John L. Rinn
Large Inergenic Non-Coding RNAs (lincRNAs)
There is growing recognition that mammalian cells produce many thousands of large ntergenic transcripts. However, the functional significance of these transcripts has been particularly controversial. While there are some well-characterized examples, the vast majority (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here, we report a new approach to identifying large non-coding RNAs (ncRNAs) by using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified ~1600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening ncRNAs (lincRNAs) exhibit strong purifying selection in their genomic loci, exonic sequences, and promoter regions with greater than 95% showing clear evolutionary conservation. We also developed a novel functional genomics approach that assigns putative functions to each lincRNA, revealing a diverse range of roles for lincRNAs in processes from ES pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NFKB, Sox2, Oc4, and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes.
LincRNAs in Cancer
The largest signature our guilt by association method revealed was comprised of hundreds of lincRNAs that all share functional associations with cell-cycle regulation, proliferation, RNA binding proteins and chromatin remodeling complexes. These results strongly suggested misregulation of lincRNAs would result in tumor formation by loss of epigenetic regulation of cell-cycle regulation genes, oncogenes and/or tumor suppressors. We have further identified candidate "onco-lincRNAs" and "tumor-suppressor lincRNAs" based on associations with key cancer pathways as well as known oncogenic and tumor-suppressor pathways, respectively. In addition, we will look for misregulation of lincRNAs that are strongly associated with cell-cycle regulation, proliferation, RNA binding proteins and chromatin remodeling complexes. By profiling lincRNAs across numerous cancer types we have rapidly identify lincRNAs that could play key roles tumor initiation and/or progression that will be explored for their functional mechanism. In summary, lincRNAs could herald a new paradigm in our understanding of cellular transformation and/or metastasis. Defining the roles of these RNA molecules in cancer could open up new avenues for better diagnostics.
LincRNAs and Chromatin Structure
Recently a lincRNA termed HOTAIR was discovered that is encoded antisense to the HOXC cluster at the exact juncture of a 40 Kb domain of heterochromatin and a 60 Kb domain of euchromatin. However, HOTAIR does not serve to regulate this boundary; Remarkably HOTAIR affects the global epigenetic state of the HOXD cluster located on a separate chromosome. HOTAIR binds the Polycomb Repressive Complex 2 (PRC2) and is required for PRC2 occupancy and histone H3 lysine-27 trimethylation of HOXD locus. Thus, transcription of large ncRNA may associate with chromatin remodeling complexes and guide them to their sites of action. We are employing a combined informatic, biochemical and immunofluorescent approaches to identify other lincRNAs associated with chromatin remodeling complexes. Together our studies aim to understand the roles of lincRNAs in portioning the genome into proper domains of euchromatin and heterochromatin.
LincRNAs in ES Pluripotency
Our "guilt by association" informatics approach revealed a striking class of lincRNAs that appear to be involved in Embryonic Stem (ES) cell pluripotency. These lincRNAs are not only exclusively expressed in ES cells but are also directly transcribed by key transcription factors (Sox2/Oct4/Nanog) that are known to regulate ES pluripotency. However, the ultimate proof that these lincRNAs are required for ES pluripotency would be to perform loss-of-function by depleting lincRNA transcripts and monitoring the resulting phenotypic changes (i.e. loss of self-renewal and/or pluripotency). Here we purpose to identify lincRNAs that are required to maintain ES pluripotency by RNAi-mediated depletion of lincRNA and monitoring the resulting phenotypic changes. To this end we are performing loss of function experiments using shRNAs targeting ES lincRNAs, to screen for ES phenotypes.
RNA Structure and Function
By grouping lincRNAs in to functional groups using our "guilt by association method" we are able to explore how structural motifs within lincRNAs are associate these functions. By using combined biochmecial and informatic methods guided by signatures of evolutionary conservation we are dissecting the key structural elements within lincRNAs that are required for their function. We are also using these same approaches to understand how lincRNAs form ribonucleic-protien complexes.