Human lncRNA mediated control of human osteogenesis

SUMMARY Epigenetic control is obligatory for human skeletal development and remodeling. Our program established multiple dimensions of epigenetic control of bone biology, including microRNAs and histone modifications. Nuclear lncRNAs that function predominantly in the organization of chromatin and regulatory networks in bone are minimally understood. During the current funding period we identified and functionally characterized a cohort of chromatin-associated long non-coding RNAs (lncRNAs) that epigenetically contribute to osteoblast phenotype development from human mesenchymal stem cells through stages of osteoblast differentiation. There is a compelling requirement to mechanistically characterize how human lncRNAs control chromatin organization and mediate competency for gene expression to initiate and sustain osteogenesis. Our working hypothesis is that specific lncRNAs function epigenetically to regulate commitment to the human osteoblast lineage and support physiological control of human osteogenesis through higher-order chromatin interactions. We characterized human chromatin-associated lncRNAs that influence osteoblast proliferation and differentiation, prioritizing three lncRNAs (MIR181A1HG, LINC01638 and LINC02341) that are expressed during human mesenchymal stem cell (hMSC) commitment to the osteoblast phenotype. LINC02341 is genetically linked (GWAS) to bone mineral density (BMD) and skeletal pathology, while all three lncRNAs have complementary roles in regulating osteogenic lineage progression. By combining in vivo and in vitro approaches to mechanistically define human lncRNA-mediated epigenetic control of hMSC commitment, our laboratory is investigating chromatin interactions and genomic organization. With longstanding and consistent contributions, we are well positioned to define chromatin architecture that establishes the osteoblast lineage. We will use state- of-the-art multidimensional chromatin conformation technologies (e.g., ATAC-seq, HiChIP) and RNA-DNA hybrid target identification (e.g., ChIRP), multispectral imaging, spatial transcriptomics, and single cell analysis of gene expression and chromatin accessibility. Using these unbiased strategies, we will discover mechanisms by which lncRNAs regulate higher order chromatin structure for hMSC commitment to the osteoblast phenotype. We will utilize CRISPR inhibition, both in vitro and in vivo, to functionally address biological and human clinical relevance by defining the lncRNA activities in promoting osteogenesis. Our strategy will pursue in parallel: in vivo xenograft evaluation of human lncRNA impact on bone formation in human MSC explants that will support species-specific representation and activities of human lncRNAs (Aim 1); regulation of chromatin organization by human lncRNAs during osteogenesis (Aim 2); and direct interactions between human lncRNAs and genomic targets (Aim 3). Impact: Our proposed studies will discover novel dimensions of osteogenesis, epigenetically mediated by long non-coding RNAs, that establish the osteoblast phenotype through chromatin interactions. These studies will advance capabilities for intervention in human skeletal disorders.