Role of SHP-1 Deregulation in HTLV-1 Leukemogenesis

描述（由申请人提供）：人t细胞嗜淋巴病毒I （HTLV-I）是成人t细胞白血病（ATL）的病原。过去的研究已经提供证据表明，IL-2受体信号的失调可能在HTLV-1导致CD4+ t细胞的永生化和致癌转化的事件中发挥重要作用。事实上，在HTLV-1转化细胞和ATL患者新分离的白血病细胞中发现了包括Jak/STAT通路在内的IL-2R复合物的组成性激活。我们和其他人最近已经证明，在HTLV-1转化的细胞中，作为IL-2R信号早期负调节因子的组成性表达的细胞质蛋白酪氨酸磷酸酶Shp-1被选择性地显著下调。Shp-1通常被募集到IL-2R上磷酸化的对接位点，在那里它可以使激活的蛋白酪氨酸（Jak）激酶去磷酸化，这些激酶是信号复合体的一部分，我们假设Shp-1下调对htlc -1白血病的发生至关重要。在本文中，我们提供了新的数据，通过ChIP实验分析证明，在HTLV-1转化的细胞中，组蛋白去乙酰化酶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抑制因子复合物的组装，该复合物涉及HDAC1、SUV39H1和Tax以及Shp-1启动子上的其他未知蛋白。在本研究中，我们将研究HTLV-1转化细胞中Shp-1启动子沉默的分子基础。具体来说，我们将首先识别和绘制Shp-1启动子中的核心启动子区域和转录因子结合位点，并确定转录因子结合是否在HTLV-1转化和ATL细胞系中发生改变。我们还将通过ChIP检测确定HTLV-1转化细胞系中是否存在转录抑制因子、甲基胞嘧啶结合蛋白或各种染色质重塑复合物与Shp-1启动子的结合。我们还将确定Shp-1核心启动子区域是否已经甲基化。我们还将鉴定新鲜CD4+ t细胞和非HTLV-1转化细胞中Shp-1启动子的“组蛋白代码”，并将该代码与HTLV-1转化和ATL细胞系进行比较。通过将这些编码与这些细胞中的Shp-1 mRNA和蛋白表达谱进行比较，我们将确定是否可以确定Shp-1表达和沉默的精确组蛋白编码。我们将把这些发现与染色质修饰蛋白的ChIP分析联系起来，以确定是否可以阐明Shp-1启动子沉默的生化机制。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，并将评估可能影响与转录因子、辅激活因子或辅抑制因子优先结合的翻译后修饰（例如乙酰化、甲基化）。最后，我们将研究组蛋白去乙酰化酶和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通路组成性激活的生物学后果。这些研究将与Shp-1基因替代研究相结合，以评估Shp-1再表达对细胞增殖、活力、细胞周期进展和凋亡的长期影响。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.