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Final Report Summary - WNTLINCS (Identification and functional and mechanistic characterization of Wnt-regulated long intergenic non-coding RNAs)

The canonical Wnt pathway is a cell signaling cascade which plays a central role in the maintenance of stem cells and the switch from proliferation to differentiation in the adult, self-renewing intestinal epithelium. It controls cell fate by regulating the activity of TCF/LEF proteins, the transcription factors at the endpoint of signaling cascade. In the absence of Wnt signaling, the levels of β-catenin, the key molecular effector of the pathway, are kept low by a destruction complex encompassing a large number of proteins - AXIN, APC and GSK3 among others. Upon physiological or mutational Wnt-pathway activation, the destruction complex is inactivated; β-catenin moves to the nucleus, binds the transcription factor TCF4, the major TCF/LEF family member in the intestine, and co-activates a transcriptional program characteristic of stem/progenitor cells. Constitutive nuclear localization of β-catenin and the subsequent aberrant transcriptional activity of the TCF4/β-catenin complex, caused by mutations in APC, AXIN or β-catenin, is the primary transforming factor in colorectal cancer.
The goal of Dr Hatzis’ laboratory is to characterize novel players at the endpoint of the Wnt pathway: novel target genes and effectors that mediate the transcriptional output of the pathway. More specifically, one aim of his laboratory is to identify long non-coding RNAs (lncRNAs) that are transcriptionally regulated by the Wnt pathway and in turn mediate part of its transcriptional output. lncRNAs are novel RNA species, defined as molecules larger than 200 nucleotides that do not encode proteins, but have diverse regulatory roles in the cell. Tens of thousands of lncRNAs have been identified in a multitude of organisms, tissues and physiological and pathological settings in recent years.
To identify such Wnt-regulated lncRNAs, the Hatzis lab took advantage of high-throughput sequencing experiments performed, among others, in colorectal carcinoma cell lines engineered to inducibly shut down the mutationally overactive Wnt pathway. RNA-seq and ChIP-seq with antibodies against the largest subunit of RNA polymerase II (polII) in these cells, before and after Wnt-pathway abrogation, reveals sites of Wnt-dependent transcription.
Many dozens of these sites correspond to long non-coding RNAs (lncRNAs) that are transcriptionally regulated by the Wnt pathway. The Hatzis laboratory has focused on the functional characterization of a number of those, with an emphasis on lncRNAs localized in the nucleus. Specifically, WiNTRLINC1, WiNTRLNC2 and WiNTRLINC3 (which stands for Wnt-regulated long (intergenic) non-coding RNA) are the focus of more intense study. For WiNTRLINC1 and WiNTRLNC2, it was established with chromatin immunoprecipitation and reporter assays that they are direct positive targets of TCF4/β-catenin in colorectal cancer cells. WiNTRLINC3, on the other hand is negatively regulated by the pathway. Knock-down experiments in CRC cells demonstrated that the absence of each lncRNA affects the expression of hundreds of genes and dramatically reduces cell growth, by inducing apoptosis and/or cell-cycle arrest.
WiNTRLINC1 positively and directly regulates the expression of one gene in particular, its close genomic neighbor ASCL2, a transcription factor that controls intestinal stem cell fate. WiNTRLINC1 interacts with TCF4/β-catenin to mediate the juxtaposition/physical contact of its own promoter with the regulatory regions of ASCL2, looping out the intervening DNA. ASCL2, in turn, regulates WiNTRLINC1 expression; the two genes thus form a feedforward regulatory loop which controls stem cell-related gene expression and is dramatically amplified in colorectal cancer and correlates with worse disease progression and other clinical features. Further analyses have uncovered the amplification of the WiNTRLINC1-ASCL2 regulatory axis in other forms of cancer and its involvement in genetic predisposition to certain cancer types; ongoing experiments are aimed at elucidating the mechanistic basis of the WiNTRLINC1-ASCL2 contribution to non-intestinal carcinogenesis.
Further ongoing work is focused on uncovering the mechanistic contribution of WiNTRLNC2 and WiNTRLINC3 to carcinogenesis in the intestine (and other tissues). Preliminary work has uncovered protein partners of the lncRNAs and is establishing their trans-acting function in the regulation of gene expression; they appear to regulate target genes at great genomic distances, conceptually complementing the cis-acting function of WiNTRLINC1.
The project on the whole has shed new light on novel players in the Wnt signaling pathway, with roles in homeostasis and carcinogenesis in the intestine and beyond. It has expanded the list of Wnt pathway target genes that are amenable to diagnostic and prognostic exploitation and therapeutic manipulation for the treatment of colorectal and other cancers.
One of the more exciting outcomes of the completed and ongoing experiments is their contribution to illuminating a fundamental problem in biology: it has recently become apparent that a much larger proportion of the eukaryotic genome (up to 90% by some estimates) is transcribed than was previously appreciated. The significance of this pervasive, developmentally and homeostatically regulated transcription of novel, mostly non-protein-coding, RNA species is the object of intense scrutiny. The WNTLINCS project has contributed to these investigations, by studying a subset of these RNA molecules, their regulation by and effect on the Wnt signaling pathway and their contribution to cellular homeostasis and carcinogenesis.

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