Research concept

Advances in high-throughput sequencing, combined with genome-wide mapping of chromatin modification signatures, have resulted in the identification of a large number of experimentally supported transcriptionally active long non-coding RNAs (ncRNAs) in multiple experimental systems. Through these sequencing efforts thousands of long ncRNAs displaying tissue specific expression have been identified. Long ncRNAs have been described in processes of gene silencing such as X-inactivation, imprinting and dosage compensation. Recent large-scale stud- ies have demonstrated that long-range transcriptional activation is another im- portant function of long ncRNAs in mammals. The mechanisms of long ncRNA regulation are starting to emerge from pioneering work, showing the role of long ncRNAs in epigentic control, long transcriptional regulation and progression of disease. Additional, recent systems scale approaches have provided evidence of essential involvement of long ncRNAs in regulating complex networks of signal- ing pathways, and important roles in regulating the p53 pathway.

Long ncRNAs transcribed from enhancers are reported to be a widespread phe- nomenon in human, and have also been observed for thousands of cases in the mouse. Functional knock-down studies in human tissue culture experiments have shown that these enhancer-derived long ncRNAs are responsible for the acti- vating function of several transcriptional enhancers previously thought to work exclusively at the DNA level. These observations reveal a novel aspect of en- hancers, placing long ncRNAs in gene regulation in a new light. Enhancers work- ing through a functional transcribed long ncRNA give the possibility of modulating their function through siRNA-based approaches. Such approaches for regula- tion of gene expression have great potential, as it allows for manipulation of the regulatory elements controlling a transcriptional network, rather than targeting single genes. The various novel aspects of long ncRNAs provide great potential for furthering the understanding of complex organisms, aspects that can be ap- plied to further the understanding of cellular and molecular biology and are very likely to provide new strategies for therapeutic approaches. A deep and thorough understanding of long ncRNA biology, biogenesis and function is a goal that we should pursue with a massive effort to expand our knowledge of molecular biol- ogy and gene regulation to the fullest extent possible. Understanding how long ncRNAs influence the regulation of cellular pathways is one of the research areas expected to impact most of the understanding of gene regulation in the next years.

 

Scientific methods and achievements/findings

The main aim of the group is to elucidate details of the molecular mechanisms of long ncRNA function in transcriptional regulation in human. The group uses large-scale approaches as RNA-sequencing and Chromosome Conformation Capture sequencing as well as traditional molecular biology and specialized RNA techniques.

Chromosome conformation capture (3C) to identify direct long ncRNA targets

Long ncRNAs have been shown to physically connect the genomic regions of regulated genes with their own genomic locus, mediating activating effects on gene expression through the direct interaction with target genes. We address di- rect and indirect targeting by long ncRNAs using state of the art approaches for chromosomal conformation, such as 3C and the larger-scale derivative 4C, to identify the genomic loci directly interacting with the long ncRNAs correlating with regulation at the transcriptional level. Using this methodology, in principle, all regions physically associated with long ncRNA genomic regions can be iden- tified.

Identifying protein complexes involved in long ncRNA function

Several protein complexes known to be involved in transcription have been identified to interact with long ncRNAs such as the PRC2 complex, hnRNP-K, WDR5 and the Mediator complex. The effects have been shown to be mutual dependent on both long ncRNAs and protein complexes. Given the largely unex- plored nature of long ncRNAs and their regulatory functions, several of the more than 500 identified RNA binding proteins are likely to play important roles in mediating long ncRNA function. We explore the functional interaction of RNA binding proteins with long ncRNAs using an siRNA screening approach coupled with a functional ncRNA reporter system that has been established in the lab. With the successful integration of various models of long ncRNA action into re- porter systems, different aspects and interaction partners of long ncRNAs can be addressed using screening for RNA binding proteins.


Delineating the extent of protein-ncRNA complexes.

Using RNA immunoprecipitation (RIP) and ChIP coupled with large-scale se- quencing along with RNA-seq, we establish functional interactions between RNA binding proteins and long ncRNAs. RIP-sequencing determines the fraction of the noncoding transcriptome associated to identified RNA binding proteins with a functional role in mediating transcriptional regulation through the complex for- mation with long ncRNAs. Identifying genomic loci bound and potentially regu- lated by long ncRNAs in complexes with RNA binding proteins are assessed by ChIP-sequencing of the identified RNA binding proteins.


Perspectives

The group is working towards a global cellular understanding of long non-coding RNAs and how they are involved in regulation of gene expression. A particular goal is to establish the molecular mechanisms of how chromatin structure is regu- lated by long ncRNAs, and how this can be applied to the enhancer-like functions that have been observed for a class of long ncRNAs.


Cooperation within the Institute

Within the institute, the Long non-coding RNA group closely cooperates with the following people and their groups: Annalisa Marsico, Martin Vingron, Bernd Timmermann.

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