The function and underlying mechanism of TET1 in myelodysplastic syndromes

项目总结（摘要）： 题目：TET 1在骨髓增生异常综合征中的功能和潜在机制。 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）血细胞减少为特征的疾病（例如，贫血、白细胞减少症和血小板减少症）。 虽然染色体异常、基因突变和一些组蛋白/DNA表观遗传学改变已被报道 在MDS中，MDS发病机制的分子机制尚未被很好地理解。TET1，The 泰特甲基胞嘧啶双加氧酶家族的创始成员，首次被鉴定为 MLL基因与急性髓细胞白血病（AML）的关系与之前认为所有三个泰特基因 (TET1/2/3）可能在癌症中起肿瘤抑制基因的作用，我们最近的工作表明TET 1是 在AML的某些亚型中过表达，并在此类AML的发展中起关键的致癌作用。 然而，TET 1在MDS中的作用仍然未知。最近，在全基因组基因表达分析中， 通过分析一个大型人类原发性MDS患者队列的数据集，我们发现TET 1也异常表达， 在分析的MDS的所有主要亚型中过表达，这在我们的内部MDS中通过qPCR证实 样品Tet 1在各种鼠MDS模型中也过表达。然后我们发现TET 1的敲除 表达显著促进MDS细胞的分化，并迫使野生型TET 1的表达（但不 催化失活的TET 1突变体）引起相反的现象。TET 1消耗也显著抑制 体内MDS进展和减少的血细胞减少。此外，我们还确定了一组潜在/候选人 TET 1在MDS中的靶基因，其中一些基因与MDS的发病机制有关。 目的/假设：TET 1通过表观遗传调节在MDS发病机制中起重要作用 一组必需的靶基因的表达。 具体目的：（1）确定TET 1是否是MDS发生和维持所必需的; (2)为了确定TET 1的强制表达是否可以促进MDS的发生和进展， TET 1的功能是否依赖于其催化活性;和（3）破译分子机制 TET 1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Tet 1/TET 1表达/功能是否是两者所必需的。 发展和维持MDS（目标1）。2)我们将对上述MDS进行功能获得性研究 模型来确定Tet 1/TET 1的强制表达是否可以促进MDS的发展和进展 通过催化活性依赖性机制（Aim 2）。3)我们将进行全基因组ChIP-seq，5 hmC-seq， 和RNA-seq，以鉴定MDS中TET 1的所有直接靶点，然后进行一组验证/功能研究， 在体外和体内的TET 1的顶级目标，阐明TET 1的作用的分子机制（目的3）。

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).