibodies were used to reverse the malignant phenotype (Drebin et al., 1984(Drebin et al., , 1985. Since then, significant efforts have been spent to improve antibodies disabling p185 her2/neu . The humanized antibody 'trastuzumab' (rhumAb 4D5 or Herceptin) (Carter et al., 1992) is already used to treat advanced breast cancer and, more recently, as an adjuvant to prevent tumor emergence (Katsumata et al., 1995;Romond et al., 2005).2C4 (Pertuzumab, Omnitarg) is a distinct recombinant humanized monoclonal antibody targeting a different epitope on the extracellular domain of the p185 her2/neu receptor. Agus et al. (2002) suggested that 2C4 disrupts the association of p185 her2/neu with all other ErbB family receptors. This hypothesis was based on the 2C4 influence on p185 her2/neu -ErbB3 association.We first identified homodimers of p185 her2/neu and heterodimers of epidermal growth factor receptor (EGFR) with p185 her2/neu some years ago (Wada et al., 1990b). Moreover, we have defined interface binding molecules that do interfere with all heteromeric associations of the ErbB family (Berezov et al., 2002). In this study, we sought to investigate the effect of 2C4 preincubation on heteromeric EGFRp185 her2/neu interactions. Here, 'preincubation' refers to contacting cells on tissue culture plate with the antibody. Cells were washed twice with phosphate-buffered saline (PBS) before they were lysed to release membrane and cytoplasmic proteins.Experiments were pe...

Many diseases are easier to treat and control when detected at an early stage of disease progression. Often, disease-related antigens or biomarkers are shed from the primary site and present in the blood. Unfortunately, there are very few tests capable of detecting these rare biomarkers in the blood. A blood test would be very useful to diagnose the disease earlier, monitor effectiveness of treatments, predict recurrence, and monitor recurrence. There is certainly a need to develop assays that are ultrasensitive, non-invasive and high-throughput. Here we describe several highly sensitive immunological assays we have developed to detect rare serum antigens. Initially we created an assay named immunodetection amplified by T7 RNA polymerase (IDAT). To enhance the effectiveness and streamline the procedure, this assay was amended to the facile amplification system termed fluorescent amplification catalyzed by T7 polymerase technique (FACTT). These assays have been used to analyze the tumor antigen HER2 and the prion protein PrP Sc . They can also be applied to other tumor markers or antigens from a variety of diseases such as cardiovascular disease, rheumatoid arthritis, Alzheimer's disease, Parkinson's disease, and hepatitis. These tests are not limited to testing only serum, but may also be applicable to detecting biomarkers in tissue, saliva, urine, cerebrospinal fluid, etc. Clearly, the FACTT-based technology represents an important step in the detection of rare molecules in fluids or tissues for a variety of diseases.

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