Ulf Andersson Ørom

Long noncoding RNAs with enhancer-like function in human cells

While the long noncoding RNAs (ncRNAs) constitute a large portion of the mammalian transcriptome, their biological functions has remained elusive. A few long ncRNAs that have been studied in any detail silence gene expression in processes such as X-inactivation and imprinting. We used a GENCODE annotation of the human genome to characterize over a thousand long ncRNAs that are expressed in multiple cell lines. Unexpectedly, we found an enhancer-like function for a set of these long ncRNAs in human cell lines. Depletion of a number of ncRNAs led to decreased expression of their neighboring protein-coding genes, including the master regulator of hematopoiesis, SCL (also called TAL1), Snai1 and Snai2. Using heterologous transcription assays we demonstrated a requirement for the ncRNAs in activation of gene expression. These results reveal an unanticipated role for a class of long ncRNAs in activation of critical regulators of development and differentiation.

Activating RNAs associate with Mediator to enhance chromatin architecture and transcription

Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms. Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions. We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis-mediated mechanism. To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo, Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

Long Noncoding RNAs Usher In a New Era in the Biology of Enhancers

Enhancer-associated long noncoding RNAs act over long distances and across chromosomes to activate transcription at distal promoters. Here, we address the latest advances made toward understanding the role of long noncoding RNA expression and the involvement of these RNAs in enhancer function through association with protein factors and modulation of chromatin structure.

Long non-coding RNAs and enhancers

Long non-coding RNAs (ncRNAs) are emerging as important regulatory factors in mammalian genomics. A number of reports within the last 2 years have identified thousands of actively expressed long ncRNA transcripts with distinct properties. The long ncRNAs show differential expression patterns and regulation in a wide variety of cells and tissues, adding significant complexity to the understanding of their biological role. Furthermore, genome-wide studies of transcriptional enhancers based on chromatin modifications and enhancer binding proteins have led to the identification of putative enhancers and provided insight into their tissue-specific regulation of gene expression. In an exciting turn of events, new evidence is indicating that long ncRNAs are associated with enhancer regions and that such non-coding transcription correlate with the increased activity of the neighboring genes. Moreover, additional experiments suggest that enhancer-function can be mediated through a transcribed long ncRNA and that this might be a common function for long ncRNAs. Here, we review recent advances made both in the genome-wide characterization of enhancers and in the identification of new classes of long ncRNAs, and discuss the functional overlap of these two classes of regulatory elements.

Identification of a long non-coding RNA-associated RNP complex regulating metastasis at the translational step

Long non‐coding RNAs (lncRNAs) are a novel class of regulatory genes that play critical roles in various processes ranging from normal development to human diseases such as cancer progression. Recent studies have shown that lncRNAs regulate the gene expression by chromatin remodelling, transcription, splicing and RNA decay control, enhancer function, and epigenetic regulation. However, little is known about translation regulation by lncRNAs. We identified a translational regulatory lncRNA (treRNA) through genome‐wide computational analysis. We found that treRNA is upregulated in paired clinical breast cancer primary and lymph‐node metastasis samples, and that its expression stimulates tumour invasion in vitro and metastasis in vivo. Interestingly, we found that treRNA downregulates the expression of the epithelial marker E‐cadherin by suppressing the translation of its mRNA. We identified a novel ribonucleoprotein (RNP) complex, consisting of RNA‐binding proteins (hnRNP K, FXR1, and FXR2), PUF60 and SF3B3, that is required for this treRNA functions. Translational suppression by treRNA is dependent on the 3′UTR of the E‐cadherin mRNA. Taken together, our study indicates a novel mechanism of gene regulation by lncRNAs in cancer progression.

Noncoding RNAs and enhancers: complications of a long-distance relationship

Spatial and temporal regulation of gene expression is achieved through instructions provided by the distal transcriptional regulatory elements known as enhancers. How enhancers transmit such information to their targets has been the subject of intense investigation. Recent advances in high throughput analysis of the mammalian transcriptome have revealed a surprising result indicating that a large number of enhancers are transcribed to noncoding RNAs. Although long noncoding RNAs were initially shown to confer epigenetic transcriptional repression, recent studies have uncovered a role for a class of such transcripts in gene-specific activation, often from distal genomic regions. In this review, we discuss recent findings on the role of long noncoding RNAs in transcriptional regulation, with an emphasis on new developments on the functional links between long noncoding RNAs and enhancers.

PROmiRNA: a new miRNA promoter recognition method uncovers the complex regulation of intronic miRNAs

The regulation of intragenic miRNAs by their own intronic promoters is one of the open problems of miRNA biogenesis. Here, we describe PROmiRNA, a new approach for miRNA promoter annotation based on a semi-supervised statistical model trained on deepCAGE data and sequence features. We validate our results with existing annotation, PolII occupancy data and read coverage from RNA-seq data. Compared to previous methods PROmiRNA increases the detection rate of intronic promoters by 30%, allowing us to perform a large-scale analysis of their genomic features, as well as elucidate their contribution to tissue-specific regulation. PROmiRNA can be downloaded from http://promirna.molgen.mpg.de.

Long noncoding RNAs as enhancers of gene expression

The human genome contains thousands of long noncoding RNAs (ncRNAs) transcribed from diverse genomic locations. A large set of long ncRNAs is transcribed independent of protein-coding genes. We have used the GENCODE annotation of the human genome to identify 3019 long ncRNAs expressed in various human cell lines and tissue. This set of long ncRNAs responds to differentiation signals in primary human keratinocytes and is coexpressed with important regulators of keratinocyte development. Depletion of a number of these long ncRNAs leads to the repression of specific genes in their surrounding locus, supportive of an activating function for ncRNAs. Using reporter assays, we confirmed such activating function and show that such transcriptional enhancement is mediated through the long ncRNA transcripts. Our studies show that long ncRNAs exhibit functions similar to classically defined enhancers, through an RNA-dependent mechanism.

Long ncRNA expression associates with tissue-specific enhancers.

Long non-coding RNAs (ncRNA) have recently been demonstrated to be expressed from a subset of enhancers and to be required for the distant regulation of gene expression. Several approaches to predict enhancers have been developed based on various chromatin marks and occupancy of enhancer-binding proteins. Despite the rapid advances in the field, no consensus how to define tissue specific enhancers yet exists. Here, we identify 2,695 long ncRNAs annotated by ENCODE (corresponding to 28% of all ENCODE annotated long ncRNAs) that overlap tissue-specific enhancers. We use a recently developed algorithm to predict tissue-specific enhancers, PreSTIGE, that is based on the H3K4me1 mark and tissue specific expression of mRNAs. The expression of the long ncRNAs overlapping enhancers is significantly higher when the enhancer is predicted as active in a specific cell line, suggesting a general interdependency of active enhancers and expression of long ncRNAs. This dependency is not identified using previous enhancer prediction algorithms that do not account for expression of their downstream targets. The predicted enhancers that overlap annotated long ncRNAs generally have a lower ratio of H3K4me1 to H3K4me3, suggesting that enhancers expressing long ncRNAs might be associated with specific epigenetic marks. In conclusion, we demonstrate the tissue-specific predictive power of PreSTIGE and provide evidence for thousands of long ncRNAs that are expressed from active tissue-specific enhancers, suggesting a particularly important functional relationship between long ncRNAs and enhancer activity in determining tissue-specific gene expression.

A long non-coding RNA links calreticulin-mediated immunogenic cell removal to RB1 transcription.

A subset of promoters bidirectionally expresses long non-coding RNAs (ncRNAs) of unknown function and protein-coding genes (PCGs) in parallel. Here, we define a set of 1107 highly conserved human bidirectional promoters that mediate the linked expression of long ncRNAs and PCGs. Depletion of the long ncRNA expressed from the RB1 promoter, ncRNA-RB1, reveals regulatory effects different from the RB1-controlled transcriptional program. ncRNA-RB1 positively regulates the expression of calreticulin (CALR) that in response to certain therapeutic interventions can translocate from the endoplasmic reticulum to the cell surface, hence activating anticancer immune responses. Knockdown of ncRNA-RB1 in tumor cells reduced expression of CALR, impaired the translocation of the protein to the cell surface upon treatment with anthracylines and consequently inhibited the cellular uptake by macrophages. In conclusion, co-transcription of ncRNA-RB1 and RB1 provides a positive link between the expression of the two tumor suppressors RB1 and the immune-relevant CALR protein. This regulatory interplay exemplifies disease-relevant co-regulation of two distinct gene products, in which loss of expression of one oncosuppressor protein entails the abolition of additional tumor-inhibitory mechanisms.