Dataset Information

Divergent Transcription of the Nkx2-5 Locus Generates Two Enhancer RNAs with Opposing Functions.

ABSTRACT: Enhancer RNAs (eRNAs) are a subset of long noncoding RNA generated from genomic enhancers: they are thought to act as potent promoters of the expression of nearby genes through interaction with the transcriptional and epigenomic machineries. In the present work, we describe two eRNAs transcribed from the enhancer of Nkx2-5-a gene specifying a master cardiomyogenic lineage transcription factor (TF)-which we call Intergenic Regulatory Element Nkx2-5 Enhancers (IRENEs). The IRENEs are encoded, respectively, on the same strand (SS) and in the divergent direction (div) respect to the nearby gene. Of note, these two eRNAs have opposing roles in the regulation of Nkx2-5: IRENE-SS acts as a canonical promoter of transcription, whereas IRENE-div represses the activity of the enhancer through recruitment of the histone deacetylase sirtuin 1. Thus, we have identified an autoregulatory loop controlling expression of the master cardiac TF NKX2-5, in which one eRNA represses transcription.

SUBMITTER: Salamon I

PROVIDER: S-EPMC7509214 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Divergent Transcription of the Nkx2-5 Locus Generates Two Enhancer RNAs with Opposing Functions.

Salamon Irene I Serio Simone S Bianco Simona S Pagiatakis Christina C Crasto Silvia S Chiariello Andrea M AM Conte Mattia M Cattaneo Paola P Fiorillo Luca L Felicetta Arianna A di Pasquale Elisa E Kunderfranco Paolo P Nicodemi Mario M Papait Roberto R Condorelli Gianluigi G

iScience 20200906 9

Enhancer RNAs (eRNAs) are a subset of long noncoding RNA generated from genomic enhancers: they are thought to act as potent promoters of the expression of nearby genes through interaction with the transcriptional and epigenomic machineries. In the present work, we describe two eRNAs transcribed from the enhancer of Nkx2-5-a gene specifying a master cardiomyogenic lineage transcription factor (TF)-which we call Intergenic Regulatory Element Nkx2-5 Enhancers (IRENEs). The IRENE</ ...[more]

PMID: 33083767

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Project description:Pervasive transcription is observed in a wide range of organisms, including humans, mice, and viruses, but the functional significance of the resulting transcripts remains uncertain. Current genetic approaches are often limited by their emphasis on protein-coding open reading frames (ORFs). We previously identified extensive pervasive transcription from the murine gammaherpesvirus 68 (MHV68) genome outside known ORFs and antisense to known genes (termed expressed genomic regions [EGRs]). Similar antisense transcripts have been identified in many other herpesviruses, including Kaposi's sarcoma-associated herpesvirus and human and murine cytomegalovirus. Despite their prevalence, whether these RNAs have any functional importance in the viral life cycle is unknown, and one interpretation is that these are merely "noise" generated by functionally unimportant transcriptional events. To determine whether pervasive transcription of a herpesvirus genome generates RNA molecules that are functionally important, we used a strand-specific functional approach to target transcripts from thirteen EGRs in MHV68. We found that targeting transcripts from six EGRs reduced viral protein expression, proving that pervasive transcription can generate functionally important RNAs. We characterized transcripts emanating from EGRs 26 and 27 in detail using several methods, including RNA sequencing, and identified several novel polyadenylated transcripts that were enriched in the nuclei of infected cells. These data provide the first evidence of the functional importance of regions of pervasive transcription emanating from MHV68 EGRs. Therefore, studies utilizing mutation of a herpesvirus genome must account for possible effects on RNAs generated by pervasive transcription. IMPORTANCE The fact that pervasive transcription produces functionally important RNAs has profound implications for design and interpretation of genetic studies in herpesviruses, since such studies often involve mutating both strands of the genome. This is a common potential problem; for example, a conservative estimate is that there are an additional 73,000 nucleotides transcribed antisense to annotated ORFs from the 119,450-bp MHV68 genome. Recognizing the importance of considering the function of each strand of the viral genome independently, we used strand-specific approaches to identify six regions of the genome encoding transcripts that promoted viral protein expression. For two of these regions, we mapped novel transcripts and determined that targeting transcripts from these regions reduced viral replication and the expression of other viral genes. This is the first description of a function for these RNAs and suggests that novel transcripts emanating from regions of pervasive transcription are critical for the viral life cycle.

Project description:BACKGROUND: A major effort of the scientific community has been to obtain complete pictures of the genomes of many organisms. This has been accomplished mainly by annotation of structural and functional elements in the genome sequence, a process that has been centred in the gene concept and, as a consequence, biased toward protein coding sequences. Recently, the explosion of transcriptome data generated and the discovery of many functional non-protein coding RNAs have painted a more detailed and complex scenario for the genome. Here we analyzed the mouse carboxypeptidase M locus in this broader perspective in order to define the mouse CPM gene structure and evaluate the existence of other transcripts from the same genomic region. RESULTS: Bioinformatic analysis of nucleotide sequences that map to the mouse CPM locus suggests that, in addition to the mouse CPM mRNA, it expresses at least 33 different transcripts, many of which seem to be non-coding RNAs. We randomly chose to evaluate experimentally four of these extra transcripts. They are expressed in a tissue specific manner, indicating that they are not artefacts or transcriptional noise. Furthermore, one of these four extra transcripts shows expression patterns that differed considerably from the other ones and from the mouse CPM gene, suggesting that there may be more than one transcriptional unit in this locus. In addition, we have confirmed the mouse CPM gene RefSeq sequence by rapid amplification of cDNA ends (RACE) and directional cloning. CONCLUSION: This study supports the recent view that the majority of the genome is transcribed and that many of the resulting transcripts seem to be non-coding RNAs from introns of genes or from independent transcriptional units. Although some of the information on the transcriptome of many organisms may actually be artefacts or transcriptional noise, we argue that it can be experimentally evaluated and used to find and define biological functional elements on the genome. Furthermore, the transcription of other functional RNAs besides the protein coding RNA from a specific genomic locus imposes extra care when designing and interpreting experiments involving genetic manipulations or expression detection and quantification.

Dataset Information

Divergent Transcription of the Nkx2-5 Locus Generates Two Enhancer RNAs with Opposing Functions.

Publications

Divergent Transcription of the <i>Nkx2-5</i> Locus Generates Two Enhancer RNAs with Opposing Functions.

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