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.
Recent discoveries have uncovered the existence of noncoding RNAs, but their function in different normal and disease processes is not well understood. We have recently described a noncoding RNA which regulates the expression and methylation of a key regulator of blood formation, C/EBPA. In this proposal, we will study this noncoding RNA, describe how it interacts with an enzyme which alters DNA, and develop methods to manipulate this noncoding RNA, which may have important uses in diseases of the blood system and leukemia.
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