Nfix regulates fetal-specific transcription in developing skeletal muscle.

Skeletal myogenesis, like hematopoiesis, occurs in successive developmental stages that involve different cell populations and expression of different genes. We show here that the transcription factor nuclear factor one X (Nfix), whose expression is activated by Pax7 in fetal muscle, in turn activates the transcription of fetal specific genes such as MCK and beta-enolase while repressing embryonic genes such as slow myosin. In the case of the MCK promoter, Nfix forms a complex with PKC theta that binds, phosphorylates, and activates MEF2A. Premature expression of Nfix activates fetal and suppresses embryonic genes in embryonic muscle, whereas muscle-specific ablation of Nfix prevents fetal and maintains embryonic gene expression in the fetus. Therefore, Nfix acts as a transcriptional switch from embryonic to fetal myogenesis.

A KAP1 phosphorylation switch controls MyoD function during skeletal muscle differentiation.

The transcriptional activator MyoD serves as a master controller of myogenesis. Often in partnership with Mef2 (myocyte enhancer factor 2), MyoD binds to the promoters of hundreds of muscle genes in proliferating myoblasts yet activates these targets only upon receiving cues that launch differentiation. What regulates this off/on switch of MyoD function has been incompletely understood, although it is known to reflect the action of chromatin modifiers. Here, we identify KAP1 (KRAB [Krüppel-like associated box]-associated protein 1)/TRIM28 (tripartite motif protein 28) as a key regulator of MyoD function. In myoblasts, KAP1 is present with MyoD and Mef2 at many muscle genes, where it acts as a scaffold to recruit not only coactivators such as p300 and LSD1 but also corepressors such as G9a and HDAC1 (histone deacetylase 1), with promoter silencing as the net outcome. Upon differentiation, MSK1-mediated phosphorylation of KAP1 releases the corepressors from the scaffold, unleashing transcriptional activation by MyoD/Mef2 and their positive cofactors. Thus, our results reveal KAP1 as a previously unappreciated interpreter of cell signaling, which modulates the ability of MyoD to drive myogenesis.

Guide Cells Support Muscle Regeneration and Affect Neuro-Muscular Junction Organization.

Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them 'guide' cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers.

Pharmacological Induction of a Progenitor State for the Efficient Expansion of Primary Human Hepatocytes.

The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration. Human HPCs from healthy donors and pediatric patients proliferated vigorously while maintaining their genomic stability and could be redifferentiated in vitro into metabolically competent cells that supported the replication of hepatitis B and delta viruses. Redifferentiation efficiency was boosted by three-dimensional culture. Finally, transcriptome analysis showed that HPCs were more closely related to mature hepatocytes than iPSC-derived hepatocyte-like cells were. Conclusion: HPC induction holds promise for a variety of applications such as ex vivo disease modeling, personalized drug testing or metabolic studies, and development of a bioartificial liver.

Polyphenic trait promotes liver cancer in a model of epigenetic instability in mice.

Hepatocellular carcinoma (HCC) represents the fifth-most common form of cancer worldwide and carries a high mortality rate attributed to lack of effective treatment. Males are 8 times more likely to develop HCC than females, an effect largely driven by sex hormones, albeit through still poorly understood mechanisms. We previously identified TRIM28 (tripartite protein 28), a scaffold protein capable of recruiting a number of chromatin modifiers, as a crucial mediator of sexual dimorphism in the liver. Trim28hep-/- mice display sex-specific transcriptional deregulation of a wide range of bile and steroid metabolism genes and development of liver adenomas in males. We now demonstrate that obesity and aging precipitate alterations of TRIM28-dependent transcriptional dynamics, leading to a metabolic infection state responsible for highly penetrant male-restricted hepatic carcinogenesis. Molecular analyses implicate aberrant androgen receptor stimulation, biliary acid disturbances, and altered responses to gut microbiota in the pathogenesis of Trim28hep-/- -associated HCC. Correspondingly, androgen deprivation markedly attenuates the frequency and severity of tumors, and raising animals under axenic conditions completely abrogates their abnormal phenotype, even upon high-fat diet challenge. CONCLUSION: This work underpins how discrete polyphenic traits in epigenetically metastable conditions can contribute to a cancer-prone state and more broadly provides new evidence linking hormonal imbalances, metabolic disturbances, gut microbiota, and cancer. (Hepatology 2017;66:235-251).

Alpha sarcoglycan is required for FGF-dependent myogenic progenitor cell proliferation in vitro and in vivo.

Mice deficient in α-sarcoglycan (Sgca-null mice) develop progressive muscular dystrophy and serve as a model for human limb girdle muscular dystrophy type 2D. Sgca-null mice suffer a more severe myopathy than that of mdx mice, the model for Duchenne muscular dystrophy. This is the opposite of what is observed in humans and the reason for this is unknown. In an attempt to understand the cellular basis of this severe muscular dystrophy, we isolated clonal populations of myogenic progenitor cells (MPCs), the resident postnatal muscle progenitors of dystrophic and wild-type mice. MPCs from Sgca-null mice generated much smaller clones than MPCs from wild-type or mdx dystrophic mice. Impaired proliferation of Sgca-null myogenic precursors was confirmed by single fiber analysis and this difference correlated with Sgca expression during MPC proliferation. In the absence of dystrophin and associated proteins, which are only expressed after differentiation, SGCA complexes with and stabilizes FGFR1. Deficiency of Sgca leads to an absence of FGFR1 expression at the membrane and impaired MPC proliferation in response to bFGF. The low proliferation rate of Sgca-null MPCs was rescued by transduction with Sgca-expressing lentiviral vectors. When transplanted into dystrophic muscle, Sgca-null MPCs exhibited reduced engraftment. The reduced proliferative ability of Sgca-null MPCs explains, at least in part, the severity of this muscular dystrophy and also why wild-type donor progenitor cells engraft efficiently and consequently ameliorate disease.

Semi-automated approaches for interrogating spatial heterogeneity of tissue samples.

Tissues are spatially orchestrated ecosystems composed of heterogeneous cell populations and non-cellular elements. Tissue components' interactions shape the biological processes that govern homeostasis and disease, thus comprehensive insights into tissues' composition are crucial for understanding their biology. Recently, advancements in the spatial biology field enabled the in-depth analyses of tissue architecture at single-cell resolution, while preserving the structural context. The increasing number of biomarkers analyzed, together with whole tissue imaging, generate datasets approaching several hundreds of gigabytes in size, which are rich sources of valuable knowledge but require investments in infrastructure and resources for extracting quantitative information. The analysis of multiplex whole-tissue images requires extensive training and experience in data analysis. Here, we showcase how a set of open-source tools can allow semi-automated image data extraction to study the spatial composition of tissues with a focus on tumor microenvironment (TME). With the use of Lunaphore COMET platform, we interrogated lung cancer specimens where we examined the expression of 20 biomarkers. Subsequently, the tissue composition was interrogated using an in-house optimized nuclei detection algorithm followed by a newly developed image artifact exclusion approach. Thereafter, the data was processed using several publicly available tools, highlighting the compatibility of COMET-derived data with currently available image analysis frameworks. In summary, we showcased an innovative semi-automated workflow that highlights the ease of adoption of multiplex imaging to explore TME composition at single-cell resolution using a simple slide in, data out approach. Our workflow is easily transferrable to various cohorts of specimens to provide a toolset for spatial cellular dissection of the tissue composition.

Liver-specific ablation of Krüppel-associated box-associated protein 1 in mice leads to male-predominant hepatosteatosis and development of liver adenoma.

UNLABELLED: The liver is characterized by sexually dimorphic gene expression translating into sex-specific differences in lipid, drug, steroid hormone, and xenobiotic metabolism, with distinct responses of males and females to environmental challenges. Here, we investigated the role of the Krüppel-associated box (KRAB)-associated protein 1 (KAP1) epigenetic regulator in this process. Liver-specific KAP1 knockout (KO) led to strikingly sexually dimorphic phenotypic disturbances, including male-predominant steatosis and hepatic tumors with up-regulation of protein kinase B and extracellular signal-related kinases 1/2 mitogen-activated protein kinase signaling. This correlated with the sex-specific transcriptional dysregulation of a wide range of metabolic genes, notably those involved in retinol and sex hormone processing as well as in detoxification. Furthermore, chromatin immunoprecipitation followed by deep sequencing indicated that a number of dysregulated genes are direct targets of the KRAB/KAP1 repression system. Those genes include sexually dimorphic cytochrome P 450 Cyp2d9, glutathione S-transferase π, Cyp2a, Cyp2b, and Cyp3a gene clusters. Additionally, we identified a male-restricted KAP1-binding site in the fat-specific protein 27 gene, correlating with its male-predominant up-regulation upon Kap1 deletion, suggesting that the latter might be an important trigger in the development of male-specific hepatosteatosis and secondary tumorigenesis. CONCLUSION: This work reveals KRAB/KAP1-mediated transcriptional regulation as a central event in metabolic control hormones, drugs, and xenobiotics in the liver and further links disturbances in these processes with hepatic carcinogenesis.

High-plex protein and whole transcriptome co-mapping at cellular resolution with spatial CITE-seq.

In this study, we extended co-indexing of transcriptomes and epitopes (CITE) to the spatial dimension and demonstrated high-plex protein and whole transcriptome co-mapping. We profiled 189 proteins and whole transcriptome in multiple mouse tissue types with spatial CITE sequencing and then further applied the method to measure 273 proteins and transcriptome in human tissues, revealing spatially distinct germinal center reactions in tonsil and early immune activation in skin at the Coronavirus Disease 2019 mRNA vaccine injection site.

Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.

Spatial-CITE-seq: spatially resolved high-plex protein and whole transcriptome co-mapping.

We present spatial-CITE-seq for high-plex protein and whole transcriptome co-mapping, which was firstly demonstrated for profiling 198 proteins and transcriptome in multiple mouse tissue types. It was then applied to human tissues to measure 283 proteins and transcriptome that revealed spatially distinct germinal center reaction in tonsil and early immune activation in skin at the COVID-19 mRNA vaccine injection site. Spatial-CITE-seq may find a range of applications in biomedical research.

Mesoangioblasts at 20: From the embryonic aorta to the patient bed.

In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded in vitro (also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.

Localization of Magic-F1 transgene, involved in muscular hypertrophy, during early myogenesis.

We recently showed that Magic-F1 (Met-activating genetically improved chimeric factor 1), a human recombinant protein derived from hepatocyte growth factor/scatter factor (HGF/SF) induces muscle cell hypertrophy but not progenitor cell proliferation, both in vitro and in vivo. Here, we examined the temporal and spatial expression pattern of Magic-F1 in comparison with Pax3 (paired box gene 3) transcription factor during embryogenesis. Ranging from 9.5 to 17.5 dpc (days post coitum) mouse embryos were analyzed by in situ hybridization using whole mounts during early stages of development (9.5-10.5-11.5 dpc) and cryostat sections for later stages (11.5-13.5-15.5-17.5 dpc). We found that Magic-F1 is expressed in developing organs and tissues of mesenchymal origin, where Pax3 signal appears to be downregulated respect to the wt embryos. These data suggest that Magic-F1 could be responsible of muscular hypertrophy, cooperating with Pax3 signal pathway in skeletal muscle precursor cells.

KAP1 targets actively transcribed genomic loci to exert pleomorphic effects on RNA polymerase II activity.

KAP1 (KRAB-associated protein 1) is best known as a co-repressor responsible for inducing heterochromatin formation, notably at transposable elements. However, it has also been observed to bind the transcription start site of actively expressed genes. To address this paradox, we characterized the protein interactome of KAP1 in the human K562 erythro-leukaemia cell line. We found that the regulator can associate with a wide range of nucleic acid binding proteins, nucleosome remodellers, chromatin modifiers and other transcription modulators. We further determined that KAP1 is recruited at actively transcribed polymerase II promoters, where its depletion resulted in pleomorphic effects, whether expression of these genes was normally constitutive or inducible, consistent with the breadth of possible KAP1 interactors. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

In vivo stem cell tracking using scintigraphy in a canine model of DMD.

One of the main challenges in cell therapy for muscle diseases is to efficiently target the muscle. To address this issue and achieve better understanding of in vivo cell fate, we evaluated the relevance of a non-invasive cell tracking method in the Golden Retriever Muscular Dystrophy (GRMD) model, a well-recognised model of Duchenne Muscular Dystrophy (DMD). Mesoangioblasts were directly labelled with 111In-oxine, and injected through one of the femoral arteries. The scintigraphy images obtained provided the first quantitative mapping of the immediate biodistribution of mesoangioblasts in a large animal model of DMD. The results revealed that cells were trapped by the first capillary filters: the injected limb and the lung. During the days following injection, radioactivity was redistributed to the liver. In vitro studies, performed with the same cells prepared for injecting the animal, revealed prominent cell death and 111In release. In vivo, cell death resulted in 111In release into the vasculature that was taken up by the liver, resulting in a non-specific and non-cell-bound radioactive signal. Indirect labelling methods would be an attractive alternative to track cells on the mid- and long-term.

Nutritional intervention with cyanidin hinders the progression of muscular dystrophy.

Muscular Dystrophies are severe genetic diseases due to mutations in structural genes, characterized by progressive muscle wasting that compromises patients' mobility and respiratory functions. Literature underlined oxidative stress and inflammation as key drivers of these pathologies. Interestingly among different myofiber classes, type I fibers display a milder dystrophic phenotype showing increased oxidative metabolism. This work shows the benefits of a cyanidin-enriched diet, that promotes muscle fiber-type switch and reduced inflammation in dystrophic alpha-sarcoglyan (Sgca) null mice having, as a net outcome, morphological and functional rescue. Notably, this benefit is achieved also when the diet is administered in dystrophic animals when the signs of the disease are seriously evident. Our work provides compelling evidence that a cyanidin-rich diet strongly delays the progression of muscular dystrophies, paving the way for a combinatorial approach where nutritional-based reduction of muscle inflammation and oxidative stress facilitate the successful perspectives of definitive treatments.

Cellular mechanisms and local progenitor activation to regulate skeletal muscle mass.

Skeletal muscle hypertrophy is a result of increased load, such as functional and stretch-overload. Activation of satellite cells and proliferation, differentiation and fusion are required for hypertrophy of overloaded skeletal muscles. On the contrary, a dramatic loss of skeletal muscle mass determines atrophy settings. The epigenetic changes involved in gene regulation at DNA and chromatin level are critical for the opposing phenomena, muscle growth and atrophy. Physiological properties of skeletal muscle tissue play a fundamental role in health and disease since it is the most abundant tissue in mammals. In fact, protein synthesis and degradation are finely modulated to maintain an appropriate muscle mass. When the molecular signaling is altered muscle wasting and weakness occurred, and this happened in most common inherited and acquired disorders such as muscular dystrophies, cachexia, and age-related wasting. To date, there is no accepted treatment to improve muscle size and strength, and these conditions pose a considerable anxiety to patients as well as to public health. Several molecules, including Magic-F1, myostatin inhibitor, IGF, glucocorticoids and microRNAs are currently investigated to interfere positively in the blueprint of skeletal muscle growth and regeneration.

ZFP30 promotes adipogenesis through the KAP1-mediated activation of a retrotransposon-derived Pparg2 enhancer.

Krüppel-associated box zinc finger proteins (KZFPs) constitute the largest family of mammalian transcription factors, but most remain completely uncharacterized. While initially proposed to primarily repress transposable elements, recent reports have revealed that KFZPs contribute to a wide variety of other biological processes. Using murine and human in vitro and in vivo models, we demonstrate here that one poorly studied KZFP, ZFP30, promotes adipogenesis by directly targeting and activating a retrotransposon-derived Pparg2 enhancer. Through mechanistic studies, we further show that ZFP30 recruits the co-regulator KRAB-associated protein 1 (KAP1), which, surprisingly, acts as a ZFP30 co-activator in this adipogenic context. Our findings provide an understanding of both adipogenic and KZFP-KAP1 complex-mediated gene regulation, showing that the KZFP-KAP1 axis can also function in a non-repressive manner.

Correction: Magic-Factor 1, a Partial Agonist of Met, Induces Muscle Hypertrophy by Protecting Myogenic Progenitors from Apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0003223.].