Noncoding RNA-DNMT1 interactions in hematopoiesis

非编码 RNA-DNMT1 在造血过程中的相互作用

• 批准号: 9087226
• 负责人: DANIEL G TENEN
• 金额: $ 26.1万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-15 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9087226
• 关键词: Aberrant DNA Methylation; Acetates; Achievement; Address; Affect; Binding; Biological; Biological Assay; Biological Process; Blood; Bone Marrow; CD34 gene; CEBPA gene; Cell Culture Techniques; Cell Line; Cell model; Cells; Chemicals; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA; DNA Methylation; DNA Modification Methylases; Development; Disease; Down-Regulation; Engineering; Enzymes; Gene Expression; Genes; Genetic Transcription; Genome; Genome engineering; Genomics; Granulopoiesis; Guide RNA; HL60; Health; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic System; Human; Immunoprecipitation; In Vitro; Investigation; Knock-out; Knowledge; Laboratories; Lead; Maps; Mediating; Messenger RNA; Methods; Methylation; Modality; Molecular Profiling; Mononuclear; Myeloid Leukemia; Nature; Noise; Oligonucleotides; Organism; Phorbols; Play; Process; RNA; Regulation; Regulatory Element; Research; Research Personnel; Role; Structure; System; Technology; Testing; Therapeutic; Translating; Tretinoin; Untranslated RNA; base; demethylation; design; knock-down; leukemia; leukemogenesis; mRNA Expression; novel; novel therapeutic intervention; nuclease; overexpression; prevent; progenitor; research study; response; small hairpin RNA; stem; tool

DESCRIPTION (provided by applicant): This proposal is submitted in response to PAS-13-031, "Stimulating Hematology Investigation: New Endeavors (SHINE) (R01)", and in response to NOT-DK-14-001, with the research objective of defining biologic function of noncoding RNAs during hematopoiesis. Several researchers in recent years have revealed that a large portion of the genome of complex organisms are transcribed but not translated. These noncoding RNAs (ncRNAs) provide an additional layer of control to many biological processes. Recently we have shown that the expression of CEBPA, a master regulator of hematopoiesis and granulopoiesis, is regulated by a ncRNA stemming from the CEBPA locus: the extra-coding (ec)CEBPA. The ecCEBPA prevents CEBPA gene locus methylation through its interaction with DNA Methyltransferase 1 (DNMT1). Our studies demonstrated that: (1) downregulation of ecCEBPA led to a decrease of CEBPA mRNA expression and to a significant increase in DNA methylation levels; and (2) overexpression of ecCEBPA resulted in increase of mRNA and concomitant decrease in DNA methylation. Given the pivotal role of CEBPA in normal hematopoiesis, seek to understand the role of ecCEBPA in hematopoietic differentiation and use the paradigm of the ecCEBPA-DNMT1 interaction as a common primary and/or secondary recognition motif to employ in order to generate a gene-specific demethylating tool. To fulfill this plan, initially, th effect of ecCEBPA downregulation will be studied during granulocytic/monocytic differentiation of laboratory cell lines and, later, primary hematopoietic progenitors (CD34+ bone marrow mononuclear cells). In parallel, the ecCEBPA-DNMT1 interaction will be dissected and mapped to identify the exact binding sequence and corresponding secondary structure. This approach will facilitate the development of an RNA-oligonucleotide based demethylating therapy. Both in vitro and cell culture assays will be applied using cell lines and primary cells. Finally, a number of RNA-oligonucleotides mimicking ecCEBPA function will be designed to induce/restore CEBPA expression. In addition, we will develop other composite RNA oligonucleotides incorporating the identified DNMT1-interacting ecCEBPA sequences to achieve gene-specific DNA methylation of other gene loci. Indeed, this motif will be engineered with specific genomic sequences that will anchor the RNA oligonucleotides to a specific locus while the DNMT1-interacting ecCEBPA sequence will act as a bait to inhibit DNMT1. Achievement of these aims will impact the basic knowledge of the functional role of the noncoding RNA ecCEBPA in hematopoietic differentiation, delineate the nature of ecCEBPA-DNMT1 interaction, and lead to a novel type of therapeutic modality.

说明(申请人提供)：本提案是应PAS-13-031《刺激性血液学研究：新的努力(SIRE)(R01)》和NOT-DK-14-001的响应而提交的，研究目的是确定非编码RNA在造血过程中的生物学功能。近年来，一些研究人员发现，复杂生物体的很大一部分基因组是转录的，但不是翻译的。这些非编码RNA(NcRNAs)为许多生物过程提供了额外的控制层。最近，我们发现CEBPA的表达是由CEBPA基因的一个ncRNA调控的，CEBPA是造血和粒细胞生成的主要调节者，它是超外编码的CEBPA。EcCEBPA通过与DNA甲基转移酶1(DNMT1)相互作用，阻止CEBPA基因位点甲基化。我们的研究表明：(1)ecCEBPA基因表达下调导致CEBPA基因表达下降，DNA甲基化水平显著升高；(2)ecCEBPA基因过表达导致基因表达增加，DNA甲基化水平降低。鉴于CEBPA在正常造血中的关键作用，试图了解ecCEBPA在造血分化中的作用，并将ecCEBPA-DNMT1相互作用的范例用作常见的初级和/或次级识别基序，以产生基因特异性的去甲基化工具。为了实现这一计划，首先将研究ecCEBPA下调在实验室细胞系粒/单核细胞分化过程中的作用，然后研究初级造血祖细胞(CD34+骨髓单个核细胞)分化过程中ecCEBPA下调的影响。同时，将对ecCEBPA-DNMT1相互作用进行解剖和映射，以确定准确的结合序列和相应的二级结构。这一方法将促进基于RNA寡核苷酸的去甲基化疗法的发展。将使用细胞系和原代细胞进行体外和细胞培养试验。最后，一个数字 将设计一系列模拟ecCEBPA功能的RNA寡核苷酸来诱导/恢复CEBPA的表达。此外，我们还将开发与识别的DNMT1相互作用的ecCEBPA序列相结合的其他复合RNA寡核苷酸，以实现其他基因座的基因特异性DNA甲基化。事实上，这个基序将被用特定的基因组序列改造，这些序列将RNA寡核苷酸锚定在特定的位点上，而与DNMT1相互作用的ecCEBPA序列将作为一个诱饵来抑制DNMT1。这些目标的实现将影响对非编码RNA ecCEBPA在造血分化中功能作用的基础知识，描述ecCEBPA-DNMT1相互作用的性质，并导致一种新型的治疗方式。