New Tools to Decipher the Role of lncRNAs and Their Protein Interactomes in Hematopoiesis

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Project Summary/Abstract Bioanalytical tools to study long noncoding RNAs (lncRNAs) and their protein interactors are desperately needed. lncRNAs are critical elements in the transcriptional regulation of gene expression. They have been shown to function through several different mechanisms. They may serve directly as gene-regulatory factors, produced by transcription at the genomic site where they act. Alternatively, lncRNAs interact with proteins to control gene expression. For example, lncRNAs can act as a “molecular sink” for proteins that interact with DNA or RNA; in this case, binding of the protein to the lncRNA competes with its interaction with its primary target. lncRNAs can also guide proteins to their DNA targets to either repress or activate transcription. They can act not only in cis, or near their site of transcription, but also in trans, at multiple genomic sites. Finally, lncRNAs can act as platforms upon which molecules can convene to perform a function as a team at a specific location and time (e.g. histone modification complexes). Each of these mechanisms requires the interaction of lncRNAs with a diverse protein cohort. Knowledge of the proteins bound to specific lncRNAs is thus essential information needed to understand mechanisms by which lncRNAs control gene expression. Unraveling this tremendous diversity of interactions demands tools capable of rapid identification and quantification of lncRNA- associated proteins. Despite the great importance of lncRNAs and the proteins that interact with them, there are significant limitations in the tools available to study them. We propose to develop and validate a suite of powerful new tools for the discovery, identification, and quantification of lncRNAs and for the comprehensive proteomic analysis of their protein interactomes. It is known that lncRNAs direct gene expression to modulate cell fate in the hematopoietic system, enabling diversification of gene programming during development. Disrupted lncRNA function can also contribute to malignant transformation in specific myeloid and lymphoid cancers. GATA factor-regulated lncRNAs, discovered in powerful genetic systems (wild type and Gata2 enhancer-mutant mice, GATA-1 genetic complementation system and GATA factor knockdowns in primary human erythroid precursor cells), control human erythroid precursor cell function and erythrocyte development. In initial profiling studies, we have identified 74 GATA factor-regulated lncRNAs. The performance of the new tools developed here will be thoroughly tested on a subset of these 74 lncRNAs to discover GATA factor-dependent regulatory circuits and networks that control hematopoiesis. The proposed research will drive state-of-the-art lncRNA identification and proteomic analysis of the lncRNA interactome, applied to one of the most important regulatory networks in hematopoiesis. We will bring a new and unprecedented level of visibility to the definition of the GATA factor-relevant lncRNA interactome, while developing powerful open-source software tools and detailed experimental protocols.