Resistance to TGFß suppression and improved anti-tumor responses in CD8+ T cells lacking PTPN22.

Transforming growth factor ß (TGFß) is important in maintaining self-tolerance and inhibits T cell reactivity. We show that CD8+ T cells that lack the tyrosine phosphatase Ptpn22, a major predisposing gene for autoimmune disease, are resistant to the suppressive effects of TGFß. Resistance to TGFß suppression, while disadvantageous in autoimmunity, helps Ptpn22 -/- T cells to be intrinsically superior at clearing established tumors that secrete TGFß. Mechanistically, loss of Ptpn22 increases the capacity of T cells to produce IL-2, which overcomes TGFß-mediated suppression. These data suggest that a viable strategy to improve anti-tumor adoptive cell therapy may be to engineer tumor-restricted T cells with mutations identified as risk factors for autoimmunity.

A Protein Interaction between ß-Catenin and Dnmt1 Regulates Wnt Signaling and DNA Methylation in Colorectal Cancer Cells.

UNLABELLED: Aberrant activation of the Wnt signaling pathway is an important step in the initiation and progression of tumor development in diverse cancers. The central effector of canonical Wnt signaling, ß-catenin (CTNNB1), is a multifunctional protein, and has been extensively studied with respect to its roles in cell-cell adhesion and in regulation of Wnt-driven transcription. Here, a novel mass spectrometry-based proteomics technique in colorectal cancer cells expressing stabilized ß-catenin, was used to identify a protein-protein interaction between ß-catenin and DNA methyltransferase I (Dnmt1) protein, the primary regulator of DNA methylation patterns in mammalian cells. Dnmt1 and ß-catenin strongly colocalized in the nuclei of colorectal cancer cells, and the interaction is mediated by the central domain of the Dnmt1 protein. Dnmt1 protein abundance is dependent upon the levels of ß-catenin, and is increased in cells expressing stabilized mutant ß-catenin. Conversely, the Dnmt1 regulates the levels of nuclear ß-catenin and ß-catenin/TCF-driven transcription. In addition, lysine-specific demethylase 1 (LSD1/KDM1A), a regulator of DNMT1 stability, was identified as a component of the Dnmt1-ß-catenin protein complex and perturbation of the Dnmt1-ß-catenin interaction altered DNA methylation. In summary, a functional protein-protein interaction was identified between two critically important oncoproteins, in turn revealing a link between Wnt signaling and downstream nuclear functions mediated by Dnmt1. IMPLICATIONS: Two critical oncoproteins, Dnmt1 and ß-catenin, mutually regulate one each other's levels and activities in colorectal cancer cells.

Different roles of the human Orc6 protein in the replication initiation process.

In eukaryotes, binding of the six-subunit origin recognition complex (ORC) to DNA provides an interactive platform for the sequential assembly of pre-replicative complexes. This process licenses replication origins competent for the subsequent initiation step. Here, we analyze the contribution of human Orc6, the smallest subunit of ORC, to DNA binding and pre-replicative complex formation. We show that Orc6 not only interacts with Orc1-Orc5 but also with the initiation factor Cdc6. Biochemical and imaging experiments reveal that this interaction is required for licensing DNA replication competent. Furthermore, we demonstrate that Orc6 contributes to the interaction of ORC with the chaperone protein HMGA1a (high mobility group protein A1a). Binding of human ORC to replication origins is not specified at the level of DNA sequence and the functional organization of origins is poorly understood. We have identified HMGA1a as one factor that might direct ORC to AT-rich heterochromatic regions. The systematic analysis of the interaction between ORC and HMGA1a revealed that Orc6 interacts with the acidic C-terminus of HMGA1a and also with its AT-hooks. Both domains support autonomous replication if targeted to DNA templates. As such, Orc6 functions at different stages of the replication initiation process. Orc6 can interact with ORC chaperone proteins such as HMGA1a to facilitate chromatin binding of ORC and is also an essential factor for pre-RC formation.

Binding of CCCTC-binding factor in vivo to the region located between Rep* and the C promoter of Epstein-Barr virus is unaffected by CpG methylation and does not correlate with Cp activity.

In this study, the binding of the insulator protein CCCTC-binding factor (CTCF) to the region located between Rep* and the C promoter (Cp) of Epstein-Barr virus (EBV) was analysed using chromatin immunoprecipitation and in vivo footprinting. CTCF binding was found to be independent of Cp usage in cell lines corresponding to the major EBV latency types. Bisulfite sequencing and an electrophoretic mobility-shift assay (using methylated and unmethylated probes) revealed that CTCF binding was insufficient to induce local CpG demethylation in certain cell lines and was unaffected by CpG methylation in the region between Rep* and Cp. In addition, CTCF binding to the latency promoter, Qp, did not correlate with Qp activity.