The promise of enhancer-associated long noncoding RNAs in cardiac regeneration.

Heart failure is a worldwide epidemic and represents a major cause of morbidity and mortality. Current clinical therapies for heart disease prolong survival by protecting the viable muscle, but they are unable to replenish lost cardiomyocytes to restore function. Over the last decade, the notion of promoting cardiac regeneration has engendered considerable research interest. New strategies envisage the transfer of stem cells into the damaged myocardium, the mobilization of cardiac precursor cells, the promotion of cardiomyocyte proliferation in situ and direct reprogramming of non-cardiac cells into electromechanically coupled cardiomyocytes. The molecular and cellular mechanisms underpinning these different regenerative avenues are under the control of integrated transcriptional programs, which are ultimately dependent on epigenomic reprogramming and reorganization of the genome nuclear architecture. Today, it is becoming evident that regulatory noncoding RNAs play fundamental roles in all these aspects of gene regulatory network activity. In particular, thousands of long noncoding RNAs are dynamically expressed across the entire genome during lineage-specific commitment, specialization, and differentiation, as well as during the response to environmental cues. Here, we review this emerging landscape, focusing particularly on a unique class of lncRNA emerging from enhancer sequences, the enhancer-associated lncRNAs, in the context of cardiac regeneration. We propose that characterizing and manipulating these enhancer-associated transcripts could provide a novel approach to awaken the dormant regenerative potential of the adult mammalian heart. Ultimately, this could lead to targeted noncoding RNA-based enhancer therapies to improve effectiveness of current regenerative strategies and provide new avenues for repair

New Lncs to mesendoderm specification.

Mammalian genomes are pervasively transcribed generating thousands of long noncoding RNAs (lncRNAs) with emergent regulatory roles. Many of these lncRNAs exhibit highly specialised expression patterns during development and typically flank and regulate key developmental factors. In this review, we discuss and summarise the latest advances in our understanding of the roles of lncRNAs during mesendoderm (ME) specification, a key step during gastrulation and the formation of the primitive streak (PS)

Comparative in silico analysis identifies bona fide MyoD binding sites within the Myocyte Stress 1 gene promoter

<p>Abstract</p> <p>Background</p> <p>Myocyte stress 1 (MS1) is a striated muscle actin binding protein required for the muscle specific activity of the evolutionary ancient myocardin related transcription factor (MRTF)/serum response factor (SRF) transcriptional pathway. To date, little is known about the molecular mechanisms that govern skeletal muscle specific expression of MS1. Such mechanisms are likely to play a major role in modulating SRF activity and therefore muscle determination, differentiation and regeneration. In this study we employed a comparative <it>in silico </it>analysis coupled with an experimental promoter characterisation to delineate these mechanisms.</p> <p>Results</p> <p>Analysis of MS1 expression in differentiating C2C12 muscle cells demonstrated a temporal differentiation dependent up-regulation in <it>ms1 </it>mRNA. An <it>in silico </it>comparative sequence analysis identified two conserved putative myogenic regulatory domains within the proximal 1.5 kbp of 5' upstream sequence. Co-transfecting C2C12 myoblasts with <it>ms1 </it>promoter/luciferase reporters and myogenic regulatory factor (MRF) over-expression plasmids revealed specific sensitivity of the <it>ms1 </it>promoter to MyoD. Subsequent mutagenesis and EMSA analysis demonstrated specific targeting of MyoD at two distinct E-Boxes (E1 and E2) within identified evolutionary conserved regions (ECRs, α and β). Chromatin immunoprecipitation (ChIP) analysis indicates that co-ordinated binding of MyoD at E-Boxes located within ECRs α and β correlates with the temporal induction in <it>ms1 </it>mRNA.</p> <p>Conclusion</p> <p>These findings suggest that the tissue specific and differentiation dependent up-regulation in <it>ms1 </it>mRNA is mediated by temporal binding of MyoD at distinct evolutionary conserved E-Boxes within the <it>ms1 </it>5' upstream sequence. We believe, through its activation of <it>ms1</it>, this is the first study to demonstrate a direct link between MyoD activity and SRF transcriptional signalling, with clear implications for the understanding of muscle determination, differentiation and regeneration.</p

Discovery and functional characterization of cardiovascular long noncoding RNAs

Recent advances in sequencing and genomic technologies have resulted in the discovery of thousands of previously unannotated long noncoding RNAs (lncRNAs). However, their function in the cardiovascular system remains elusive. Here we review and discuss considerations for cardiovascular lncRNA discovery, annotation and functional characterization. Although we primarily focus on the heart, the proposed pipeline should foster functional and mechanistic exploration of these transcripts in various cardiovascular pathologies. Moreover, these insights could ultimately lead to novel therapeutic approaches targeting lncRNAs for the amelioration of cardiovascular diseases including heart failure

Comparative transcriptome profiling of the injured zebrafish and mouse hearts identifies miRNA-dependent repair pathways.

The adult mammalian heart has poor regenerative capacity. In contrast, the zebrafish heart retains a robust capacity for regeneration into adulthood. These distinct responses are consequences of a differential utilization of evolutionary-conserved gene regulatory networks in the damaged heart. To systematically identify miRNA-dependent networks controlling cardiac repair following injury, we performed comparative gene and miRNA profiling of the cardiac transcriptome in adult mice and zebrafish. Using an integrated approach, we show that 45 miRNA-dependent networks, involved in critical biological pathways, are differentially modulated in the injured zebrafish vs. mouse hearts. We study, more particularly, the miR-26a-dependent response. Therefore, miR-26a is down-regulated in the fish heart after injury, whereas its expression remains constant in the mouse heart. Targets of miR-26a involve activators of the cell cycle and Ezh2, a component of the polycomb repressive complex 2 (PRC2). Importantly, PRC2 exerts repressive functions on negative regulators of the cell cycle. In cultured neonatal cardiomyocytes, inhibition of miR-26a stimulates, therefore, cardiomyocyte proliferation. Accordingly, miR-26a knockdown prolongs the proliferative window of cardiomyocytes in the post-natal mouse heart. This novel strategy identifies a series of miRNAs and associated pathways, in particular miR-26a, which represent attractive therapeutic targets for inducing repair in the injured heart

Functional importance of cardiac enhancer-associated noncoding RNAs in heart development and disease.

The key information processing units within gene regulatory networks are enhancers. Enhancer activity is associated with the production of tissue-specific noncoding RNAs, yet the existence of such transcripts during cardiac development has not been established. Using an integrated genomic approach, we demonstrate that fetal cardiac enhancers generate long noncoding RNAs (lncRNAs) during cardiac differentiation and morphogenesis. Enhancer expression correlates with the emergence of active enhancer chromatin states, the initiation of RNA polymerase II at enhancer loci and expression of target genes. Orthologous human sequences are also transcribed in fetal human hearts and cardiac progenitor cells. Through a systematic bioinformatic analysis, we identified and characterized, for the first time, a catalog of lncRNAs that are expressed during embryonic stem cell differentiation into cardiomyocytes and associated with active cardiac enhancer sequences. RNA-sequencing demonstrates that many of these transcripts are polyadenylated, multi-exonic long noncoding RNAs. Moreover, knockdown of two enhancer-associated lncRNAs resulted in the specific downregulation of their predicted target genes. Interestingly, the reactivation of the fetal gene program, a hallmark of the stress response in the adult heart, is accompanied by increased expression of fetal cardiac enhancer transcripts. Altogether, these findings demonstrate that the activity of cardiac enhancers and expression of their target genes are associated with the production of enhancer-derived lncRNAs

A transcribed enhancer dictates mesendoderm specification in pluripotency.

Enhancers and long noncoding RNAs (lncRNAs) are key determinants of lineage specification during development. Here, we evaluate remodeling of the enhancer landscape and modulation of the lncRNA transcriptome during mesendoderm specification. We sort mesendodermal progenitors from differentiating embryonic stem cells (ESCs) according to Eomes expression, and find that enhancer usage is coordinated with mesendoderm-specific expression of key lineage-determining transcription factors. Many of these enhancers are associated with the expression of lncRNAs. Examination of ESC-specific enhancers interacting in three-dimensional space with mesendoderm-specifying transcription factor loci identifies MesEndoderm Transcriptional Enhancer Organizing Region (Meteor). Genetic and epigenetic manipulation of the Meteor enhancer reveal its indispensable role during mesendoderm specification and subsequent cardiogenic differentiation via transcription-independent and -dependent mechanisms. Interestingly, Meteor-deleted ESCs are epigenetically redirected towards neuroectodermal lineages. Loci, topologically associating a transcribed enhancer and its cognate protein coding gene, appear to represent therefore a class of genomic elements controlling developmental competence in pluripotency