Mario Looso

A de novo assembly of the newt transcriptome combined with proteomic validation identifies new protein families expressed during tissue regeneration.

BackgroundNotophthalmus viridescens, an urodelian amphibian, represents an excellent model organism to study regenerative processes, but mechanistic insights into molecular processes driving regeneration have been hindered by a paucity and poor annotation of coding nucleotide sequences. The enormous genome size and the lack of a closely related reference genome have so far prevented assembly of the urodelian genome.ResultsWe describe the de novo assembly of the transcriptome of the newt Notophthalmus viridescens and its experimental validation. RNA pools covering embryonic and larval development, different stages of heart, appendage and lens regeneration, as well as a collection of different undamaged tissues were used to generate sequencing datasets on Sanger, Illumina and 454 platforms. Through a sequential de novo assembly strategy, hybrid datasets were converged into one comprehensive transcriptome comprising 120,922 non-redundant transcripts with a N50 of 975. From this, 38,384 putative transcripts were annotated and around 15,000 transcripts were experimentally validated as protein coding by mass spectrometry-based proteomics. Bioinformatical analysis of coding transcripts identified 826 proteins specific for urodeles. Several newly identified proteins establish novel protein families based on the presence of new sequence motifs without counterparts in public databases, while others containing known protein domains extend already existing families and also constitute new ones.ConclusionsWe demonstrate that our multistep assembly approach allows de novo assembly of the newt transcriptome with an annotation grade comparable to well characterized organisms. Our data provide the groundwork for mechanistic experiments to answer the question whether urodeles utilize proprietary sets of genes for tissue regeneration.

On Marathons and Sprints: An Integrated Quantitative Proteomics and Transcriptomics Analysis of Differences Between Slow and Fast Muscle Fibers

Skeletal muscle tissue contains slow as well as fast twitch muscle fibers that possess different metabolic and contractile properties. Although the distribution of individual proteins in fast and slow fibers has been investigated extensively, a comprehensive proteomic analysis, which is key for any systems biology approach to muscle tissues, is missing. Here, we compared the global protein levels and gene expression profiles of the predominantly slow soleus and fast extensor digitorum longus muscles using the principle of in vivo stable isotope labeling with amino acids based on a fully lysine-6 labeled SILAC-mouse. We identified 551 proteins with significant quantitative differences between slow soleus and fast extensor digitorum longus fibers out of >2000 quantified proteins, which greatly extends the repertoire of proteins differentially regulated between both muscle types. Most of the differentially regulated proteins mediate cellular contraction, ion homeostasis, glycolysis, and oxidation, which reflect the major functional differences between both muscle types. Comparison of proteomics and transcriptomics data uncovered the existence of fiber-type specific posttranscriptional regulatory mechanisms resulting in differential accumulation of Myosin-8 and α-protein kinase 3 proteins and mRNAs among others. Phosphoproteome analysis of soleus and extensor digitorum longus muscles identified 2573 phosphosites on 973 proteins including 1040 novel phosphosites. The in vivo stable isotope labeling with amino acids-mouse approach used in our study provides a comprehensive view into the protein networks that direct fiber-type specific functions and allows a detailed dissection of the molecular composition of slow and fast muscle tissues with unprecedented resolution.

RBM24 is a major regulator of muscle-specific alternative splicing.

Cell-type-specific splicing generates numerous alternatively spliced transcripts playing important roles for organ development and homeostasis, but only a few tissue-specific splicing factors have been identified. We found that RBM24 governs a large number of muscle-specific splicing events that are critically involved in cardiac and skeletal muscle development and disease. Targeted inactivation of RBM24 in mice disrupted cardiac development and impaired sarcomerogenesis in striated muscles. In vitro splicing assays revealed that recombinant RBM24 is sufficient to promote muscle-specific exon inclusion in nuclear extracts of nonmuscle cells. Furthermore, we demonstrate that binding of RBM24 to an intronic splicing enhancer (ISE) is essential and sufficient to overcome repression of exon inclusion by an exonic splicing silencer (ESS) containing PTB and hnRNP A1/A2 binding sites. Introduction of ESS and ISE converted a constitutive exon into an RMB24-dependent alternative exon. We reason that RBM24 is a major regulator of alternative splicing in striated muscles.

ErbB-2 signals through Plexin-B1 to promote breast cancer metastasis

Diagnosis of metastatic breast cancer is associated with a very poor prognosis. New therapeutic targets are urgently needed, but their development is hampered by a lack of understanding of the mechanisms leading to tumor metastasis. Exemplifying this is the fact that the approximately 30% of all breast cancers overexpressing the receptor tyrosine kinase ErbB-2 are characterized by high metastatic potential and poor prognosis, but the signaling events downstream of ErbB-2 that drive cancer cell invasion and metastasis remain incompletely understood. Here we show that overexpression of ErbB-2 in human breast cancer cell lines leads to phosphorylation and activation of the semaphorin receptor Plexin-B1. This was required for ErbB-2-dependent activation of the pro-metastatic small GTPases RhoA and RhoC and promoted invasive behavior of human breast cancer cells. In a mouse model of ErbB-2-overexpressing breast cancer, ablation of the gene encoding Plexin-B1 strongly reduced the occurrence of metastases. Moreover, in human patients with ErbB-2-overexpressing breast cancer, low levels of Plexin-B1 expression correlated with good prognosis. Our data suggest that Plexin-B1 represents a new candidate therapeutic target for treating patients with ErbB-2-positive breast cancer.

Prmt5 is a regulator of muscle stem cell expansion in adult mice

Skeletal muscle stem cells (MuSC), also called satellite cells, are indispensable for maintenance and regeneration of adult skeletal muscles. Yet, a comprehensive picture of the regulatory events controlling the fate of MuSC is missing. Here, we determine the proteome of MuSC to design a loss-of-function screen, and identify 120 genes important for MuSC function including the arginine methyltransferase Prmt5. MuSC-specific inactivation of Prmt5 in adult mice prevents expansion of MuSC, abolishes long-term MuSC maintenance and abrogates skeletal muscle regeneration. Interestingly, Prmt5 is dispensable for proliferation and differentiation of Pax7+ myogenic progenitor cells during mouse embryonic development, indicating significant differences between embryonic and adult myogenesis. Mechanistic studies reveal that Prmt5 controls proliferation of adult MuSC by direct epigenetic silencing of the cell cycle inhibitor p21. We reason that Prmt5 generates a poised state that keeps MuSC in a standby mode, thus allowing rapid MuSC amplification under disease conditions.

Targeted Ablation of Periostin-Expressing Activated Fibroblasts Prevents Adverse Cardiac Remodeling in Mice

RATIONALE Activated cardiac fibroblasts (CF) are crucial players in the cardiac damage response; excess fibrosis, however, may result in myocardial stiffening and heart failure development. Inhibition of activated CF has been suggested as a therapeutic strategy in cardiac disease, but whether this truly improves cardiac function is unclear. OBJECTIVE To study the effect of CF ablation on cardiac remodeling. METHODS AND RESULTS We characterized subgroups of murine CF by single-cell expression analysis and identified periostin as the marker showing the highest correlation to an activated CF phenotype. We generated bacterial artificial chromosome-transgenic mice allowing tamoxifen-inducible Cre expression in periostin-positive cells as well as their diphtheria toxin-mediated ablation. In the healthy heart, periostin expression was restricted to valvular fibroblasts; ablation of this population did not affect cardiac function. After chronic angiotensin II exposure, ablation of activated CF resulted in significantly reduced cardiac fibrosis and improved cardiac function. After myocardial infarction, ablation of periostin-expressing CF resulted in reduced fibrosis without compromising scar stability, and cardiac function was significantly improved. Single-cell transcriptional analysis revealed reduced CF activation but increased expression of prohypertrophic factors in cardiac macrophages and cardiomyocytes, resulting in localized cardiomyocyte hypertrophy. CONCLUSIONS Modulation of the activated CF population is a promising approach to prevent adverse cardiac remodeling in response to angiotensin II and after myocardial infarction.

Advanced Identification of Proteins in Uncharacterized Proteomes by Pulsed in Vivo Stable Isotope Labeling-based Mass Spectrometry

Despite progress in the characterization of their genomes, proteomes of several model organisms are often only poorly characterized. This problem is aggravated by the presence of large numbers of expressed sequence tag clones that lack homologues in other species, which makes it difficult to identify new proteins irrespective of whether such molecules are involved in species-specific biological processes. We have used a pulsed stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry method, which is based on the detection of paired peptides after [13C6]lysine incorporation into proteins in vivo, to greatly increase the confidence of protein identification in cross-species database searches. The method was applied to identify nearly 3000 proteins in regenerating tails of the urodele amphibian Notophthalmus viridescens, which possesses outstanding capabilities in the regeneration of complex tissues. We reason that pulsed in vivo SILAC represents a versatile tool to identify new proteins in species for which only limited sequence information exists.

Long Noncoding RNA MANTIS Facilitates Endothelial Angiogenic Function

Background: The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and functionally important endothelial lncRNAs. Methods: Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression. Results: A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding. Conclusion: MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.

Integrative ''Omics''-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses

Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level (“expressome”). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions.

High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX

The eukaryotic genome is organized into chromatins, the physiological template for DNA-dependent processes including replication, recombination, repair, and transcription. Chromatin-mediated transcription regulation involves DNA methylation, chromatin remodeling, and histone modifications. However, chromatin also contains non-histone chromatin-associated proteins, of which the high-mobility group (HMG) proteins are the most abundant. Although it is known that HMG proteins induce structural changes of chromatin, the processes underlying transcription regulation by HMG proteins are poorly understood. Here we decipher the molecular mechanism of transcription regulation mediated by the HMG AT-hook 2 protein (HMGA2). We combined proteomic, ChIP-seq, and transcriptome data to show that HMGA2-induced transcription requires phosphorylation of the histone variant H2AX at S139 (H2AXS139ph; γ-H2AX) mediated by the protein kinase ataxia telangiectasia mutated (ATM). Furthermore, we demonstrate the biological relevance of this mechanism within the context of TGFβ1 signaling. The interplay between HMGA2, ATM, and H2AX is a novel mechanism of transcription initiation. Our results link H2AXS139ph to transcription, assigning a new function for this DNA damage marker. Controlled chromatin opening during transcription may involve intermediates with DNA breaks that may require mechanisms that ensure the integrity of the genome.