Role of SHP-1 Deregulation in HTLV-1 Leukemogenesis

描述(申请人提供)：人类T细胞嗜淋巴病毒I型(HTLV-I)是成人T细胞白血病(ATL)的病原体。过去的研究已经证明，IL-2受体信号通路的失控可能在HTLV-1导致的CD4+T细胞永生化和致癌转化中发挥重要作用。事实上，在HTLV-1转化细胞和ATL患者新鲜分离的白血病细胞中发现了包括Jak/STAT通路在内的IL-2R复合体的结构性激活。我们和其他人最近证明了SHP-1，一种结构性表达的胞浆蛋白酪氨酸磷酸酶，作为IL-2R信号的早期负调节因子，在HTLV-1转化的细胞中选择性地显著下调。SHP-1通常被招募到IL-2R上的磷酸化对接位置，在那里它可以去磷酸化作为信号复合体一部分的激活的蛋白酪氨酸(JAK)激酶，我们假设SHP-1的下调在HTLV-1白血病的发生中具有核心重要性。在这个方案中，我们提供了新的数据，通过芯片分析表明，在HTLV-1转化细胞中，组蛋白脱乙酰酶HDAC1、组蛋白H3甲基转移酶SUV39H1和HTLV-1 Tax都与SHP-1启动子结合。因此，SHP-1启动子沉默的分子基础似乎是在SHP-1启动子上组装了一个涉及HDAC1、SUV39H1和TAX等未知蛋白的阻遏复合体。在这项建议中，我们将研究这些HTLV-1转化细胞中SHP-1启动子沉默的分子基础。具体地说，我们将首先鉴定和定位SHP-1启动子中的核心启动子区域和转录因子结合部位，并确定在HTLV-1转化和ATL细胞系中转录因子结合是否发生改变。我们还将通过芯片检测来确定在HTLV-1转化的细胞系中是否存在转录抑制物、甲基胞嘧啶结合蛋白或各种染色质重塑复合体与SHP-1启动子结合。我们还将确定SHP-1核心启动子区域是否已甲基化。我们还将在新鲜的CD4+T细胞和未转化的HTLV-1细胞中鉴定SHP-1启动子的“组蛋白密码”，并将其与HTLV-1转化和ATL细胞系进行比较。通过将这些密码与这些细胞中SHP-1的mRNA和蛋白质表达谱进行比较，我们将确定是否可以识别SHP-1表达和沉默的精确组蛋白密码。我们将把这些发现与染色质修饰蛋白的芯片分析相关联，以确定是否可以阐明SHP-1启动子沉默所涉及的生化机制。我们还将确定导致HTLV-1 Tax与SHP-1启动子结合的分子相互作用，并将评估翻译后对Tax的修饰(例如乙酰化、甲基化)，这些修饰可能会影响与转录因子、辅活化子或辅阻遏子的优先结合。最后，我们将检验组蛋白脱乙酰酶和DNA甲基转移酶抑制剂对HTLV-1转化细胞系中SHP-1启动子激活的影响。我们还将研究这种抑制对SHP-1表达和JAK/STAT通路结构性激活的生物学后果。这些研究将与SHP-1基因替换研究相结合，以评估SHP-1重新表达对细胞增殖、活力、细胞周期进展和细胞凋亡的长期影响。这些研究将为HTLV-1白血病发生的生化机制提供新的见解，并有助于开发这种致命疾病的新治疗方法。

DESCRIPTION (provided by applicant): Human T-cell lymphotropic virus I (HTLV-I) is the etiological agent for Adult T-cell Leukemia (ATL). Past studies have provided evidence that deregulation of IL-2 receptor signaling may play an important role in the events that lead to immortalization and oncogenic transformation of CD4+ T-cells by HTLV-1. Indeed, constitutive activation of the IL-2R complex including the Jak/STAT pathway is found in HTLV-1 transformed cells and freshly isolated leukemic cells from ATL patients. We and others have recently demonstrated that Shp-1, a constitutively expressed cytosolic protein tyrosine phosphatase that functions as an early negative regulator of IL-2R signaling is selectively and markedly downregulated in HTLV-1 transformed cells. Shp-1 is normally recruited to phosphorylated docking sites on IL-2R where it can then dephosphorylate activated protein tyrosine (Jak) kinases that are part of the signaling complex and we hypothesize that SHP-1 downregulation is of central importance to HTLV-1 leukemogenesis. In this proposal we provide new data which demonstrates by ChIP assay analysis that both the histone deacetylase HDAC1, the histone H3 methyl transferase SUV39H1 and HTLV-1 Tax are bound to the Shp-1 promoter in the HTLV-1 transformed cells. Thus, the molecular basis of Shp-1 promoter silencing appears to be the assembly of a repressor complex involving HDAC1, SUV39H1 and Tax and other unknown proteins at the Shp-1 promoter. In this proposal we will investigate the molecular basis of Shp-1 promoter silencing in these HTLV-1 transformed cells. Specifically, we will first identify and map the core promoter region and transcription factor binding sites in the Shp-1 promoter and determine if transcription factor binding is altered in HTLV-1 transformed and ATL cell lines. We will also determine by ChIP assay if there is binding of transcriptional repressors, methyl-cytosine binding proteins or various chromatin-remodeling complexes to the Shp-1 promoter in the HTLV-1 transformed cell lines. We will also determine if the Shp-1 core promoter region has been methylated. We will also identify the "histone code" of the Shp-1 promoter in fresh CD4+ T-cells and non-HTLV-1 transformed cells and compare this code to HTLV-1 transformed and ATL cell lines. By comparing these codes to the Shp-1 mRNA and protein expression profiles in these cells we will determine if the precise histone codes for Shp-1 expression and silencing can be identified. We will correlate these findings to the ChIP assay analyses of chromatin modifying proteins to determine if the biochemical mechanisms involved in Shp-1 promoter silencing can be elucidated. We will also determine the molecular interactions that lead to HTLV-1 Tax binding to the Shp-1 promoter and will evaluate post-translational modifications to Tax (e.g. acetylation, methylation) that may influence preferential binding to transcription factors, coactivators or corepressors. Finally, we will examine the effects of inhibitors of histone deacetylase and DNA methyltransferase on activation of the Shp-1 promoter in HTLV-1 transformed cell lines. We will also investigate the biological consequences of this inhibition on Shp-1 expression and constitutive activation of the Jak/STAT pathway. These studies will be coupled with Shp-1 gene replacement studies to evaluate the longer term consequences of Shp-1 re-expression on cell proliferation, viability, cell cycle progression and apoptosis. These studies should provide new insight into the biochemical mechanisms of HTLV-1 leukemogenesis and aid in the development of new treatments for this fatal disease.