We are interested in molecular biology of programmed cell death and cell cycle control, T lymphocyte development and regulation of T-cell receptor gene rearrangement and expression. We are using mouse and human cell lines as well as transgenic and knock-out mice to study these processes.
Programmed cell death or apoptosis is a general phenomenon which occurs in many organisms during development. It is charcterized by chromatin condensation and subsequent cleavage of the genomic DNA into nucleosome-size fragments. In the mouse and human, apoptosis is an integral part of brain development, of lymphocyte development and of several other developmental processes. In the immune system, apoptosis is essential in elimination of cells that recognize self (negative selection). Problems with development of the immune system may lead to autoimmune diseases or cancer.
We are interested in the molecular mechanisms of cell death and have used T lymphocytes as a model system. Apoptosis in T cells can be induced using a variety of means (glucocorticoid, anti-T-cell receptor antibody, anti-fas, etc.). Different pathways are likely to exist for apoptosis caused by these various inducers as not all of them require de novo protein synthesis. Apoptosis by antibody specific for T-cell receptor (anti-TCR) in T cell hybridomas and immature T cells may be related to negative selection and its process requires new gene transcription. We have shown that one of the genes that is rapidly induced during this activation induced apoptosis is Nur77, an orphan steroid receptor, Nur77 is turn on rapidly within 20 minutes of induction by anti-TCR during apoptosis. Although its deduced protein structure is similar to the glucocorticoid receptor, Nur77 does not need any steroid to be active. Dominant negative mutant of Nur77 gene product is required for anti-TCR apoptosis. Various immunosuppressive drugs, which inhibit anti-TCR apoptosis also negatively regulate Nur77 protein expression. More significantly, dominant negative Nur77 can inhibit negative selection when introduced into mice. Constitutive Nur77 expression leads to massive apoptosis. Currently, we are addressing the following questions: What are the genes regulated by Nur77? What upstream events control Nur77 and other cell death genes (bcl-2, bax, ice, c-myc, etc.)? Is phosphorylation of Nur77 crucial for its function?
We are also interested in the signalling events involved in fas mediated apoptosis. Both cytoplasmic tails of fas and p55 TNF receptors contain a conserved domain (the death domain). This domain can presumably transmit a signal, which results in apoptosis. Using this domain as a bait in a yeast 2 hybrid screen, we have isolated several fas-interacting proteins. Characterization of these proteins is in progress to define their role in apoptosis.
Cell cycle control in apoptosis. In addition to apoptosis, anti-TCR signals in T cell hybridomas also lead to G1 arrest. How G1 arrest is linked to apoptosis is an interesting question. We have recently isolated a novel p19 cell cycle inhibitor, based on its ability to associate with Nur77 in a yeast 2 hybrid system. p19 consists of 4 ankyrin repeats and is homologous to the p16ink4 tumor suppressor gene. p19 associates with the G1 cyclin dependent kinase cdk4/cdk6 in vitro and in vivo but not with other cdk proteins or the cyclins. Overexpression of p19 leads to inhibition of the cyclinD/cdk4 kinase activity. Further biochemical and genetic experiments are aimed at understanding mechanisms of cell cycle regulation by p19 and the relationship between cell cycle arrest and apoptosis.
Regulation of rearrangement and expression of T-cell antigen receptor genes (TCR). Based on T cell receptor surface expression and function, T cells can be divided into 2 separate lineages (alpha-beta and gamma-delta T cells). TCR is a heterodimeric protein composed of either alpha-beta or gamma-delta subunits with which T cells can recognize foreign antigen. Each of the genes encoding all four TCR is comprised of several gene segments (V:variable, J:joining, C:constant) which rearrange during development. The process of V(D)J rearrangement is developmentally and tissue-sepcifically regulated. Studies using transgenic mice and cell lines suggest that chromatin accessibility plays a crucial role in this process. The TCR alpha-delta locus is particularly interesting as the delta is located within the alpha gene segments. Differential chromatin accessibility must occur to account for the differential developmental activation of the alpha and delta TCR genes. We have found 8 DNase I hypersensitive sites at 3' of the T cell receptor alpha-delta locus, which contain an LCR activity in transgenic mice. We put forward an LCR competition model to explain the differential accessibility of the alpha versus delta region during T cell development. In this model, the relative gene order to LCR plays an important role in regulation of chromatin structure. Current experiments using knock-out and transgenic mice are underway to test this model.