The function and underlying mechanism of TET1 in myelodysplastic syndromes

项目摘要(摘要)： 标题：TET1在骨髓增生异常综合征中的作用和潜在机制。 背景：骨髓增生异常综合征(MDS)是一组异质性克隆性造血干细胞 以外周血(PB)细胞减少症(如贫血、白细胞减少和血小板减少)为特征的疾病。 尽管已经报道了染色体异常、基因突变和一些组蛋白/DNA表观遗传学变化 在MDS中，MDS发病的分子机制尚不清楚。Tet1，The Tet甲基胞嘧啶双加氧酶家族的创始成员，最初被确定为 急性髓系白血病(AML)的MLL基因与之前的想法相反，这三个Tet基因都是 (Tet1/2/3)可能在癌症中发挥抑癌基因的作用，我们最近的工作表明TET1是 在某些亚型的AML中过度表达，在此类AML的发生发展中起着关键的致癌作用。 然而，TET1在MDS中的作用仍不清楚。最近，在分析一种全基因组的基因表达时 对大量人类原发MDS患者的数据集进行分析后，我们发现TET1也发生了异常 在分析的所有主要MDS亚型中都过表达，这在我们内部的MDS中得到了qPCR的证实 样本。Tet1在各种小鼠MDS模型中也有过表达。然后我们展示了TET1的敲除 表达显著促进MDS细胞分化，并强制表达野生型TET1(但不 催化失活的TET1突变体)导致了相反的现象。Tet1的耗尽也明显受到抑制 体内MDS进展和减少的细胞减少。此外，我们已经确定了一组潜在的/候选的 TET1在MDS中的靶基因，其中一些基因参与了MDS的发病机制。 目的/假设：Tet1通过表观遗传调控在MDS发病机制中发挥重要作用 一组重要的靶基因的表达。 具体目标：(1)确定MDS的开发和维护是否需要TET1； (2)确定强制表达TET1是否能促进MDS的发生和发展； TET1功能是否依赖于其催化活性；以及(3)破译分子机制(S) TET1在MDS中的病理作用。 研究设计：1)我们将在遗传动物MDS模型和患者来源的MDS模型中进行功能丧失研究 异种移植(PDX)MDS模型，以确定两者是否都需要Tet1/TET1表达/功能 MDS的开发和维护(目标1)。2)我们将与上述MDS进行功能收益研究 确定强制表达Tet1/TET1是否可以促进MDS发生和进展的模型 通过催化活性依赖机制(目标2)。3)我们将进行全基因组芯片序列，5hmC-seq， 和RNA-seq来确定MDS中TET1的所有直接靶点，随后对SET进行验证/功能研究 目的3)研究TET1在体内外的最高靶点，阐明TET1‘S作用的分子机制。

PROJECT SUMMARY (ABSTRACT): Title: The function and underlying mechanism of TET1 in myelodysplastic syndromes. Background: Myelodysplastic syndromes (MDS) is a heterogenous group of clonal hematopoietic stem cell disorders, characterized by peripheral blood (PB) cytopenias (e.g., anemia, leukopenia, and thrombocytopenia). Although chromosomal abnormalities, gene mutations and some histone/DNA epigenetic changes have been reported in MDS, the molecular mechanisms underlying the pathogenesis of MDS have not been well understood. TET1, the founding member of the TET methylcytosine dioxygenase family, was first identified as a fusion partner of the MLL gene in acute myeloid leukemia (AML). In contrast to the previous thought that all three TET genes (TET1/2/3) may function as tumor suppressor genes in cancers, our recent work showed that TET1 is overexpressed in certain subtypes of AMLs, and plays a critical oncogenic role in the development of such AMLs. However, the role of TET1 in MDS remains unknown. Recently, in analysis of a genome-wide gene expression profiling dataset of a large cohort of human primary MDS patients, we found that TET1 is also aberrantly overexpressed in all the major subtypes of MDS analyzed, which was confirmed by qPCR in our in-house MDS samples. Tet1 is also overexpressed in various murine MDS models. We then showed that knockdown of TET1 expression substantially promoted differentiation of MDS cells, and forced expression of wild-type TET1 (but not catalytic inactive TET1 mutant) caused the opposite phenomenon. TET1 depletion also significantly inhibited MDS progression and diminished cytopenias in vivo. Furthermore, we have identified a set of potential/candidate target genes of TET1 in MDS, and some of them have been implicated in the pathogenesis of MDS. Objective/Hypothesis: TET1 plays an essential role in MDS pathogenesis through epigenetically regulating expression of a set of essential target genes. Specific Aims: (1) To determine whether TET1 is required for the development and maintenance of MDS; (2) To determine whether forced expression of TET1 can promote MDS development and progression, and whether TET1 function is dependent on its catalytic activity; and (3) To decipher the molecular mechanism(s) underlying the pathological role of TET1 in MDS. Study Design: 1) We will conduct loss-of-function studies in genetic animal MDS models and patient-derived xeno-transplantation (PDX) MDS models to determine whether Tet1/TET1 expression/function is required for both development and maintenance of MDS (Aim 1). 2) We will conduct gain-of-function studies with the above MDS models to determine whether forced expression of Tet1/TET1 can promote MDS development and progression via a catalytic activity-dependent mechanism (Aim 2). 3) We will perform genome-wide ChIP-seq, 5hmC-seq, and RNA-seq to identify all direct targets of TET1 in MDS, followed by the validation/functional studies of a set of top targets of TET1 in vitro and in vivo, to elucidate the molecular mechanism underlying TET1’s role (Aim 3).