SATB1-protein Interaction/function at the BcL2 mbr/MAR

描述(由申请者提供)：该奖项将允许候选人在希奥多·G·克朗蒂里斯位于希望之城国家医学中心的实验室获得额外一年的细胞生物学和成像技术以及定向突变技术方面的指导培训。经过这段时间的指导培训后，候选人将留在希望之城担任研究科学家的职位，主要负责研究工作。这种培训和知识的获得对于她未来的目标是了解Bcl2t(14；18)易位的机制至关重要，Bcl2t(14；18)易位是滤泡性淋巴瘤发病的一个诱发因素。紧接在Bcl2主断点区下游的是一个富含AT的区域，它结合了一种胸腺细胞特异性的富含AT的序列结合蛋白L(SATB 1)和MAR相关蛋白。SATB 1过表达下调Bcl2的表达。在体内，连接介导的聚合酶链式反应(LM-PCR)的DNase I足迹显示了Bcl2 MBR的结构性和细胞型特异性蛋白结合，以及依赖于SATB1的DNA螺旋构型的改变。最近，酵母双杂交系统(Y2H)被用来鉴定大量与SATB 1相互作用的蛋白质。其中许多蛋白质的所谓功能(S)对MAR相关蛋白(即染色质重塑和转录)似乎是合理的。出乎意料的是，SATB 1显示了与蛋白质修饰因子SUMO-1以及额外的SUMO-1酶分子的排他性相互作用。我们建议将我们的研究扩展如下：(1)通过生化、生物物理和免疫学方法表征SATB1与SUMO-1修饰蛋白以及特定转录和染色质重塑因子的相互作用。(2)对Bcl2 MBR/MAR进行定向突变，以确定哪些DNA序列参与了MAR的功能。使用这些靶细胞系来建立SATB 1和MARBc12对特定蛋白质结合的贡献，以及对Bcl2 MBR/MAR染色质构象和DNA拓扑的贡献。我们预计，这些研究的成功完成将阐明导致疾病状态的细胞特异性蛋白质和DNA功能的机制。

DESCRIPTION (provided by applicant): This award will allow the candidate to acquire an additional year of mentored training in techniques of cell biology and imaging technology, and targeted mutagenesis, in the laboratory of Theodore G. Krontiris at the City of Hope National Medical Center. After this period of mentored training, the candidate will remain at the City of Hope in a Research Scientist position, with responsibilities directed primarily towards research. This training and acquisition of knowledge is essential for her future goals directed towards understanding the mechanism of the Bcl2 t(14;18) translocation which is an instigating factor in the onset of follicular lymphoma. Immediately downstream of the Bcl2 major breakpoint region (mbr) is an AT-rich region that binds special AT-rich sequence binding protein l (SATB1), a 'thymocyte-specific', and MAR associating protein. SATB 1 over-expression down-regulates Bcl2 expression. In vivo DNase I footprinting by ligation-mediated PCR (LM-PCR) revealed constitutive and cell-type specific protein binding at the Bcl2 mbr and also an altered DNA helical configuration dependent upon SATB1. More recently, the yeast two-hybrid system (y2h) was used to identify numerous proteins that interact with SATB 1. The purported function(s) of many of these proteins appears logical for MAR-associating proteins (i.e., chromatin remodeling and transcription). Unexpectedly, SATB 1 demonstrated exclusive interaction with SUMO-1, a protein modifying factor, and with additional SUMO-1 enzymatic molecules. We propose to extend our studies as follows: (1) Characterize, through biochemical, biophysical, and immunological approaches, the interaction of SATB1 with SUMO-1 modifying proteins and with select transcription and chromatin remodeling factors. (2) Use targeted mutagenesis of the Bcl2 mbr/MAR to establish which DNA sequences contribute to MAR function. Use these targeted cell lines to establish the contribution of SATB 1 and MARBc12 to specific protein binding, and to chromatin conformation and DNA topology at the Bcl2 mbr/MAR. We anticipate that successful completion of these studies will elucidate mechanisms that contribute to cell-specific protein and DNA functions that lead to disease state.