Linking RNA to chromatin landscape and organization

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BACKGROUND. Important technological advances in the genomics field have led to the realization that mammalian genomes are much more extensively transcribed than originally thought. Thousands of long non-coding RNAs (lncRNAs) have now been identified, and although the biological relevance of many of them has been demonstrated, the mechanisms by which they regulate the expression of genes remain mostly unknown. Identifying these will help understand why genes are expressed at a given time, and provide insight into the biological contribution and significance of lncRNAs in gene regulation at large. Progress in this field has been hampered by the lack of robust methods to identify bona fide lncRNA chromatin targets. Here, we suggest using the HOTAIRM1 lncRNA as a discovery tool to characterize how these transcripts may regulate gene expression genome-wide during cellular differentiation. Last year, we reported that HOTAIRM1 controls gene expression through changes in chromatin structure and spatial organization. We found that HOTAIRM1 expression is necessary to physically dissociate genes along the HOXA cluster and uncouple their expression. Our preliminary fluorescence in situ hybridization (FISH) data revealed partially overlapping nuclear localizations of HOTAIRM1 variants, with punctate patterns suggesting that they regulate the expression of many genes across the genome. We now suggest developing two novel technologies to find lncRNA gene targets genome-wide. My research will be useful to study any type of non-coding RNAs, and through my program, I will train students from all levels to conduct research at the bench and analyze scientific data computationally. SPECIFIC AIMS. Aim 1. Identify new HOTAIRM1-regulated genes with TIERI. We propose developing a new methodology to identify direct gene targets of given lncRNAs genome-wide. We named this technique TIERI' for “Targeted In situ Extraction of RNA-chromatin Interactions”, and designed it to circumvent many of the known deficiencies of current methods. We will use retinoic acid-induced HOTAIRM1 in NT2-D1 cells to establish and optimize this technique, and identify new genomic regions controlled by this lncRNA. Aim 2. Adapt 2C-ChIP to profile the physical distribution of HOTAIRM1 along target genomic regions. Recently, we devised carbon copy-ChIP (2C-ChIP) to profile ChIP signal from defined genomic regions with deep sequencing. This technique is quantitative and can achieve very high resolutions over large genomic regions. We propose combining 2C-ChIP with TIERI to quantitatively map the actual physical distribution of HOTAIRM1 along the HOXA cluster and new target regions found in Aim 1 with TIERI.