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: 9279117
• 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 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9279117
• 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; Hematological Disease; Hematology; Hematopoiesis; Hematopoietic; Hematopoietic System; Hematopoietic stem cells; Human; Immunoprecipitation; Impairment; 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; experimental study; granulocyte; knock-down; leukemia; leukemogenesis; mRNA Expression; monocyte; novel; novel therapeutic intervention; nuclease; overexpression; prevent; public health relevance; 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.

描述（由申请人提供）：该提案是针对PAS-13-031的响应，“刺激血液学研究：新事业（Shine）（Shine）（R01）”，以及响应于NOT-DK-14-001，其研究目的是定义造血期间不编码RNA的生物学功能的研究目标。近年来，一些研究人员透露，复杂生物体的大部分基因组被转录但未翻译。这些非编码RNA（NCRNA）为许多生物过程提供了额外的控制层。最近，我们表明，Cebpa的表达是造血和颗粒的主要调节剂，受到CEBPA基因座的NCRNA的调节：外部编码（EC）CEBPA。 ECCEBPA通过与DNA甲基转移酶1（DNMT1）的相互作用来防止CEBPA基因基因座甲基化。我们的研究表明：（1）eCceBPA的下调导致CEBPA mRNA表达的降低，并显着增加DNA甲基化水平； （2）eCCEBPA的过表达导致mRNA的增加和DNA甲基化的伴随降低。鉴于CEBPA在正常造血疾病中的关键作用，试图了解eCCEBPA在造血分化中的作用，并使用ECCEBPA-DNMT1相互作用的范式作为常见的主要和/或次要识别型基因，以便将基因的特异性脱甲基化工具用于基因。为了实现该计划，最初，将在实验室细胞系的粒细胞/单核细胞分化过程中研究EcceBPA下调的TH效应，然后研究原发性造血祖细胞（CD34+骨髓单核细胞）。同时，将解剖并映射ECCEBPA-DNMT1相互作用，以识别确切的结合序列和相应的二级结构。这种方法将促进基于RNA-寡核苷酸的脱甲基疗法的发展。在体外和细胞培养测定中均将使用细胞系和原代细胞应用。最后，一个数字 模仿eCceBPA功能的RNA-寡核苷酸的作品将旨在诱导/恢复CEBPA表达。此外，我们还将开发其他复合RNA寡核苷酸，其中融合了已鉴定的DNMT1相互作用的ECCEBPA序列，以实现其他基因基因座的基因特异性DNA甲基化。实际上，该基序将通过特定的基因组序列进行设计，这些序列将将RNA寡核苷酸固定在特定基因座上，而DNMT1相互作用的ECCEBPA序列将充当抑制DNMT1的诱饵。这些目标的实现将影响对非编码RNA eCCEBPA在造血分化中的功能作用的基本知识，描述EcceBPA-DNMT1相互作用的性质，并导致一种新型的治疗方式。