Isabelle Plaisance

Panagiotis Chouvardas

Yuliangzi Sun

Mohamed Nemir

Parisa Aghagolzadeh

Sophie Shen

Jun Woo

Shim

Francesca Rochais

Rory Johnson

Nathan Palpant

Thierry Pedrazzini
email: thierry.pedrazzini@chuv.ch

A transposable element into the human long noncoding RNA CARMEN is a switch for cardiac precursor cell specification

Aims:

The major cardiac cell types composing the adult heart arise from common multipotent precursor cells. Cardiac lineage decisions are guided by extrinsic and cell-autonomous factors, including recently discovered long noncoding RNAs (lncRNAs). The human lncRNA CARMEN , which is known to dictate specification towards the cardiomyocyte (CM) and the smooth muscle cell (SMC) fates, generates a diversity of alternatively spliced isoforms.

Methods and Results:

The CARMEN locus can be manipulated to direct human primary cardiac precursor cells (CPCs) into specific cardiovascular fates. Investigating CARMEN isoform usage in differentiating CPCs represents therefore a unique opportunity to uncover isoform-specific function in lncRNAs. Here, we identify one CARMEN isoform, CARMEN-201, to be crucial for SMC commitment.

CARMEN-201 activity is encoded within an alternatively-spliced exon containing a MIRc short interspersed nuclear element. This element binds the transcriptional repressor REST (RE1 Silencing Transcription Factor), targets it to cardiogenic loci, including ISL1, IRX1, IRX5, and SFRP1, and thereby blocks the CM gene program. In turn, genes regulating SMC differentiation are induced.

Conclusions: These data show how a critical physiological switch is wired by alternative splicing and functional transposable elements in a long noncoding RNA. They further demonstrated the crucial importance of the lncRNA isoform CARMEN-201 in SMC specification during heart development.

INTRODUCTION

The mammalian heart is composed of several cell types that derives from mesodermal progenitor cells of the first and second heart field 1 . The distinct lineages arise from multipotent cardiovascular precursors [START_REF] Kattman | Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages[END_REF][START_REF] Moretti | Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification[END_REF][START_REF] Wu | Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart[END_REF] . In this framework, very few studies have evaluated transcriptional regulation in primary human cardiac precursor cells (CPCs). We have previously derived clonal populations of CPCs from the fetal and the adult human heart [START_REF] Gonzales | Isolation of cardiovascular precursor cells from the human fetal heart[END_REF][START_REF] Plaisance | Cardiomyocyte lineage specification in adult human cardiac precursor cells via modulation of enhancer-associated long noncoding RNA expression[END_REF] . Their comparison allows for the dissection of the molecular mechanisms controlling cardiac specification and differentiation. Understanding the processes regulating cardiac cell programming and reprogramming provides also novel insights for the treatment of cardiovascular disease.

Next generation sequencing coupled to assessment of the epigenetic landscape has shown that mammalian genomes produce thousands of noncoding transcripts. Of these, long noncoding (lnc)RNAs represent the most heterogeneous and diverse class of RNA molecules. LncRNAs can be multiexonic, spliced, capped and polyadenylated . In this case, lncRNAs partner with proteins such as chromatin remodelers to modify the local chromatin environment at target locations. In the cardiovascular system, several lncRNAs have been identified as key players of cell differentiation and homoeostasis.

Braveheart, Fendrr and Meteor have been involved in mesoderm and cardiac differentiation [START_REF] Klattenhoff | Braveheart, a long noncoding RNA required for cardiovascular lineage commitment[END_REF][START_REF] Grote | The tissue-specific lncRNA Fendrr is an essential regulator of heart and body wall development in the mouse[END_REF][START_REF] Alexanian | A transcribed enhancer dictates mesendoderm specification in pluripotency[END_REF] . Myheart controls CM hypertrophy, and Wisper is a regulator of cardiac fibroblasts, critical for the development of fibrosis [START_REF] Han | A long noncoding RNA protects the heart from pathological hypertrophy[END_REF][START_REF] Micheletti | The long noncoding RNA Wisper controls cardiac fibrosis and remodeling[END_REF] . Then, SMILR and SENCR have been associated with SMC proliferation and differentiation [START_REF] Bell | Identification and initial functional characterization of a human vascular cell-enriched long noncoding RNA[END_REF][START_REF] Ballantyne | Smooth Muscle Enriched Long Noncoding RNA (SMILR) Regulates Cell Proliferation[END_REF] .

The way in which functions are encoded in lncRNAs' sequences remains enigmatic. It is thought that lncRNAs comprise modular assemblages of functional elements, composed of structural motifs that interact with proteins or other nucleic acids [START_REF] Kirk | Functional classification of long non-coding RNAs by k-mer content[END_REF] . Recent studies have implicated repetitive transposable elements as a source of functional lncRNA domains [START_REF] Carlevaro-Fita | Ancient exapted transposable elements promote nuclear enrichment of human long noncoding RNAs[END_REF][START_REF] Kapusta | Transposable elements are major contributors to the origin, diversification, and regulation of vertebrate long noncoding RNAs[END_REF] . In this context, lncRNA loci can produce a . Nevertheless, the miRNA precursor represents only one CARMEN isoform among several others. All other isoforms are lncRNA splice variants, with no apparent coding potential, for which functions remain to be fully defined. We showed previously that three isoforms were involved in cardiac specification in human fetal CPCs

21

. Moreover, we reported that one isoform, referred earlier as to CARMEN-7 (formally CARMEN-201 or ENST00000505254), was differentially expressed in CPCs committing to the CM vs. the SMC lineage 6 . Interestingly, the CARMEN locus can be manipulated to direct CPCs into specific cardiovascular fates. Investigating CARMEN isoform usage in differentiating CPCs represents therefore a unique opportunity to uncover isoformspecific function in lncRNAs. Here, we demonstrate that CARMEN-201 controls specification into the SMC lineage in human CPCs, and that this function is encoded within an alternatively-spliced exon containing a MIRc short interspersed nuclear element.

METHODS

Methods are described in details in the Supplementary Information available online.

Human cardiac precursor cells (CPCs)

Fetal heart biopsies were collected at 5 weeks of gestation following abortion, and adult atrial appendages were obtained from patients undergoing cardiac surgery. CPCs were isolated by enzymatic digestion as previously described 5, 6 .

Human plasma samples

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Plasma samples were collected from patients admitted to the Lausanne University Hospital with a diagnosis of myocardial infarction.

Study approval

The study was approved by the Lausanne University Hospital Ethics Committee and the Swiss Ethics Committee (Human cardiac precursor cells: Protocols 22/03 and 178/09; Human plasma from patients with myocardial infarction: Protocol PB_2018-00231 and 94/15), and was conducted according to the Declaration of Helsinki. Written informed consent was obtained from all patients included in the study.

GapmeR-mediated knockdown

CARMEN-201 silencing was obtained by adding CARMEN-201-specific GapmeRs targeting Exon 2 to

CPCs at a final concentration of 20 nM.

siRNA-mediated knockdown

siRNA transfection was performed using RNAiMax (ThermoFisher). CPCs were transfected with the indicated siRNA at a final concentration of 10 nM.

RNA extraction, RT-PCR and real time PCR

Total RNA from plasma and from cultured cells was extracted using the miRNeasy Serum/Plasma Advanced kit and the miRNeasy kit (Qiagen).

Absolute quantification of CARMEN isoform expression

pBluescript SK+ plasmids containing CARMEN-201, CARMEN-205 or CARMEN-217 cDNA were synthesized (GenScript, USA). Data were converted into transcript copy per cell, assuming 100% efficiency in conversion of RNA into cDNA.

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Subcellular fractionation

Cells were harvested and lysed. The lysate was centrifuged at 3800g for 2 min. The supernatant represented the cytoplasmic fraction. The pellet was used to produce the nuclear fraction.

CRISPR/Cas9-mediated Exon 2 deletion

A CRISPR/Cas9-D10A Nickase-based strategy was used to delete Exon 2 in the CARMEN gene.

CRISPR-On activation

We used the CRISPR/dCas9-based Synergistic Activator Mediator (SAM) gain of function system to activate CARMEN isoform expression in fetal CPCs

23

.

RNA sequencing

Sequencing libraries were prepared according to Illumina RNA Seq library kit instructions. Libraries were sequenced with the Illumina HiSeq2000 (100bp; PE).

TRIAGE analysis

The Transcriptional Regulatory Inference Analysis from Gene Expression (TRIAGE) analysis, providing a means to identify cell type-specific regulatory genes has been described 24 .

Uniform Manifold Approximation and Projection (UMAP) density plots

We used the data provided by Asp et al., 2019 (accession number is European Genome-phenome Archive (EGA): EGAS00001003996). The UMAP were generated using Nebulosa package.

Lentiviral vectors

The SIN-cppt-CMV-EGFP-WHV plasmid was a kind gift of Dr.

Tandem mass spectrometry

For identifying CARMEN-201 protein partners, tryptic peptide mixtures were injected on an Ultimate RSLC 3000 nanoHPLC system (Dionex, Sunnyvale, CA, USA).

Western blotting

Proteins associated to biotinylated C-201 transcript were resolved by SDS-PAGE and electroblotted onto PVDF membranes (GE Healthcare).

RNA Immunoprecipitation (RIP)

The RIP experiment was conducted as described 14 .

RIP following by sequencing

RIP was performed using anti-REST IgG. REST-associated transcripts were purified using the RNeasy isolation kit (Qiagen). Sequencing libraries were prepared according to Illumina RNA Seq library kit instructions. Libraries were sequenced with the Illumina HiSeq2000 (100bp; PE).

Chromatin immunoprecipitation followed by real-time quantitative PCR (ChIP-qPCR)

ChIP-qPCR was performed using the Pierce magnetic ChIP Kit (Thermo Scientific) and the ChIPAb+ REST Kit (Millipore) according to the manufacturers' instructions.

A C C E P T E

Masson's trichome staining

Paraffin tissue sections were also processed for Masson's trichrome staining and analyzed with a Zeiss Axioscan Z1 (Carl Zeiss).

Statistics

All data were collected from at least 3 independent experiments, performed at least in triplicates. Data throughout the paper are expressed as mean ± SEM. Statistical analysis: ANOVA with post-hoc Tukey.

RESULTS

CARMEN isoforms are differentially expressed in CPCs committing to the CM vs. the SMC fate.

To study the importance of CARMEN isoforms in cardiac differentiation, we took advantage of primary CPCs isolated from the human heart. We isolated CPCs from the fetal heart at 5 weeks of gestation, hereafter referred to as fetal CPCs S1b).

Absolute quantification confirmed C-201 represented the main isoform in adult CPCs but the least abundant in fetal CPCs (Fig. 1d), suggesting its involvement in SMC differentiation. All CARMEN isoforms but the miRNA precursor (C-215) were more abundant in the nucleus than the cytoplasm, a feature compatible with the postulated function of lncRNAs as regulators of gene expression (Fig. 1e; Supplemental Fig. S1c).

CARMEN-201 controls SMC specification via its second exon

We next evaluated the involvement of C-201 in SMC differentiation using a knockdown approach.

Antisense oligonucleotides (GapmeRs) were designed to target the C-201 second exon, uniquely present in this isoform (Fig. 1c). C-201 was downregulated in adult CPCs following GapmeR transfection (Supplemental Fig. S2a). The anti-C-201 GapmeRs affected no other isoforms, demonstrating the specificity of the approach. C-201 silencing had no effects on differentiation of fetal CPCs into CMs but completely blocked the capacity of adult CPCs to produce SMCs (Supplemental Fig. S2b andc). To explore the importance of the C-201 second exon in SMC specification, we produce adult CPCs lacking the exon using CRISPR/Cas9 deletion. Guide (g)RNAs were designed to remove the second exon without affecting any other exons (Supplemental Fig. S3a). C-201 Exon 2-deleted adult CPC clones were derived.

Importantly, the C-201 isoform was still expressed in adult CPCs lacking C-201 Exon 2. However, the transcript was reduced by the size of the second exon (Supplemental Fig. S3b). Endogenous C-201 expression was similarly induced in both wild-type and deleted adult CPC (201Ex2) clones during differentiation as detected by using a primer pair amplifying the 3' end of the transcript (Fig. 1f; primer pair P1). The deletion of the second exon was verified using two primer pairs spanning the exon (i.e. P2; P3).

Next, we evaluated the capacity of deleted adult CPCs to produce SMCs. Measurement of marker gene ). Endogenous C-201 expression was downregulated in fetal CPCs in the absence of gRNA but markedly increased when the gRNA was expressed (Fig. 2a). Compared to the large C-201 induction, the other CARMEN isoforms, as well as the two hosted miRNAs, were marginally activated (Fig. 2a; Supplemental Fig. S3c). We tested therefore the effects of C-201 manipulation on the fate of normally cardiogenic fetal CPCs. Strikingly, cells with forced C-201 transcription produced large amount of SMCs, indicating that C-201 expression was sufficient to adopt a SMC fate (Fig. 2b-c).

Production of CMs was minimally affected, likely reflecting differentiation of untransfected fetal CPCs (Supplemental Fig. S3d-e). Globally, expression of C-201 was found to be necessary and sufficient for inducing SMC specification in CPCs.

The CARMEN-201 second exon contains a functional transposable element that drives SMC commitment

The results above prompted us to evaluate the role of the C-201 second exon in SMC specification. We looked at its primary structure (397 nucleotides) and detected a short interspersed nuclear element (SINE), which was identified as a 126 nucleotide-long Mammalian-wide Interspersed Repeat (MIR)c element. Of note, the exon is highly conserved in primates but not found in other species (Fig. 2d). Intriguingly, MIRc is part of a catalog of predicted Repeat Insertion Domains of LncRNAs (RIDLs) promoting nuclear localization 18 , a feature consistent with the pronounced nuclear enrichment of C-201 (Fig. 1e). The exon also contains a partial ALU sequence.

To study the possible role of the MIRc element in determining SMC specification, we produced two lentiviral vectors for overexpressing the entire Exon 2 in fetal CPCs (Supplemental Fig. S4a). The first version contained wild-type MIRc sequences (C-201 Ex2) whereas, in the second vector, the whole MIRc

Identification of upstream regulators of CM and SMC specification in human CPCs

To better understand the processes leading to a switch in cell identity, we profiled the CPC transcriptomes under different experimental conditions. Principal component analysis (PCA) was conducted to evaluate differentiation when C-201 Exon 2 was expressed and not expressed (Fig. 3a). We next analyzed the transcriptomic data in details (Fig. 3b; Supplemental Fig. S4e). and associated biological pathways (GO BP) using all identified TRIAGE candidates validated their functional roles in CM and SMC commitment (Supplemental Fig. S5a).

Samples of differentiating fetal

To further investigate the association of CARMEN expression with blood vessel development in vivo, we used data reporting the comprehensive transcriptional analysis of the embryonic human heart at the single-cell level at different stages of gestation 28 . To understand how CARMEN expression was related to gene network changes, we performed a correlation analysis of CARMEN against all genes in all cells across all developmental time points (Fig. 3e). CARMEN abundance was associated preferentially with pericyte and SMC gene programs, and not with the expression of CM or endothelial cell markers. We next reanalyzed data generated at 6.5 weeks post-conception 28 , an important point in development bound by REST with similar length distribution and orientation. Nevertheless, the enrichment is also higher when exploring repeat-containing genes in general, suggesting that global REST binding to RNA molecules could require additional transposable elements (Supplemental Fig. S6e; Supplemental Table S3).

To evaluate the role of REST in C-201-mediated CPC specification, we first used a knockdown approach. Fetal CPCs were transfected with control or REST siRNA, and induced to differentiate into SMC following C-201 Exon 2 overexpression (Fig. 4g-h). Under control conditions, C-201 Exon 2 expression forced CPCs to adopt a SMC specification. In sharp contrast, REST knockdown abolished the capacity of C-201-expressing CPCs to commit to the SMC lineage. To confirm these results, we tested the effects of REST silencing in adult CPCs spontaneously differentiating into SMC (Supplemental Fig. S6f).

Again, in the absence of REST, adult CPCs were unable to produce a SMC progeny. Interestingly, C-201 appeared downregulated in differentiating REST-deficient CPCs. This was also confirmed using RNA BaseScope in situ hybridization in adult CPCs (Supplemental Fig. S6g). The resetting of C-201 expression following REST knockdown mimicked therefore what observed in differentiating fetal CPCs (Supplemental Fig. S1a-b). Moreover, the cellular distribution of the C-201 isoform was modified following REST silencing (Supplemental Fig. S6h). Significant cytoplasmic enrichment was evident in the absence of REST, in contrast to what measured under basal conditions. This observation suggested therefore that REST, which carries a nuclear localization signal, might contribute to retain C-201 in the nucleus via its capacity to bind the MIRc element in the second exon.

CARMEN-201 inhibits the CM fate via REST-mediated repression of cardiogenic transcription factor expression

The association of C-201 with REST suggested a mechanism involving the targeting of the Several DBDs were predicted in C-201, in particular in the sequences spanning the second exon (Fig. 5a).

As control, we performed a similar analysis for C-205 and C-217. The C-205 transcript was found to contain distinct DBDs (Fig. 5a) whereas C-217 was not predicted to contain significant DBDs (not shown).

In total, 447 gene promoters were identified as potentially bound by C-201, and 387 by C-205 respectively. Among those, 337 were uniquely associated with C-201. These genes were related to GO Biological Processes defining striated muscle contraction (Fig. 5b). In order to identify relevant targets of C-201/REST action, we crossed the list of C-201-bound genes as predicted by TDF with the list of TRIAGE candidates and the list of validated cardiac genes (Human Protein Atlas -ENSEMBL) (Fig. 5c; Supplemental Table S4). Hypergeometric tests explored the significance of the overlaps and revealed four primary candidates: IRX1; IRX5; ISL1 and SFRP1. ISL1 is a member of the LIM homeodomain family of transcription factor, crucial for the development of the SHF. The two Iroquois homeobox transcription factors IRX1 and IRX5 have been involved in developmental patterning in the embryonic heart. Finally, SFRP1 is a modulator of the WNT pathway that plays important roles in cardiac specification and differentiation. Importantly, single-cell analysis demonstrated that these factors were not expressed in CARMEN-expressing cells in the embryonic human heart at 6.5 weeks of gestation (Supplemental Fig. S5b). Importantly, all four genes contained REST binding sites as determined by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) in a study interrogating REST binding in various cell types (Supplemental Fig. S7a; Gene Expression Omnibus: GSM803369; GSM1010735; silencing. Consistently, REST occupancy at candidate promoters in adult CPCs was blunted following GapmeR-mediated C-201 knockdown (Figure 5d).

To validate the relevance of ISL1, IRX1, IRX5 and SFRP1 in CPC specification, we first determined expression in fetal CPCs induced to differentiate into SMC following forced C-201 expression using CRISPR-On (Fig. 5e). Each candidate was downregulated after induction of C-201 expression. We then measured expression in adult CPC clones lacking C-201 exon 2 (201Ex2), i.e. not able to activate a SMC gene program. ISL1, IRX1, IRX5 and SFRP1 expression was restored in these cells during differentiation as compared to what observed in wild-type cells (Fig. 5f). Furthermore, we evaluated expression after manipulating REST. We observed re-expression of the four factors in differentiating adult CPCs after REST knockdown (Supplemental Fig. S7c). Moreover, REST silencing allowed also reexpression of ISL1, IRX1, IRX5 and SFRP1 in fetal CPCs overexpressing C-201 Exon 2 (Fig. 5g).

Altogether, these findings supported that the four candidates were under control by C-201 via RESTmediated repression.

ISL1, IRX1, IRX5 and SFRP1 silencing promotes the SMC fate

We next proceeded to validate the importance of ISL1, IRX1, IRX5 and SFRP1 in controlling specification into the CM vs. the SMC lineage. To mimic REST-mediated repression, we used a siRNA approach to knockdown each candidate in fetal CPCs normally committing to the CM lineage. We first evaluated the effects of individual factor silencing on the capacity of fetal CPCs to produce a functional progeny. Knocking down either IRX1, IRX5 or SFRP1 did not affect other candidate gene expression but ISL1 knockdown slightly decreased IRX1, IRX5 and SFRP1 levels (Supplemental Fig. S8a-d (Supplemental Fig. S8e-h), which are known to mark bipotential cardiac precursors giving raise to CMs and SMCs

33

. Nevertheless, manipulating each candidates separately has little impact on SMC gene expression (Supplemental Fig. S8i-l). In fact, ISL1 knockdown had even a negative effect on late SMC maker expression. This suggested that ISL1 operated at the onset of CPC specification but was also necessary for late-stage differentiation.

We tested therefore combinations of siRNAs targeting IRX1, IRX5 and SFRP1 (Fig. 6e). As a positive control for the activation of the SMC gene program, C-201 Exon 2-overexpressing fetal CPCs were included in the experiment. Each combination was associated with a large induction of epicardial and SMC gene expression as compared to individual knockdown, with maximal impact achieved when all three factors were downregulated simultaneously. This manipulation was as potent as C-201 Exon 2 overexpression in inducing SMC genes in normally cardiogenic CPCs. Accordingly, massive SMC differentiation was observed by immunostaining following IRX1, IRX5 and SFRP1 knockdown (Fig. 6f).

Interestingly, commitment occurred at the expense of the cardiogenic lineage (Fig. 6e andf) but had no impact on endothelial cell production (not shown). Our data indicated therefore that IRX1, IRX5 and SFRP1 downregulation was sufficient to redirect fetal CPCs into the epicardial and the SMC lineages.

Nevertheless, as mentioned above, ISL1 expression appeared necessary during SMC differentiation. To formally demonstrate this point, we performed an additional experiment in which the four factors were silenced together (Supplemental Fig. S8m). In this case, ISL1 silencing produced a slight negative effect on SMC marker expression induced by combined IRX1, IRX5 and SFRP1 knockdown, sustaining a role for ISL1 in the late stage of SMC differentiation.

Interestingly, manipulating IRX1, IRX5 or SFRP1 had a striking effect on CARMEN isoform expression (Fig. 6g). Indeed, while endogenous C-201 was downregulated during specification in differentiating fetal CPCs, its expression was reactivated after IRX5 and SFRP1 knockdown, and even more so when IRX5 and SFRP1 were silenced together, suggesting C-201 was negatively regulated by the two cardiogenic factors. Remarkably, C-217 expression demonstrated a mirror image, consistent with coordinated regulation of the two isoforms and suggesting a switch might operate during SMC specification. C-205 was not modulated under these different conditions. In an attempt to evaluate the relevance of our findings in disease, we queried the association of CARMEN with cardiovascular traits using CTG-VIEW (https://view.genoma.io). We identified important phenotypes related to cardiovascular conditions as strongly associated with CARMEN (Fig. 7a). This prompted us to investigate whether C-201 was differentially expressed in the damaged myocardium. We first used RNA BaseScope in situ hybridization to localize C-201 expression in the human heart. Samples were collected from explanted hearts of transplant patients, and expression was compared in CMs vs.

mural cells (Fig. 7b-e). C-201 was found uniquely expressed in mural cells of large coronary vessels.

Immunostaining for VIMENTIN (marking endothelial cells and fibroblasts) and smooth-muscle myosin heavy chain (SMMHC; marking SMCs) supported C-201 expression being associated primarily with SMCs (Fig. 7f; Supplemental Fig. S8n). C-201 expression seemed equally distributed in the ventricular and atrial vasculature. Next, we measured C-201 expression in the blood of patients experiencing acute coronary syndrome, with no prior history of cardiac disease. Plasma samples were obtained during angioplasty that took place less than 1 hours after myocardial infarction, and at 24 and 48 hours thereafter (Figure 7g).

Individuals were classified based on the presence or absence of ST elevation, namely STEMI and non-STEMI (NSTEMI). C-201 was not expressed immediately after infarction, suggesting that the transcript was not induced under basal conditions. However, the amounts of transcript dramatically increased after one and two days. Importantly, circulating C-201 concentrations were more elevated in STEMI vs. non-STEMI patients. Altogether, it suggested that C-201 was expressed in large vessels of the heart and responded acutely to hemodynamic stress with an expression being proportional to the severity of the disease.

DISCUSSION

In this study, we characterized for the first time the role of lncRNA isoforms in cell fate determination through a systematic examination of the human CARMEN locus. In primary human CPCs committing to the SMC lineage, the C-201 isoform associates with REST via its MIRc element, targets the repressor to important cardiogenic loci, namely IRX1, IRX5, SFRP1 and ISL1, represses their expression

7 .of transcription 8 .

 78 Current estimates predict the existence of approximately 200,000 lncRNAs in human, of which very few have been fully characterized. LncRNAs are implicated in a variety of functions that define cell identity and behavior. They exert Cis-and Trans-regulatory functions, controlling chromatin remodeling and transcription. Cis-acting lncRNAs operate at a close vicinity to their site On the other hand, Trans-acting lncRNAs leave their site of transcription and exert functions at remote locations in the genome 9

5 variety of transcripts through alternative splicing 7 , 20 . 21 .

 572021 It has been speculated that these alternative isoforms, by containing different combinations of exon sequences, thereby exert diverse functions. Some years ago, we identified CARMEN, a conserved lncRNA essential for cardiogenesis CARMEN has been previously referred to as MIR143HG because the locus hosts MIR-143 and MIR-145, two microRNAs (miRNAs) important for SMC differentiation[START_REF] Vacante | The function of miR-143, miR-145 and the MiR-143 host gene in cardiovascular development and disease[END_REF]

  Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvac191/6941187 by Universitätsbibliothek Bern user on 21 December 2022 CVR-2022-0360 11 expression as well as immunostaining demonstrated that adult CPCs lacking C-201 Exon 2 lost their ability to differentiate into SMCs (Fig. 1f-h). CARMEN-201 induction is sufficient to trigger a SMC gene program in undifferentiated fetal CPCs To evaluate the capacity of C-201 to redirect fetal CPCs into the SMC lineage, we used a CRISPR-On approach. We targeted transcriptional activators (dCas9-VP64; MS2-p65-HSF1), 200 bp upstream of the C-201 transcriptional start site (TSS) via expression of a modified gRNA containing MS2 aptamers (SAM system; 23

  sequence was scrambled (C-201 mutEx2), thus maintaining length and sequence composition (Supplemental Fig.S4a). Endogenous C-201 expression was downregulated in differentiating fetal CPCs as expected (Fig.2e; P1). However, significant wild-type and mutated C-201 Exon 2 expression was measured in the respective transduced groups as judged by using Exon 2-specific primers (P3). We next evaluated SMC and CM differentiation (Fig.2f-g; Supplemental Fig.S4b-d). Non-transduced fetal CPCs differentiated into CMs. In sharp contrast, overexpression of wild-type C-201 Exon 2 forced fetal CPCs to adopt a SMC fate, as evidenced by expression of SMC markers and immunostaining. Importantly, when the MIRc element was mutated (C-201 mutEx2), SMC differentiation was not observed, supporting a critical role for this transposable element in the capacity of C-201 to direct CPCs into the SMC lineage.

  CPCs (None) revealed temporal changes (d0 / Expansion; d1; d7) characterizing CM specification. In contrast, fetal CPCs overexpressing wild-type C-201 Exon 2 deviated significantly from the original differentiation track. Importantly, CPC samples with overexpression of the MIRc-mutated C-201 Exon 2 were transcriptionally similar to untransfected samples, indicating again that the transposable element was necessary for SMC commitment.

14 (

 14 corresponding to the formation of the cardiac vasculature. Newly generated Uniform Manifold Approximation and Projection (UMAP) density plots revealed lineage-specific markers, reflecting the cellular diversity of the embryonic human heart including epicardial cells (WT1; TCF21; TBX18), pericytes MCAM; CSPG4; ACTA2; PDGFRB), and SMCs (GATA6; TAGLN; CNN1; MYH11) (Fig.3f). CARMEN was found expressed preferentially in these cells, substantiating an important role for this locus in pericyte and SMC specification from the epicardium during development of the human heart. In contrast, CMs and endothelial cells, marked by TBX5; GATA4; NKX2-5; MYH6 and PECAM; KDR, did not expressed CARMEN at this developmental stage (Supplemental Fig.S5b). Interestingly, ALDH1A2 and the COUP transcription factor NR2F2, critical for atrial identity, were also expressed in CARMEN-expressing cells. The MIRc element is a binding module for the RE1 Silencing Transcription Factor Many lncRNAs function through interacting with proteins. To identify C-201 protein partners, we performed a RNA pulldown assay. Biotinylated C-201 Exon 2 was used as a bait to purify C-201associated proteins from adult CPC lysates. As control, we used a mutated C-201 Exon 2 lacking the MIRc element. Proteins were identified by mass spectrometry. Three proteins were detected as significantly associated to C-201 Exon 2, namely the transcriptional repressor RE1 Silencing Transcription Factor (REST; aka Neuron Restrictive Silencer Factor), the RNA methyltransferase NOP2/Sun RNA Methyltransferase 6 (NSUN6), and the Replication Protein A1 (RPA1), a protein implicated in stabilization of single-stranded DNA (Supplemental Fig. S6a; Supplemental TableS2). We first confirmed the association of each protein with C-201 by pulling down the full C-201 transcript and quantifying the amount of bound proteins by Western blotting (Fig.4a-c; see full unedited gels in Supplementary Information). An antisense C-201 transcript was used as control. The results demonstrated the specific interaction of C-201 with REST, NSUN6 and RPA1 in proliferating and differentiating adult CPCs. We next performed a RNA immunoprecipitation assay (RIP) using antibodies directed against REST, NSUN6 and RPA1 respectively (Fig.4d-f). Quantitative measurement of bound CARMEN isoforms confirmed the interaction of C-201 with REST, NSUN6 and RPA1 during CPC expansion and differentiation. No other CARMEN isoforms were found associated to REST (Supplemental Fig.S6b). In contrast, small amounts of C-205 were detected as bound to NSUN6, and C-217 appeared to interact with both NSUN6 and RPA1 (Supplemental Fig.S6c-d). Finally, to determine whether REST possessed intrinsic propensity to bind MIRc-containing transcripts, we performed a REST RIP coupled to RNA profiling. REST-bound transcripts were found significantly enriched in sequences containing a MIRc element as compared to transcripts not Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvac191/6941187 by Universitätsbibliothek Bern user on 21 December 2022 CVR-2022-0360 15

29 ,

 29 repressor to important regulatory loci to control cell fate in differentiating CPCs. In addition, RPA1, a C-201 protein partner, has been implicated in RNA:DNA triple helix stabilization indicating that C-201 could interact with DNA sequences at target promoters. Thus, we took advantage of Triplex Domain Finder (TDF), an application developed to detect DNA binding domains (DBDs) in lncRNAs 30 . TDF identifies also the DNA regions bound by the selected lncRNAs, i.e. gene promoters containing binding Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvac191/6941187 by Universitätsbibliothek Bern user on 21 December 2022 CVR-2022-0360 16 sites for the lncRNA DBDs. Because REST had been associated with repression, we sought to identify C-201 target promoters within the list of downregulated genes following C-201 Exon 2 overexpression.

GSM1010804) 31 .A

 31 We evaluated therefore REST occupancy at the promoters of IRX1, IRX5, ISL1 and SFRP1 in differentiating fetal and adult CPCs by ChIP-quantitative real-time (q)PCR. As expected, REST occupied the promoter of the different candidate gene solely in adult CPCs expressing C-201, and not in fetal CPCs (Supplemental Fig.S7b). In these experiments, we used GAPDH as a negative control (REST occupancy in neither fetal nor adult CPCs) and SYN1 as a positive control (REST occupancy in both fetal and adult CPCs). Then, to formally demonstrate the dependence of REST targeting to IRX1; IRX5; ISL1 and SFRP1 on C-201 action, we performed an additional experiment in adult CPCs with or without C-201 Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvac191/6941187 by Universitätsbibliothek Bern user on 21 December 2022

32 .

 32 ), suggesting ISL1 lies upstream of these factors in the cardiac regulatory network, in accordance with its role as a pioneer transcription factor in the developing heart We then investigated re-specification of fetal CPCs into the SMC fate following siRNA-mediated silencing of each factor individually or in combination. In differentiating fetal CPCs, knocking down either IRX1, IRX5, ISL1 or SFRP1 restored epicardial gene expression, i.e. WT1, TCF21 and TBX18, confirming that downregulation of these critical cardiogenic factors was a mandatory step in reprogramming CPCs into the smooth muscle fate (Fig.6a-d). In addition, individual factor silencing also resulted in the reexpression of GATA6, HAND2, PDGFRA and TBX20, Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvac191/6941187 by Universitätsbibliothek Bern user on 21 December 2022 CVR-2022-0360 18

  expression is increased in response to myocardial infarction in humans

Finally 25 .

 25 , C-201 could represent an interesting biomarker for assessing the extent of cardiovascular damage in various pathological situations.Altogether, this work demonstrates how a biological switch is encoded in lncRNA sequence to regulate cardiovascular specification. We have linked two key phenomena, namely alternative splicing and the presence of deeply-conserved transposable elements. LncRNAs display far greater levels of alternative splicing, although it has not been clear whether this reflects relaxed constraint or regulated production of isoforms with distinct functions Here, we have shown an example where these two processes converge to produce functional transcript isoforms, and provided the first physiological role for a transposable element acting via a lncRNA during heart development.FUNDINGThis work was supported by grants from the Swiss National Science Foundation (T.P.; Grant No. CRSII5-1_173738 and 31003A_182322).

AAA

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Figure 1 .

 1 Figure 1. CARMEN-201 controls SMC specification via its second exon (a-b) Representative images and quantification of ACTN2-positive TNNI-positive CMs and SMMHCpositive SMCs in differentiating fetal and adult CPC cultures. Scale bar: 50 µm. (c) Annotated CARMEN isoforms. The CARMEN-201 second exon is highlighted in red. (d) Absolute quantification of CARMEN-201 (C-201), CARMEN-205 (C-205) and CARMEN-217 (C-217) in differentiating fetal and adult CPCs. (e) Nuclear and cytoplasmic levels of CARMEN-201, CARMEN-205, CARMEN-217, ACTB, and NEAT. (f) Expression of CARMEN-201, CARMEN-205, CARMEN-217, SMC markers (MYH11; CNN1; TAGLN), and CM markers (MYH6; MYH7) in adult WT and 201Ex2 CPC clones lacking CARMEN-201 Exon 2. (g-h) Representative images and quantification of SMMHC-positive CNN1-positive TAGLN-positive SMCs in cultures of differentiating adult WT or 201Ex2 CPC clones. Scale bar: 50 µm. Data represent means ± SEM; *p < 0.05 as compared to fetal CPCs in expansion; § p < 0.05 compared to the indicated conditions (n=3-6). ANOVA with post-hoc Tukey. See also Supplemental Fig. S1, S2 and S3.

Figure 2 .

 2 Figure 2. The CARMEN-201 Exon 2 contains a functional transposable element implicated in SMC specification. (a) Expression of CARMEN isoforms and (b) SMC markers (MYH11; CNN1; TAGLN; CALD1) in differentiating fetal cells either untransfected (None), transfected with the SAM system in the absence of gRNA (SAM) or with the SAM system with a gRNA targeting sequences upstream the CARMEN TSS (SAM/gRNA). (c) Representative images and quantification of SMCs in cultures of differentiating fetal CPCs transfected as in (a). Scale bar: 50 µm. (d) Position of the MIRc transposable element in the CARMEN-201 second exon, and sequence conservation. (e) Expression of CARMEN-201 using either a primer pair specific for the endogenous transcript (P1) or the exogenous exon 2 (P3), and (f) SMC markers (MYH11; CNN1; TAGLN; CALD1) in differentiating fetal CPCs either not transduced (None), transduced with a lentiviral vector encoding CARMEN(C)-201 Ex2 or transduced with a lentiviral vector encoding a mutated C-201 Ex2 (C-201 mutEx2). (g) Representative images and quantification of SMMHC-positive CNN1-positive SMCs in cultures of differentiating fetal CPCs transfected as in (f). Scale

RNA Pulldown and identification of CARMEN-201 protein partners

  The pBluescript SK+ plasmids containing C-201 Ex2 or C-201 mutEx2 were used to synthetize biotinylated probes. Precleared lysate was incubated with either no probe, biotinylated C-201 Ex2 or biotinylated mutC-201 Ex2. Proteins were loaded on 12% SDS-PAGE.

	CVR-2022-0360
	Nicole Deglon (University of Lausanne, Lausanne, Switzerland). EGFP sequences were replaced by either the wild-type CARMEN-201 Exon 2 or a mutated Exon 2 containing scrambled MIRc sequences. A C C E P T E D M A N U S C R I P T	Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvac191/6941187 by Universitätsbibliothek Bern user on 21 December 2022
	8

  specification (see Graphical Abstract). Conversely, in CPCs adopting a CM fate, C-201 is not expressed, allowing IRX1, IRX5, ISL1 and SFRP1 expression and the subsequent activation of the cardiogenic program. Importantly, the MIRc-containing exon in C-201 is found in primates but not in other mammals, suggesting that MIRc-mediated functions controlling commitment into the SMC lineage has been integrated in the CARMEN locus only recently in evolution. The CARMEN locus has been expression during the development of the heart also reflects the role of REST in cell fate determination. REST binds C-201 but not C-205 and C-217, further substantiating the importance of the C-201/REST complex for SMC differentiation. C-201 appears to be also indirectly under control by REST. C-201 relocalizes into the cytoplasm upon REST silencing. Therefore, the nuclear enrichment of C-201 could depend in part on its binding to REST. In this vein, REST binds C-201 via the MIRc repeat in the second exon, which was recently demonstrated to be associated with transcript nuclear localization 18 . CARMEN isoforms were found differentially expressed depending on the cardiac pathology, exemplifying again the complexity of the regulation of the CARMEN locus. Here, we show that C-201 levels increase in the blood during the acute phase of myocardial infarction. The likely source of circulating C-201 is the damaged heart. However, we cannot rule out the possibility that hemodynamic stress also stimulates release from the peripheral vasculature. Nevertheless, assuming a cardiac origin for C-201, its expression in CPCs could be part of the healing process initiated following injury. In this scenario, CPCs expressing C-201 would be specified preferentially into the SMC lineage. Increased myocardial tissue perfusion has been reported in cell-based regenerative therapies for heart disease. However, clinical trials failed to demonstrate functional improvement. This can be expected if precursors are diverted from the cardiogenic lineage secondary to C-201 expression. Our data propose therefore a mean to improve CM production via modulating C-201 expression in cardiac precursors.

	CVR-2022-0360 CVR-2022-0360 CVR-2022-0360
	A C C E P T E D M A N U S C R I P T 21 . Our data suggest that coordinate regulation of C-201 and C-217 expression takes place during specification, providing a plausible mechanism for controlling specification. CARMEN isoforms appear to share a single promoter. Yet, additional transcription start sites have been recently detected in the C-201 isoform, suggesting transcriptional regulation might control C-201 expression in differentiating SMCs 34 . Multipotent cardiovascular precursor cells expressing ISL1 give rise to both CMs and SMCs 2-4 . In this context, our TRIAGE analysis identifies key cardiac TFs as regulators of fetal CPC differentiation. On the other hand, CPCs respecified into the SMC lineage after C-201 Exon 2 overexpression express a different gene program. Induction of GATA6, HAND2, PDGFRA and TBX20 in CPCs is a characteristic feature of cardiovascular intermediates capable of producing CMs and SMCs 33 . We show also that commitment to the SMC lineage is characterized by the stepwise expression of markers of epicardium-derived cells (EPDCs) such as WT1, MEOX1, KRT19 and TBX18, and pericytes such as MCAM, CSPG4, ACTA2 and PDGFRB. During development, EPDCs establish the subepicardial mesenchyme, then migrate into the myocardium. These cells represent a known source of pericytes and SMCs for the forming coronary vasculature 35 . In addition, genetic tracing experiments suggest that epicardial cells can also give rise to a myocardial progeny 36-38 . Along the same line, a recent single-cell analysis identifies the juxta-cardiac field (JCF) contributing to both EPDCs and CMs 39 . Trajectory analysis revealed a link between precursors from the JCF and the posterior SHF, supporting the postulated developmental origin of CARMEN-expressing CPCs. Of note, TRIAGE identifies IRX1 and IRX5 as important regulators of cardiogenesis. IRX1 is detected in the trabeculated and compact myocardium of the developing ventricular septum whereas IRX5 demonstrates a subendocardial to subepicardial gradient of expression 40 . Consistently, our experiments show that knocking down IRX1 and IRX5 in fetal CPCs allows A C C E P T E D M A N U S C R I P T antagonist. Downregulation of the WNT pathway is an important step in establishing cardiac fates 41 . In specifying CPCs, C-201 acts via REST-mediated repression. A role for C-201 in blocking REST activity during SMC determination, for instance via sequestering REST, is unlikely since REST silencing abolishes SMC commitment in C-201-expressing CPCs. Consistently, REST acts as a transcriptional repressor in the developing heart, where it is thought to repress adult cardiac gene expression 42-44 . Accordingly, blockade of REST in the heart leads to cardiac dysfunction 45 . In addition, our results suggest that temporal REST 46 . Mechanistically, lncRNA m5C modification could be involved in transcript structure and stability 47 . C-201 associates also to RPA1. Importantly, RPA1 binds RNA with high affinity and promotes R-loop formation with homologous DNA 29 . RNA-DNA hybrids initiate cellular processes regulating transcription and genome dynamics, two important determinants of cell specification 48, 49 . Thus, RPA1 might contribute to effective targeting of REST at regulatory loci via its capacity to stabilize C-201/promoter association, a feature consistent with the predicted DNA binding domains in C-201. CARMEN is expressed in adult tissues, particularly in the heart and the vasculature, reflecting expression in CMs and SMCs 50 . An increasing body of evidence suggests CARMEN is associated with pathological states in the cardiovascular system 22 . Relevant to the present work, CARMEN was recently demonstrated to regulate SMC differentiation and proliferation in atherosclerotic plaques 34 . Unstable regions, in which high proliferation of dedifferentiated SMCs is observed, were characterized with A C C E P T E D M A N U S C R I P T and promotes SMC involved in cardiogenesis, implicating however other isoforms than C-201 maintain a cardiogenic identity in committed precursors. C-201 targets also SFRP1, a known WNT human hearts 21 . Interestingly, human	Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvac191/6941187 by Universitätsbibliothek Bern user on 21 December 2022 Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvac191/6941187 by Universitätsbibliothek Bern user on 21 December 2022 Downloaded from https://academic.oup.com/cardiovascres/advance-article/doi/10.1093/cvr/cvac191/6941187 by Universitätsbibliothek Bern user on 21 December 2022
	reexpression of epicardial and SMC markers, suggesting expression of the two factors is sufficient to
	20 21 22

Then, C-201 associates with NSUN6 and RPA1. A recent study demonstrated a role for NSUN6 in methylating mRNAs and lncRNAs, such as MALAT1, NEAT1 AND XIST decreased CARMEN levels. Consistently, SMCs adopting a synthetic phenotype characterized Carmen knockout mice. We have demonstrated previously that CARMEN is induced in the stressed mouse and

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ACKNOWLEDGEMENTS

We express our gratitude to Dr Gabriel Cuellar Partida, University of Queensland, Brisbane, Australia, for his help in analyzing the GWAS data. We are grateful to Dr Nicole Deglon and Dr Maria Del Rey, University of Lausanne Medical School, Switzerland, for the plasmids used to prepare lentiviral vectors. We thank the Genomic Technologies Facility, the Protein Analysis Facility, the Cellular Imaging Facility and the Mouse Pathology Facility at the University of Lausanne, Switzerland, for providing expertise in transcriptomics, proteomics, imaging and histology respectively.

AUTHORS CONTRIBUTION

T.P. conceived the project, designed experiments and wrote the paper. I.S. designed and performed all wet-lab experiments, with help from M.N., P.A. and F.A.. P.C. and R.J. conducted the bioinformatic analyses. Y.S., S.S., W.J.S. and N.P. performed the TRIAGE, GWAS and single-cell analyses. F.R. provided critical human material for RNA FISH experiment, and her expertise in heart development.

CONFLICT OF INTEREST

T.P. is co-founder of Haya Therapeutics, Epalinges, Switzerland

DATA AVAILABILITY

All transcriptomic data has been deposited to GEO with the identifier GSE199930. See also Supplemental Fig. S7.