How broad is the selfing syndrome? Insights from convergent evolution of gene expression across species and tissues in the Capsella genus

The shift from outcrossing to selfing is one of the main evolutionary transitions in plants. It is accompanied by profound effects on reproductive traits, the so-called selfing syndrome. Because the transition to selfing also implies deep genomic and ecological changes, one also expects to observe a genomic selfing syndrome. We took advantage of the three independent transitions from outcrossing to selfing in the Capsella genus to characterize the overall impact of mating system change on RNA expression, in flowers but also in leaves and roots. We quantified the extent of both selfing and genomic syndromes, and tested whether changes in expression corresponded to adaptation to selfing or to relaxed selection on traits that were constrained in outcrossers. Mating system change affected gene expression in all three tissues but more so in flowers than in roots and leaves. Gene expression in selfing species tended to converge in flowers but diverged in the two other tissues. Hence, convergent adaptation to selfing dominates in flowers, whereas genetic drift plays a more important role in leaves and roots. The effect of mating system transition is not limited to reproductive tissues and corresponds to both adaptation to selfing and relaxed selection on previously constrained traits

Myocardin regulates exon usage in smooth muscle cells through induction of splicing regulatory factors

Differentiation of smooth muscle cells (SMCs) depends on serum response factor (SRF) and its co-activator myocardin (MYOCD). The role of MYOCD for the SMC program of gene transcription is well established. In contrast, the role of MYOCD in control of SMC-specific alternative exon usage, including exon splicing, has not been explored. In the current work we identified four splicing factors (MBNL1, RBPMS, RBPMS2, and RBFOX2) that correlate with MYOCD across human SMC tissues. Forced expression of MYOCD family members in human coronary artery SMCs in vitro upregulated expression of these splicing factors. For global profiling of transcript diversity, we performed RNA-sequencing after MYOCD transduction. We analyzed alternative transcripts with three different methods. Exon-based analysis identified 1637 features with differential exon usage. For example, usage of 3 ' exons in MYLK that encode telokin increased relative to 5 ' exons, as did the 17 kDa telokin to 130 kDa MYLK protein ratio. Dedicated event-based analysis identified 239 MYOCD-driven splicing events. Events involving MBNL1, MCAM, and ACTN1 were among the most prominent, and this was confirmed using variant-specific PCR analyses. In support of a role for RBPMS and RBFOX2 in MYOCD-driven splicing we found enrichment of their binding motifs around differentially spliced exons. Moreover, knockdown of either RBPMS or RBFOX2 antagonized splicing events stimulated by MYOCD, including those involving ACTN1, VCL, and MBNL1. Supporting an in vivo role of MYOCD-SRF-driven splicing, we demonstrate altered Rbpms expression and splicing in inducible and SMC-specific Srf knockout mice. We conclude that MYOCD-SRF, in part via RBPMS and RBFOX2, induce a program of differential exon usage and alternative splicing as part of the broader program of SMC differentiation.Peer reviewe

High-throughput muscle fiber typing from RNA sequencing data

Background: Skeletal muscle fiber type distribution has implications for human health, muscle function, and performance. This knowledge has been gathered using labor-intensive and costly methodology that limited these studies. Here, we present a method based on muscle tissue RNA sequencing data (totRNAseq) to estimate the distribution of skeletal muscle fiber types from frozen human samples, allowing for a larger number of individuals to be tested. Methods: By using single-nuclei RNA sequencing (snRNAseq) data as a reference, cluster expression signatures were produced by averaging gene expression of cluster gene markers and then applying these to totRNAseq data and inferring muscle fiber nuclei type via linear matrix decomposition. This estimate was then compared with fiber type distribution measured by ATPase staining or myosin heavy chain protein isoform distribution of 62 muscle samples in two independent cohorts (n = 39 and 22). Results: The correlation between the sequencing-based method and the other two were r(ATpas) = 0.44 [0.13-0.67], [95% CI], and r(myosin) = 0.83 [0.61-0.93], with p = 5.70 x 10(-3) and 2.00 x 10(-6), respectively. The deconvolution inference of fiber type composition was accurate even for very low totRNAseq sequencing depths, i.e., down to an average of similar to 10,000 paired-end reads. Conclusions: This new method (https://github.com/OlaHanssonLab/PredictFiberType) consequently allows for measurement of fiber type distribution of a larger number of samples using totRNAseq in a cost and labor-efficient way. It is now feasible to study the association between fiber type distribution and e.g. health outcomes in large well-powered studies.Peer reviewe

High-throughput muscle fiber typing from RNA sequencing data

Background Skeletal muscle fiber type distribution has implications for human health, muscle function, and performance. This knowledge has been gathered using labor-intensive and costly methodology that limited these studies. Here, we present a method based on muscle tissue RNA sequencing data (totRNAseq) to estimate the distribution of skeletal muscle fiber types from frozen human samples, allowing for a larger number of individuals to be tested. Methods By using single-nuclei RNA sequencing (snRNAseq) data as a reference, cluster expression signatures were produced by averaging gene expression of cluster gene markers and then applying these to totRNAseq data and inferring muscle fiber nuclei type via linear matrix decomposition. This estimate was then compared with fiber type distribution measured by ATPase staining or myosin heavy chain protein isoform distribution of 62 muscle samples in two independent cohorts (n = 39 and 22). Results The correlation between the sequencing-based method and the other two were rATPas = 0.44 [0.13–0.67], [95% CI], and rmyosin = 0.83 [0.61–0.93], with p = 5.70 × 10–3 and 2.00 × 10–6, respectively. The deconvolution inference of fiber type composition was accurate even for very low totRNAseq sequencing depths, i.e., down to an average of ~ 10,000 paired-end reads. Conclusions This new method (https://github.com/OlaHanssonLab/PredictFiberType) consequently allows for measurement of fiber type distribution of a larger number of samples using totRNAseq in a cost and labor-efficient way. It is now feasible to study the association between fiber type distribution and e.g. health outcomes in large well-powered studies.journal articl

The genetic consequences of dog breed formation-Accumulation of deleterious genetic variation and fixation of mutations associated with myxomatous mitral valve disease in cavalier King Charles spaniels

Selective breeding for desirable traits in strictly controlled populations has generated an extraordinary diversity in canine morphology and behaviour, but has also led to loss of genetic variation and random entrapment of disease alleles. As a consequence, specific diseases are now prevalent in certain breeds, but whether the recent breeding practice led to an overall increase in genetic load remains unclear. Here we generate whole genome sequencing (WGS) data from 20 dogs per breed from eight breeds and document a similar to 10% rise in the number of derived alleles per genome at evolutionarily conserved sites in the heavily bottlenecked cavalier King Charles spaniel breed (cKCs) relative to in most breeds studied here. Our finding represents the first clear indication of a relative increase in levels of deleterious genetic variation in a specific breed, arguing that recent breeding practices probably were associated with an accumulation of genetic load in dogs. We then use the WGS data to identify candidate risk alleles for the most common cause for veterinary care in cKCs-the heart disease myxomatous mitral valve disease (MMVD). We verify a potential link to MMVD for candidate variants near the heart specific NEBL gene in a dachshund population and show that two of the NEBL candidate variants have regulatory potential in heartderived cell lines and are associated with reduced NEBL isoform nebulette expression in papillary muscle (but not in mitral valve, nor in left ventricular wall). Alleles linked to reduced nebulette expression may hence predispose cKCs and other breeds to MMVD via loss of papillary muscle integrity

Whole genome resequencing of Heliconius butterflies revolutionizes our view of the level of admixture between species

The theory of "genomic islands of speciation" has been extensively debated during the last decade. This thesis not only supports this theory, but provides evidence that challenges previous beliefs on the level of admixture between species. The recently published Heliconius melpomene genome project reported apparent genomic paraphyly of H. pardalinus with regard to H. elevatus (Heliconius Genome Consortium 2012). Here, we investigate this pair of butterfly species more fully, firstly by using whole-genome resequence data, and secondly by analyzing additional geographic populations of both species, as well as outgroup taxa. Using a nuclear whole-genome phylogenetic analysis we also confirm that H. elevatus is paraphyletic. The genome-wide phylogenetic signal in H. pardalinus and H. elevatus does not indicate expected mutual monophyly of each species as it seems strongly distorted by a high level of admixture. However, several regions of the genome remain differentiated and do show the presumably original phylogenetic signal with mutual monophyly of H. pardalinus and H. elevatus. The genomic background is so homogenized that its level of differentiation (FST ~ 0.03) virtually implies panmixia. The pattern of a high level of homogenization across the genome with several regions of differentiation was consistent with a number of other statistics such as absolute divergence Dxy, nucleotide polymorphism π, number of fixed differences and with a sliding window phylogeny. The identified genomic islands of divergence comprise genes responsible for wing-patterning and chemosensation in Heliconius and some of these genes are found to be under positive selection, suggesting possible candidates of speciation

Genome evolution and adaptation of a successful allopolyploid, Capsella bursa-pastoris

The term allopolyploid refers to an organism that originated through hybridization and increased its ploidy level by retaining the unreduced genomes of its parents. Both hybridization and polyploidy usually have negative consequences for the organism. However, there are species that not only survive these modifications but even thrive and can outcompete their diploid relatives. There are many intuitive explanations for the success of polyploids, but the number of empirical studies is limited. The shepherd's purse (Capsella bursa-pastoris) is an emerging model for studying a successful allopolyploid species. C. bursa-pastoris occurs worldwide, whereas its parental species, Capsella grandiflora and Capsella orientalis, have more limited distribution range. C. grandiflora is confined to Northern Greece and Albania, and C. orientalis is found only in the steppes of Central Asia. We described the genetic variation within C. bursa-pastoris and showed that it is not homogeneous across Eurasia but rather subdivided into three genetically distinct populations: one comprises accessions from Europe and Eastern Siberia, the second one is located in Eastern Asia and the third one groups accessions around the Middle East. Reconstruction of the colonization history suggested that this species originated in the Middle East and subsequently spread to Europe and Eastern Asia. This colonization was probably human-mediated. Interestingly, these three populations survive in different environmental conditions, and yet most gene expression differences between them could be explained by neutral processes. We also found that despite a common history within one species, the two subgenomes retained differences already present between the parental species. In particular, the genetic load was still higher on the subgenome inherited from C. orientalis than on the one inherited from C. grandiflora. The two subgenomes were also differentially influenced by introgression and selection in the three genetic clusters. Gene expression variation was highly correlated between the two subgenomes but the total level of expression showed variation in parental dominance across flower, leaf, and root tissues. This thesis for the first time shows that the evolutionary pathways of allopolyploids may differ not only on the species level but also between populations within one species. It also supports the theory that alloploidy provides an increased amount of genetic material that enables evolutionary flexibility

Genome evolution and adaptation of a successful allopolyploid, Capsella bursa-pastoris

The term allopolyploid refers to an organism that originated through hybridization and increased its ploidy level by retaining the unreduced genomes of its parents. Both hybridization and polyploidy usually have negative consequences for the organism. However, there are species that not only survive these modifications but even thrive and can outcompete their diploid relatives. There are many intuitive explanations for the success of polyploids, but the number of empirical studies is limited. The shepherd's purse (Capsella bursa-pastoris) is an emerging model for studying a successful allopolyploid species. C. bursa-pastoris occurs worldwide, whereas its parental species, Capsella grandiflora and Capsella orientalis, have more limited distribution range. C. grandiflora is confined to Northern Greece and Albania, and C. orientalis is found only in the steppes of Central Asia. We described the genetic variation within C. bursa-pastoris and showed that it is not homogeneous across Eurasia but rather subdivided into three genetically distinct populations: one comprises accessions from Europe and Eastern Siberia, the second one is located in Eastern Asia and the third one groups accessions around the Middle East. Reconstruction of the colonization history suggested that this species originated in the Middle East and subsequently spread to Europe and Eastern Asia. This colonization was probably human-mediated. Interestingly, these three populations survive in different environmental conditions, and yet most gene expression differences between them could be explained by neutral processes. We also found that despite a common history within one species, the two subgenomes retained differences already present between the parental species. In particular, the genetic load was still higher on the subgenome inherited from C. orientalis than on the one inherited from C. grandiflora. The two subgenomes were also differentially influenced by introgression and selection in the three genetic clusters. Gene expression variation was highly correlated between the two subgenomes but the total level of expression showed variation in parental dominance across flower, leaf, and root tissues. This thesis for the first time shows that the evolutionary pathways of allopolyploids may differ not only on the species level but also between populations within one species. It also supports the theory that alloploidy provides an increased amount of genetic material that enables evolutionary flexibility

Genome evolution and adaptation of a successful allopolyploid, Capsella bursa-pastoris

The term allopolyploid refers to an organism that originated through hybridization and increased its ploidy level by retaining the unreduced genomes of its parents. Both hybridization and polyploidy usually have negative consequences for the organism. However, there are species that not only survive these modifications but even thrive and can outcompete their diploid relatives. There are many intuitive explanations for the success of polyploids, but the number of empirical studies is limited. The shepherd's purse (Capsella bursa-pastoris) is an emerging model for studying a successful allopolyploid species. C. bursa-pastoris occurs worldwide, whereas its parental species, Capsella grandiflora and Capsella orientalis, have more limited distribution range. C. grandiflora is confined to Northern Greece and Albania, and C. orientalis is found only in the steppes of Central Asia. We described the genetic variation within C. bursa-pastoris and showed that it is not homogeneous across Eurasia but rather subdivided into three genetically distinct populations: one comprises accessions from Europe and Eastern Siberia, the second one is located in Eastern Asia and the third one groups accessions around the Middle East. Reconstruction of the colonization history suggested that this species originated in the Middle East and subsequently spread to Europe and Eastern Asia. This colonization was probably human-mediated. Interestingly, these three populations survive in different environmental conditions, and yet most gene expression differences between them could be explained by neutral processes. We also found that despite a common history within one species, the two subgenomes retained differences already present between the parental species. In particular, the genetic load was still higher on the subgenome inherited from C. orientalis than on the one inherited from C. grandiflora. The two subgenomes were also differentially influenced by introgression and selection in the three genetic clusters. Gene expression variation was highly correlated between the two subgenomes but the total level of expression showed variation in parental dominance across flower, leaf, and root tissues. This thesis for the first time shows that the evolutionary pathways of allopolyploids may differ not only on the species level but also between populations within one species. It also supports the theory that alloploidy provides an increased amount of genetic material that enables evolutionary flexibility

Regulation of the Muscarinic M-3 Receptor by Myocardin-Related Transcription Factors

Myocardin-related transcription factors (MRTFs: myocardin/MYOCD, MRTF-A/MRTFA, and MRTF-B/MRTFB) are co-factors of serum response factor (SRF) that activate the smooth muscle cell (SMC) gene program and that play roles in cardiovascular development and mechanobiology. Gain and loss of function experiments have defined the SMC gene program under control of MRTFs, yet full understanding of their impact is lacking. In the present study, we tested the hypothesis that the muscarinic M-3 receptor (CHRM3) is regulated by MRTFs together with SRF. Forced expression of MYOCD (8d) in human coronary artery (SMC) followed by RNA-sequencing showed increased levels of M-2, M-3, and M-5 receptors (CHRM2: 2-fold, CHRM3: 16-fold, and CHRM5: 2-fold). The effect of MYOCD on M-3 was confirmed by RT-qPCR using both coronary artery and urinary bladder SMCs, and correlation analyses using human transcriptomic datasets suggested that M-3 may also be regulated by MRTF-B. Head-to-head comparisons of MYOCD, MRTF-A and MRTF-B, argued that while all MRTFs are effective, MRTF-B is the most powerful transactivator of CHRM3, causing a 600-fold increase at 120h. Accordingly, MRTF-B conferred responsiveness to the muscarinic agonist carbachol in Ca2+ imaging experiments. M-3 was suppressed on treatment with the MRTF-SRF inhibitor CCG-1423 using SMCs transduced with either MRTF-A or MRTF-B and using intact mouse esophagus in culture (by 92 +/- 2%). Moreover, silencing of SRF with a short hairpin reduced CHRM3 (by >60%) in parallel with alpha-actin (ACTA2). Tamoxifen inducible knockout of Srf in smooth muscle reduced Srf (by 54 +/- 4%) and Chrm3 (by 41 +/- 6%) in the urinary bladder at 10days, but Srf was much less reduced or unchanged in aorta, ileum, colon, trachea, and esophagus. Longer induction (21d) further accentuated the reduction of Chrm3 in the bladder and ileum, but no change was seen in the aorta. Single cell RNA-sequencing revealed that Mrtfb dominates in ECs, while Myocd dominates in SMCs, raising the possibility that Chrm3 may be driven by Mrtfb-Srf in the endothelium and by Myocd-Srf in SMCs. These findings define a novel transcriptional control mechanism for muscarinic M-3 receptors in human cells, and in mice, that could be targeted for therapy.Peer reviewe