It is now well-established that noncoding regions of the genome have a vital role to play in gene expression. Genome-wide approaches have led to the identification of an abundance of noncoding RNAs, from microRNAs to long non-coding RNAs (lncRNAs). LncRNAs were initially discovered in the 1980’s but the extent of their transcription was only realized over twenty years later with the advent of improved high-throughput sequencing technologies. Subsequent research has revealed the breadth of cellular processes they are involved in. Maite Huarte from the University of Navarra, Spain and colleagues probe how the novel lncRNA Pint is a target of p53 – a transcription factor that is central to cellular homeostasis and tumour suppression. Huarte explains more about their findings, published in a recent Genome Biology study, that demonstrates how Pint, p53 and epigenetic silencing are all connected.
Your group studies long noncoding RNAs (lncRNAs). What led to your interest in this particular field of research?
I was always interested in how cells manage to execute their gene expression programs with such precision. For this reason I initiated my postdoctoral research at Harvard Medical School, USA in the field of epigenetics. At that time, evidence of the extraordinary richness of the noncoding genome started to emerge and the first exciting examples of lncRNAs involved in epigenetic regulation appeared. This is when I decided to wholly dedicate my studies into lncRNAs and joined John Rinn’s laboratory at the Broad Institute of MIT and Harvard. Now I have established an independent group in Spain dedicated to the research of lncRNAs in cancer, where we have performed this study.
Could you explain what lncRNAs are and why they are important?
LncRNAs are RNA molecules longer than 200 nucleotides that lack functional open reading frames – the reason why they have been long considered part of the ‘genomic junk’. Now we know that lncRNAs are present at virtually every aspect of cell biology, but our knowledge of them is still very limited. The few available studies show that lncRNAs are highly diverse and can modulate gene expression through multiple mechanisms. For instance, lncRNAs can act as molecular scaffolds that hold and guide chromatin complexes, enhance gene transcription, interfere with the transcription machinery, or even maintain the structure of nuclear speckles. Additionally, some may work post-transcriptionally as regulators of splicing, mRNA decay, protein translation, or as molecular decoys for microRNAs. Importantly, alterations in lncRNAs are observed in many diseases, including cancer. For this reason they are emerging as attractive therapeutic targets.
In your study you describe a novel lncRNA, Pint, that you studied in mouse cells. What were the main findings of these experiments? What did you uncover about the function of Pint?
We have identified Pint, a ubiquitously expressed lincRNA (long intergenic noncoding RNA) that is finely controlled by the tumor suppressor p53. We found that in mouse cells, Pint regulates the expression of genes of the TNF-b, MAPK and p53 pathways, promoting proliferation and survival. We therefore decided to investigate the mechanism of gene regulation by Pint, and found that it has an effect on the epigenome: Pint is a nuclear lincRNA that interacts with Polycomb Repressive Complex 2 (PRC2), an important epigenetic regulator, and is required for PRC2 targeting of specific genes for their repression. In fact, we observed that Pint functional activity is highly dependent on PRC2 expression.
How did you identify human PINT?
After finding Pint in the mouse genome, we wondered if an equivalent lincRNA exists in human cells. Indeed, we identified a human transcript that is produced from the human syntenic region. Despite relatively low sequence homology, human PINT presents suggestive analogies with the mouse lincRNA (Pint): PINT is similarly regulated by p53, and its expression correlates with the same cellular pathways as the mouse ortholog, including the p53 pathway. Interestingly, PINT is under-expressed in colon cancer, while its enforced expression inhibits the proliferation of tumor cells. These data are consistent with a possible role of PINT as a tumor suppressor lncRNA.
What do we know about lncRNAs relationship with the p53 pathway? How can this knowledge be useful to us?
We know that p53 controls the expression of many lncRNAs, and we are only starting to understand the biological meaning of this. Our work is revealing that these p53-regulated lncRNAs can effectively fine-tune the p53 response. Our findings help understand how lncRNAs contribute to gene networks in general and, in the particular case of p53, have important implications: lncRNAs have an impact in the way our cells respond to stresses that trigger cancer, and this should be taken into account in the design of novel therapeutic approaches.
You discovered that human PINT can behave like a tumor suppressor. Do you think there is diagnostic and/or therapeutic potential here?
The lncRNA field is still in its early stages, but we can already foresee that lncRNAs like PINT may offer some advantages as prognostic biomarkers. For instance, as opposed to mRNAs, the lncRNA itself is a functional molecule and its expression levels may be better indicators of the disease. Additionally, although the possibility is still distant, therapeutic expression of PINT may carry fewer negative effects than that of protein coding genes, given that it regulates specific facets of p53 and PRC2.
What are your plans for future studies?
There are still many unresolved questions that we continue to investigate. First of all, regarding the mechanism of gene repression: How can Pint-PRC2 specifically recognize and target specific genes? Are there other proteins/RNAs involved in this repressive complex? Another aspect that interests us is the role of human PINT as a tumor suppressor: How does PINT loss affect the biology of a tumor? Is the mechanism conserved between mouse and human? These are some of the questions that we would like to answer with our future work.
Maite Huarte obtained her PhD in the laboratory of Amelia Nieto at the National Center for Biotechnology (CSIC), Universidad Autónoma de Madrid, Spain, where she studied influenza virus-host interactions. Huarte then moved to the USA for her postdoctoral training at Harvard University, USA and later at the Massachusetts Institute of Technology, USA in the laboratories of Yang Shi and John Rinn, respectively. During this time she identified new histone demethylase enzymes and investigated their relationship with epigenetic regulation, as well as the role of long non-coding RNAs (lncRNAs) in gene expression. Since 2011 Huarte has been the director of the ‘Regulation of Gene Expression’ laboratory in the Oncology Department of Center for Applied Medical Research, University of Navarra, Spain. Her research interests center on how lncRNAs contribute to the mechanisms of gene regulation at the epigenetic and non-epigenetic levels in cancer cells and combines both molecular and cell biology techniques with functional genomics, in vivo studies and the analysis of patient samples.
Pint lincRNA connects the p53 pathway with epigenetic silencing by the Polycomb repressive complex 2
Marin-Bejar O, Marchese FP, Athie A, Sanchez Y, Gonzalez J, Segura V, Huang L, Moreno I et al.