Noncoding RNA-DNMT1 interactions in hematopoiesis

描述（由申请人提供）：本提案是为了响应PAS-13-031“刺激血液学研究：新的努力（SHINE）（R 01）"和响应NOT-DK-14-001而提交的，研究目的是定义造血过程中非编码RNA的生物学功能。近年来，一些研究人员发现，复杂生物体的大部分基因组被转录，但不被翻译。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，我们已经表明，CEBPA的表达，造血和粒细胞生成的主调节器，是由来自CEBPA基因座的ncRNA调节：额外编码（ec）CEBPA。ecCEBPA通过与DNA甲基转移酶1（DNMT 1）的相互作用防止CEBPA基因位点甲基化。我们的研究表明：（1）下调ecCEBPA导致CEBPA mRNA表达降低，DNA甲基化水平显著升高;（2）过表达ecCEBPA导致mRNA表达升高，DNA甲基化水平降低。鉴于CEBPA在正常造血中的关键作用，寻求理解ecCEBPA在造血分化中的作用，并使用ecCEBPA-DNMT 1相互作用的范例作为共同的初级和/或二级识别基序，以产生基因特异性去甲基化工具。为了实现这一计划，最初，将在实验室细胞系的粒细胞/单核细胞分化期间研究ecCEBPA下调的作用，随后研究原代造血祖细胞（CD 34+骨髓单核细胞）。同时，ecCEBPA-DNMT 1相互作用将被解剖和映射，以确定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-寡核苷酸的去甲基化治疗的发展。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 将设计模拟ecCEBPA功能的RNA-寡核苷酸以诱导/恢复CEBPA表达。此外，我们还将开发其他复合RNA寡核苷酸，其中包含已鉴定的DNMT 1相互作用ecCEBPA序列，以实现其他基因位点的基因特异性DNA甲基化。实际上，该基序将用特定的基因组序列进行工程化，所述基因组序列将RNA寡核苷酸锚于特定的基因座，而与DNMT 1相互作用的ecCEBPA序列将充当诱饵以抑制DNMT 1。这些目标的实现将影响非编码RNA ecCEBPA在造血分化中的功能作用的基本知识，描绘ecCEBPA-DNMT 1相互作用的性质，并导致一种新型的治疗方式。

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.