Author Correction: Impaired cAMP-PKA-CREB1 signalling drives mitochondrial dysfunction in skeletal muscle during cancer cachexia.
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Published Erratum↗
Biomedical subjects
Vittorio Sartorelli
Publications and source records attributed to Vittorio Sartorelli.
The DND1-NANOS3 complex shapes the primordial germ cell transcriptome via a heptanucleotide sequence in mRNA 3' UTRs.
The RNA-binding proteins DND1 and NANOS3 are essential for primordial germ cell survival1-5. Their co-immunoprecipitation and overlapping loss-of-function phenotypes suggest joint function6-8, yet how they co-regulate target mRNAs remains unclear. Here, we developed Tandem PAR-CLIP and identified a DND1-NANOS3 ribonucleoprotein that specifically recognizes an AUGAAUU heptanucleotide on target mRNAs, termed the NANOS3-dependent DND1 Recognition Element (N3-DRE). mRNAs containing 3'-UTR N3-DREs are aberrantly upregulated in DND1- or NANOS3-deficient germ cells and encode key cell-cycle and epigenome regulators, such as CDK1. Genome editing showed that the N3-DRE is essential for Cdk1 repression in mouse PGCs in vivo. A 1.7-Å crystal structure of the ternary complex of DND1, NANOS3, and CDK1-N3-DRE RNA revealed a continuous RNA-binding surface that confers high-affinity, sequence-specific recognition. Together, these findings define the molecular and functional basis of N3-DRE-mediated mRNA regulation in germ cell development. Moreover, we provide a paradigm of two RNA-binding proteins with low (DND1) or no (NANOS3) intrinsic sequence-specificity, jointly building a high-information-content RNA sequence motif that is different from the sum of their individual preferences. Because RNA-binding protein specificities are typically studied individually9-13, rather than in the context of ribonucleoproteins, this type of "two-factor authorization" may be an underappreciated mechanism to protect posttranscriptional gene regulatory networks from aberrant expression of an individual ribonucleoprotein component.
Journal Article↗
A deep learning framework for denoising and ordering scRNA-seq data using adversarial autoencoder with dynamic batching.
Single-cell RNA sequencing (scRNA-seq) provides high resolution of cell-to-cell variation in gene expression and offers insights into cell heterogeneity, differentiating dynamics, and disease mechanisms. However, technical challenges such as low capture rates and dropout events can introduce noise in data analysis. Here, we present a deep learning framework, called the dynamic batching adversarial autoencoder (DB-AAE), for denoising scRNA-seq datasets. First, we describe steps to set up the computing environment, training, and tuning. Then, we depict the visualization of the denoising results. For complete details on the use and execution of this protocol, please refer to Ko et al.1.
Deep Learning↗
Nuclear envelope dystrophies show a transcriptional fingerprint suggesting disruption of Rb-MyoD pathways in muscle regeneration.
Mutations of lamin A/C (LMNA) cause a wide range of human disorders, including progeria, lipodystrophy, neuropathies and autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD). EDMD is also caused by X-linked recessive loss-of-function mutations of emerin, another component of the inner nuclear lamina that directly interacts with LMNA. One model for disease pathogenesis of LMNA and emerin mutations is cell-specific perturbations of the mRNA transcriptome in terminally differentiated cells. To test this model, we studied 125 human muscle biopsies from 13 diagnostic groups (125 U133A, 125 U133B microarrays), including EDMD patients with LMNA and emerin mutations. A Visual and Statistical Data Analyzer (VISDA) algorithm was used to statistically model cluster hierarchy, resulting in a tree of phenotypic classifications. Validations of the diagnostic tree included permutations of U133A and U133B arrays, and use of two probe set algorithms (MAS5.0 and MBEI). This showed that the two nuclear envelope defects (EDMD LMNA, EDMD emerin) were highly related disorders and were also related to fascioscapulohumeral muscular dystrophy (FSHD). FSHD has recently been hypothesized to involve abnormal interactions of chromatin with the nuclear envelope. To identify disease-specific transcripts for EDMD, we applied a leave-one-out (LOO) cross-validation approach using LMNA patient muscle as a test data set, with reverse transcription-polymerase chain reaction (RT-PCR) validations in both LMNA and emerin patient muscle. A high proportion of top-ranked and validated transcripts were components of the same transcriptional regulatory pathway involving Rb1 and MyoD during muscle regeneration (CRI-1, CREBBP, Nap1L1, ECREBBP/p300), where each was specifically upregulated in EDMD. Using a muscle regeneration time series (27 time points) we develop a transcriptional model for downstream consequences of LMNA and emerin mutations. We propose that key interactions between the nuclear envelope and Rb and MyoD fail in EDMD at the point of myoblast exit from the cell cycle, leading to poorly coordinated phosphorylation and acetylation steps. Our data is consistent with mutations of nuclear lamina components leading to destabilization of the transcriptome in differentiated cells.
Biopsy↗
Follistatin induction by nitric oxide through cyclic GMP: a tightly regulated signaling pathway that controls myoblast fusion.
The mechanism of skeletal myoblast fusion is not well understood. We show that endogenous nitric oxide (NO) generation is required for myoblast fusion both in embryonic myoblasts and in satellite cells. The effect of NO is concentration and time dependent, being evident only at the onset of differentiation, and direct on the fusion process itself. The action of NO is mediated through a tightly regulated activation of guanylate cyclase and generation of cyclic guanosine monophosphate (cGMP), so much so that deregulation of cGMP signaling leads to a fusion-induced hypertrophy of satellite-derived myotubes and embryonic muscles, and to the acquisition of fusion competence by myogenic precursors in the presomitic mesoderm. NO and cGMP induce expression of follistatin, and this secreted protein mediates their action in myogenesis. These results establish a hitherto unappreciated role of NO and cGMP in regulating myoblast fusion and elucidate their mechanism of action, providing a direct link with follistatin, which is a key player in myogenesis.
Animals↗
The RNA helicases p68/p72 and the noncoding RNA SRA are coregulators of MyoD and skeletal muscle differentiation.
MyoD regulates skeletal myogenesis. Since proteins associated with MyoD exert regulatory functions, their identification is expected to contribute important insights into the mechanisms governing gene expression in skeletal muscle. We have found that the RNA helicases p68/p72 are MyoD-associated proteins and that the noncoding RNA SRA also immunoprecipitates with MyoD. In vitro and in vivo experiments indicated that both p68/p72 and SRA are coactivators of MyoD. RNA interference toward either p68/p72 or SRA prevented proper activation of muscle gene expression and cell differentiation. Unexpectedly, reducing the levels of p68/p72 proteins impaired recruitment of the TATA binding protein TBP; RNA polymerase II; and the catalytic subunit of the ATPase SWI/SNF complex, Brg-1, and hindered chromatin remodeling. These findings reveal that p68/p72 play a critical role in promoting the assembly of proteins required for the formation of the transcription initiation complex and chromatin remodeling.
Amino Acid Sequence↗
Fgfr4 is required for effective muscle regeneration in vivo. Delineation of a MyoD-Tead2-Fgfr4 transcriptional pathway.
Fgfr4 has been shown to be important for appropriate muscle development in chick limb buds; however, Fgfr4 null mice show no phenotype. Here, we show that staged induction of muscle regeneration in Fgfr4 null mice becomes highly abnormal at the time point when Fgfr4 is normally expressed. By 7 days of regeneration, differentiation of myotubes became poorly coordinated and delayed by both histology and embryonic myosin heavy chain staining. By 14 days much of the muscle was replaced by fat and calcifications. To begin to dissect the molecular pathways involving Fgfr4, we queried the promoter sequences for transcriptional factor binding sites and tested candidate regulators in a 27-time point regeneration series. The Fgfr4 promoter region contained a Tead protein binding site (M-CAT 5'-CATTCCT-3'), and Tead2 showed induction during regeneration commensurate with Fgfr4 regulation. Co-transfection of Tead2 and Fgfr4 promoter reporter constructs into C2C12 myotubes showed Tead2 to activate Fgfr4, and mutation of the M-CAT motif in the Fgfr4 promoter abolished these effects. Immunostaining for Tead2 showed timed expression in myotube nuclei consistent with the mRNA data. Query of the expression timing and genomic sequences of Tead2 suggested direct regulation by MyoD, and consistent with this, MyoD directly bound to two strong E-boxes in the first intron of Tead2 by chromatin immunoprecipitation assay. Moreover, co-transfection of MyoD and Tead2 intron reporter constructs into 10T1/2 cells activated reporter activity in a dose-dependent manner. This activation was greatly reduced when the two E-boxes were mutated. Our data suggest a novel MyoD-Tead2-Fgfr4 pathway important for effective muscle regeneration.
Animals↗
A p38-dependent pathway regulates DeltaNp63 DNA binding to p53-dependent promoters in UV-induced apoptosis of keratinocytes.
The p53 protein plays a pivotal role in determining the quality of the response to DNA damage through its transcriptional activity. Upon DNA damage, p53 is activated by post-translational modifications, binds its cognate sequences on the promoters of its target genes and stimulates transcription. In proliferating keratinocytes, the activity of p53 is blunted by its inhibitor DeltaNp63alpha. Here, we describe a novel mechanism through which DeltaNp63 functions in order to prevent the survival and propagation of ultraviolet (UV)-damaged keratinocytes. We found that UVB stimulation induces the rapid phosphorylation of DeltaNp63, which precedes DeltaNp63 transcriptional downregulation and protein degradation, which is mediated by the p38 MAPK. Phosphorylated DeltaNp63 has a lower affinity for p53REs and detaches from cell cycle arrest and apoptotic promoters, thus allowing the rapid activation of p53-dependent transcriptional apoptotic program.
Apoptosis↗
Mechanisms underlying the transcriptional regulation of skeletal myogenesis.
During skeletal myogenesis, chromatin-modifying enzymes are engaged at discrete genomic regions by transcription factors that recognize sequence-specific DNA motifs located at muscle gene regulatory regions. The composition of the chromatin-bound protein complexes and their temporally and spatially regulated recruitment influence gene expression. Recent findings are consistent with the concept that chromatin modifiers play an important role in regulating skeletal muscle gene expression and cellular differentiation.
Animals↗
The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation.
The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression. We report that Ezh2 expression was developmentally regulated in the myotome compartment of mouse somites and that its down-regulation coincided with activation of muscle gene expression and differentiation of satellite-cell-derived myoblasts. Increased Ezh2 expression inhibited muscle differentiation, and this property was conferred by its SET domain, required for the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 was associated with the transcriptional regulator YY1. Both Ezh2 and YY1 were detected, with the deacetylase HDAC1, at genomic regions of silent muscle-specific genes. Their presence correlated with methylation of K27 of histone H3. YY1 was required for Ezh2 binding because RNA interference of YY1 abrogated chromatin recruitment of Ezh2 and prevented H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and YY1 dissociated from muscle loci, H3-K27 became hypomethylated and MyoD and SRF were recruited to the chromatin. These findings suggest the existence of a two-step activation mechanism whereby removal of H3-K27 methylation, conferred by an active Ezh2-containing protein complex, followed by recruitment of positive transcriptional regulators at discrete genomic loci are required to promote muscle gene expression and cell differentiation.
Animals↗
Molecular and cellular determinants of skeletal muscle atrophy and hypertrophy.
The maintenance of adult skeletal muscle mass is ensured by physical exercise. Accordingly, physiological and pathological situations characterized by either impaired motor neuron activity, reduced gravity (microgravity during space flights), or reduced physical activity result in loss of muscle mass. Furthermore, a plethora of clinical conditions, including cancer, sepsis, diabetes, and AIDS, are associated with varying degrees of muscle atrophy. The cellular and molecular pathways responsible for maintaining the skeletal muscle mass are not well defined. Nonetheless, studies aimed at the understanding of the mechanisms underlying either muscular atrophy or hypertrophy have begun to identify the physiological determinants and clarify the molecular pathways responsible for the maintenance of muscle mass.
Animals↗
Deacetylase recruitment by the C/H3 domain of the acetyltransferase p300.
The balance between acetylation and deacetylation of histone and nonhistone proteins controls gene expression in a variety of cellular processes, with transcription being activated by acetyltransferases and silenced by deacetylases. We report here the formation and enzymatic characterization of a complex between the acetyltransferase p300 and histone deacetylases. The C/H3 region of p300 was found to co-purify deacetylase activity from nuclear cell extracts. A prototype of class I histone deacetylases, HDAC1, interacts with p300 C/H3 domain in vitro and in vivo. The p300-binding protein E1A competes with HDAC1 for C/H3 binding; and, like E1A, HDAC1 overexpression interferes with either activation of Gal4p300 fusion protein or p300-dependent co-activation of two C/H3-binding proteins, MyoD and p53. The exposure to deacetylase inhibitors could reverse the dominant-negative effect of a C/H3 fragment insulated from the rest of the molecule, on MyoD- and p53-dependent transcription, whereas inhibition by E1A was resistant to trichostatin A. These data support the hypothesis that association between acetyltransferases and deacetylases can control the expression of genes implicated in cellular growth and differentiation, and suggest that the dominant-negative effect of the p300 C/H3 fragment relies on deacetylase recruitment.
Acetyltransferases↗
Regulation of the p300 HAT domain via a novel activation loop.
The transcriptional coactivator p300 is a histone acetyltransferase (HAT) whose function is critical for regulating gene expression in mammalian cells. However, the molecular events that regulate p300 HAT activity are poorly understood. We evaluated autoacetylation of the p300 HAT protein domain to determine its function. Using expressed protein ligation, the p300 HAT protein domain was generated in hypoacetylated form and found to have reduced catalytic activity. This basal catalytic rate was stimulated by autoacetylation of several key lysine sites within an apparent activation loop motif. This post-translational modification and catalytic regulation of p300 HAT activity is conceptually analogous to the activation of most protein kinases by autophosphorylation. We therefore propose that this autoregulatory loop could influence the impact of p300 on a wide variety of signaling and transcriptional events.
Acetyl Coenzyme A↗
Deacetylase inhibitors increase muscle cell size by promoting myoblast recruitment and fusion through induction of follistatin.
Fusion of undifferentiated myoblasts into multinucleated myotubes is a prerequisite for developmental myogenesis and postnatal muscle growth. We report that deacetylase inhibitors favor the recruitment and fusion of myoblasts into preformed myotubes. Muscle-restricted expression of follistatin is induced by deacetylase inhibitors and mediates myoblast recruitment and fusion into myotubes through a pathway distinct from those utilized by either IGF-1 or IL-4. Blockade of follistatin expression by RNAi-mediated knockdown, functional inactivation with either neutralizing antibodies or the antagonist protein myostatin, render myoblasts refractory to HDAC inhibitors. Muscles from animals treated with the HDAC inhibitor trichostatin A display increased production of follistatin and enhanced expression of markers of regeneration following muscle injury. These data identify follistatin as a central mediator of the fusigenic effects exerted by deacetylase inhibitors on skeletal muscles and establish a rationale for their use to manipulate skeletal myogenesis and promote muscle regeneration.
Animals↗
Tax relieves transcriptional repression by promoting histone deacetylase 1 release from the human T-cell leukemia virus type 1 long terminal repeat.
Expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated by the viral transcriptional activator Tax. Tax activates viral transcription through interaction with the cellular transcription factor CREB and the coactivators CBP/p300. In this study, we have analyzed the role of histone deacetylase 1 (HDAC1) on HTLV-1 gene expression from an integrated template. First we show that trichostatin A, an HDAC inhibitor, enhances Tax expression in HTLV-1-transformed cells. Second, using a cell line containing a single-copy HTLV-1 long terminal repeat, we demonstrate that overexpression of HDAC1 represses Tax transactivation. Furthermore, a chromatin immunoprecipitation assay allowed us to analyze the interaction of transcription factors, coactivators, and HDACs with the basal and activated HTLV-1 promoter. We demonstrate that HDAC1 is associated with the inactive, but not the Tax-transactivated, HTLV-1 promoter. In vitro and in vivo glutathione S-transferase-Tax pull-down and coimmunoprecipitation experiments demonstrated that there is a direct physical association between Tax and HDAC1. Importantly, biotinylated chromatin pull-down assays demonstrated that Tax inhibits and/or dissociates the binding of HDAC1 to the HTLV-1 promoter. Our results provide evidence that Tax interacts directly with HDAC1 and regulates binding of the repressor to the HTLV-1 promoter.
Animals↗
Sir2 regulates skeletal muscle differentiation as a potential sensor of the redox state.
Sir2 is a NAD(+)-dependent histone deacetylase that controls gene silencing, cell cycle, DNA damage repair, and life span. Prompted by the observation that the [NAD(+)]/[NADH] ratio is subjected to dynamic fluctuations in skeletal muscle, we have tested whether Sir2 regulates muscle gene expression and differentiation. Sir2 forms a complex with the acetyltransferase PCAF and MyoD and, when overexpressed, retards muscle differentiation. Conversely, cells with decreased Sir2 differentiate prematurely. To inhibit myogenesis, Sir2 requires its NAD(+)-dependent deacetylase activity. The [NAD(+)]/[NADH] ratio decreases as muscle cells differentiate, while an increased [NAD(+)]/[NADH] ratio inhibits muscle gene expression. Cells with reduced Sir2 levels are less sensitive to the inhibition imposed by an elevated [NAD(+)]/[NADH] ratio. These results indicate that Sir2 regulates muscle gene expression and differentiation by possibly functioning as a redox sensor. In response to exercise, food intake, and starvation, Sir2 may sense modifications of the redox state and promptly modulate gene expression.
Acetyltransferases↗
Stage-specific modulation of skeletal myogenesis by inhibitors of nuclear deacetylases.
Nuclear acetyltransferases promote and deacetylases inhibit skeletal muscle-gene expression, suggesting the potential effectiveness of deacetylase inhibitors (DIs) in modulating skeletal myogenesis. Surprisingly, previous studies have indicated that DIs suppress myogenesis. The recent observations that histone deacetylases associate with the muscle-regulatory proteins MyoD and MEF2C only in undifferentiated myoblasts prompted us to evaluate the effect of DIs at distinct stages of the myogenic program. We found that exposure of established rodent and human muscle cells to distinct DIs has stage-specific effects. Exposure of undifferentiated skeletal myoblasts to DIs, followed by incubation in differentiation medium, enhanced the expression of muscle-specific reporters and increased the levels of endogenous muscle proteins, leading to a dramatic increase in the formation of multinucleated myotubes. By contrast, simultaneous exposure of muscle cells to differentiation medium and DIs inhibited the myogenic program. Likewise, embryos exposed in utero to nonteratogenic doses of DI at the early stages of somitic myogenesis (embryonic day 8.5) exhibited an increased number of somites and augmented expression of a muscle-specific transgene as well as endogenous muscle genes. The functional effects induced by DIs were mirrored by changes in the state of acetylation of histones present at a muscle-gene enhancer and of MyoD itself. These results represent the first evidence that DIs can enhance muscle differentiation and suggest the rationale for their use in manipulating adult and embryonic skeletal myogenesis.
Acetylation↗
Slug is a novel downstream target of MyoD. Temporal profiling in muscle regeneration.
Temporal expression profiling was utilized to define transcriptional regulatory pathways in vivo in a mouse muscle regeneration model. Potential downstream targets of MyoD were identified by temporal expression, promoter data base mining, and gel shift assays; Slug and calpain 6 were identified as novel MyoD targets. Slug, a member of the snail/slug family of zinc finger transcriptional repressors critical for mesoderm/ectoderm development, was further shown to be a downstream target by using promoter/reporter constructs and demonstration of defective muscle regeneration in Slug null mice.
Animals↗