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Regolazione funzionale dei geni mef2ca e mef2cb durante lo sviluppo embrionale di zebrafish Danio rerio tramite il processo di splicing.
No full textLa famiglia dei fattori trascrizionali MEF2 (Myocite Enhancer Factor-2) è composta da quattro membri (MEF2A-B-C-D) la cui funzione durante lo sviluppo e nel mantenimento dei tessuti sono state ampliamente descritte. Tra i quattro membri della famiglia, MEF2C svolge un ruolo fondamentale durante la miogenesi del tessuto muscolare cardiaco e scheletrico e la sua attività può essere finemente regolata da eventi di splicing alternativo che sono conservati tra i vertebrati. Il pesce Danio rerio (zebrafish) possiede due geni paraloghi di mef2c, mef2ca e mef2cb che derivano da un evento di duplicazione genomica specifico dei Teleostei. Sebbene entrambi i paraloghi siano espressi precocemente nei precursori cellulari cardiaci e muscolari, l'analisi del profilo di espressione genica indica che mef2ca ha una espressione predominante durante lo sviluppo che è prevalente nel tessuto muscolare scheletrico, mentre mef2cb è maggiormente espresso nel tessuto cardiaco. Al fine di caratterizzare la funzione di mef2ca e mef2cb durante lo sviluppo embrionale di zebrafish sono state condotte analisi di RT-PCR agli stadi di 12, 24, 48 e 72 hpf (hours post fertilization). Tramite queste analisi ho osservato eventi di splicing che coinvolgono la regione degli esoni 4, 5 e 6, portando alla produzione di tre diverse varianti di mRNA che differiscono in base alla presenza o assenza dell'esone 5 o di una regione più ampia che comprende parte dell'esone 4 e parte dell'esone 6. I dati ottenuti dimostrano che a partire da 24 hpf i trascritti che presentano l'esone 5 nella propria sequenza codificante risultano predominanti rispetto alle altre varianti di splicing, questo dato acquisisce importanza considerando che questo specifico stadio di sviluppo è caratterizzato dall'inizio del differenziamento terminale del tessuto scheletrico muscolare. Inoltre, dati ottenuti in vitro dimostrano che le isoforme di mef2ca che mancano dell'esone 5 mef2ca mostrano una significativa riduzione della capacità transattivante. Mediante saggi di in situ hybridization a 24 hpf è stata rilevata un'arrichita espressione nel tessuto muscolare scheletrico dei trascritti contenenti esone 5, i quali mostrano una localizzazione specifica alle estremità della fibra muscolare nel punto di giunzione tra i somiti, regione in cui l'espressione dei trascritti di mef2c è circoscritta solo in fasi più tardive dello sviluppo. L'iniezione dell' mRNA codificante per l'isoforma di mef2ca contenente l'esone 5 influenza drammaticamente il progredire dello sviluppo embrionale. Infatti, a seguito dell'iniezione, una porzione di embrioni presenta un fenotipo caratterizzato dalla formazione di un doppio asse dorsale; tale osservazione è coerente con la maggiore attività della isoforma contenente l'esone 5 di indurre un aumento dell'espressione di geni profondamente coinvolti nella definizione dorso-ventrale dell'embrione quali nodal related 1 (ndr1), goosecoid (gsc) e chordin (chd). Al contrario le analisi condotte non hanno rilevato la presenza di trascritti di mef2cb che manchino dell'esone 5, tuttavia l'isoforma di splicing di mef2cb predominante durante lo sviluppo presenta nella sua sequenza codificante una porzione dell'introne 5 derivante da un processo di esonizzazione. L'iniezione di questa isoforma è in grado di indurre la formazione ectopica di tessuto muscolare scheletrico nel mesoderma della testa. Questo lavoro evidenzia quindi l'importanza del meccanismo di splicing alternativo nel modulare e conferire attività differenti ai fattori mef2ca e mef2cb durante lo sviluppo embrionale di Danio rerio.MEF2 (Myocite Enhancer Factor-2) family of transcription factors has four member (MEF2A-B-C-D) whose functions during body development and manteinance are well-established. Among the members, mef2c has a unique role during cardiac and skeletal myogenesis and several reports described how alternative splicing can regulate its activity and that those splicing patterns are conserved among vertebrates. Zebrafish Danio rerio possess two mef2c paralogues, mef2ca and mef2cb that arise from a Teleost-specific genome duplication event. Both genes are expressed early in the embryo in myogenic and cardiac precursors, however expression pattern analysis indicates that among the two paralogues mef2ca expression is higher throughout development and mainly in skeletal muscle whereas mef2cb transcripts are detected in heart primordium. With the goal to gain insight into the function of mef2ca and mef2cb I analyzed their mRNA splicing patterns during embrionic development, focusing the analysis at 12, 24, 48 and 72 hpf (hours post fertilization). RT-PCR analysis revealed the occurence of alternative splicing events, within exon 4, 5 and 6 of mef2ca, that give rise to three different splicing variants. My work demonstrates that inclusion of exon 5 becomes predominant starting from 24 hpf, a developmental stage charachterized by the onset of skeletal muscle terminal differentiation. I further demostrate that Mef2ca isoforms lacking this sequence are significantly less efficient in activating Mef2-dependent transcription, therefore exon 5 inclusion enhances Mef2ca transcriptional activity. By in situ RNA hybridization at 24 hpf I found that the expression of transcripts containing exon 5 is enriched in skeletal muscle showing a specific localisation at fiber ends, facing somite-somite junctions where mef2ca expression is restricted at later stages. Furthermore in-vivo mRNA injection of full-lenght mef2ca dramatically affected embryonic development. Coherently with the occurence of double axis phenotype in a fraction of injected embryos, full-lenght mef2ca has the ability to drive, directly or non-directly, expression of nodal related 1(ndr1), goosecoid (gsc) and chordin (chd), which are deeply involved in the regulation of early dorso-ventral patterning of the embryo. By contrast regulated alternative splicing on mef2cb mRNA to exclude exon 5 during development was not observed. However, I demonstrate that a longer splicing isoform derived from exonization of a fragment of intron 5 is predominant throughout development. Intriguingly, mRNA injection of this long isoform is able to induce ectopic muscle in head mesoderm. My work provide evidence that both zebrafish mef2ca and mef2cb function is regulated through splicing events occuring within exon 5 region. In conclusion alternative splicing confers unique activities to mef2ca and mef2cb paralogues during Danio rerio development
Phosphorylation-dependent degradation of MEF2C contributes to regulate G2/M transition
No full textThe Myocyte Enhancer Factor 2C (MEF2C) transcription factor plays a critical role in skeletal muscle differentiation, promoting muscle-specific gene transcription. Here we report that in proliferating cells MEF2C is degraded in mitosis by the Anaphase Promoting Complex/Cyclosome (APC/C) and that this downregulation is necessary for an efficient progression of the cell cycle. We show that this mechanism of degradation requires the presence on MEF2C of a D-box (R-X-X-L) and 2 phospho-motifs, pSer98 and pSer110. Both the D-box and pSer110 motifs are encoded by the ubiquitous alternate α1 exon. These two domains mediate the interaction between MEF2C and CDC20, a co-activator of APC/C. We further report that in myoblasts, MEF2C regulates the expression of G2/M checkpoint genes (14-3-3γ, Gadd45b and p21) and the sub-cellular localization of CYCLIN B1. The importance of controlling MEF2C levels during the cell cycle is reinforced by the observation that modulation of its expression affects the proliferation rate of colon cancer cells. Our findings show that beside the well-established role as pro-myogenic transcription factor, MEF2C can also function as a regulator of cell proliferation
Regulation of Mef2c activity by alternative splicing in zebrafish muscle development
No full textTeleost fish possess two Mef2c gene paralogues: mef2ca and mef2cb. In zebrafish, Danio rerio, the Mef2cs proteins function to promote cardiomyogenic differentiation and myofibrilogenesis in nascent skeletal muscle fibers. In mouse and human, an array of MEF2C proteins are generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform in vivo. We found that the transcripts of zebrafish mef2ca and mef2cb are alternatively spliced, the resulting splice variants have differing biological activity. Among the two mef2c paralogues, mef2ca is more abundantly expressed in developing skeletal muscle and the splicing pattern of its transcript is tuned through zebrafish development. At 24 hours post fertilization (hpf), a stage when mef2ca activity is required for proper myogenesis, we found the expression of a highly active full length protein. At earlier stages mef2ca activity is attenuated by AS, indeed a high proportion of the mef2ca transcripts lack part of the transcriptional activation domain. We present evidence that the developmentally regulated AS of mef2ca transcripts is important for a correct development of the zebrafish embryos
A POTENTIAL ROLE OF MEF2C IN THE MYOGENIC PROGRESSION BESIDE TERMINAL DIFFERENTIATION
No full textMEF2C belongs to the family of Myocyte Enhancer Factor 2 transcription factors, which activate the muscle-specific gene expression program. Its activity is finely modulated at several levels but some aspects of this regulation still remains uncharacterized; for example, MEF2C is already expressed in proliferating myoblasts, but it is transcriptionally silent unless the cells are stimulated to withdraw the cell cycle and differentiate. Phosphorylation of MEF2 factors at so-called Ser/Thr-Pro motifs can modulate protein function through the induction of conformational changes by the peptidyl–prolyl cis/trans isomerase Pin1. This regulatory mechanism is based on the physical interaction between Pin1 and the Ser98 and Ser110 phosphoacceptor sites located in the alternative spliced exon α1 of MEF2C. The MEF2C/Pin1 interaction results in the repression of MEF2C stability and transcriptional activity, inhibiting muscle differentiation.
We investigated the function of this regulatory mechanism in primary myogenic stem cells (SCs) combining the analysis of the dynamics of MEF2C phosphorylation with the study of the alternative splicing pattern.
We demonstrated that the conditions necessary for the interaction between MEF2C and Pin1 are satisfied exclusively in proliferating SCs, where:
Pin1 expression is upregulated
MEF2C isoform containing the exon α1 specifically appears in response to activation signals
MEF2C phosphorylation on the Pin1-binding sites occurs specifically in proliferating SCs
Indeed we showed that MEF2C and Pin1 can interact in the nuclei of C2C7 and SCs-derived myoblasts. Overall we provide evidence that this interaction not only would serve as a failsafe mechanism to keep silent the MEF2C-dependent transcription of muscle specific genes in proliferating SCs but we hypothesize that the expression of the MEF2Cα1 isoform phosphorylated on Ser98 and Ser110 and the interaction with Pin1 might actively contribute to promote SCs proliferation, allowing the expansion of the activated SCs pool and avoiding their premature differentiation
VCP AND AUTOPHAGOLYSOSOMAL PATHWAY: GUARDIANS OF PROTEOSTASIS AND STRESS GRANULE DYNAMICS. UNRAVELING THEIR IMPLICATIONS IN ALS
No full textAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease comprising clinically indistinguishable sporadic (s) and familial (f) forms, associated with an aberrant behavior of a number of gene products (SOD1, TDP-43, FUS, UBQLN2, VCP, FIG4, CHMP2B, SQSTM1, C9orf72). Most of these proteins are involved in protein degradation via the ubiquitin proteasome (UPP) or the autophagolysosomal (APLP) pathways, as well as in RNA processing or stress granule (SG) response. In ALS, motoneurons accumulate protein aggregates that contain RNA-binding proteins markers of SGs. Thus, proteostasis (mediated by the protein quality control, PQC) and ribostasis (involving SGs) may be interconnected and their imbalance may participate to ALS. At present, it is largely unknown whether interplay between PQC (chaperones, UPP and APLP) and SG dynamics exists and to what extent deregulated PQC may affect SGs, thereby contributing to ALS. Notably, valosin containing protein (VCP) assists with autophagy SG clearance (1) and we found that inhibition of autophagy, lysosomes and depletion of VCP alter SG size, number and composition, pointing to APLP and chaperone-assisted degradation as interconnected processes. These data imply that imbalances in proteostasis and deregulated APLP, which occur in ALS, will affect SG morphology and composition; thus, defective SG response may also contribute to ALS.
Here, we will: 1) dissect how interplay between PQC, VCP and SGs occurs, identifying new players involved in this process and specific clients whose VCP-assisted/APLP-mediated degradation affects SGs; 2) identify the functional consequences of impaired SG response; 3) test whether boosting chaperone-assisted degradation or client targeting may rescue SG morphology and composition. Using motoneuronal ALS cell models we will characterize the impact of ALS mutants of VCP on SG and proteostasis. In parallel, we will characterize and compare SG morphology and composition in fibroblasts and lymphoblasts derived from ALS patients with mutations in VCP, TDP-43, FUS, SOD1 and C9orf72, as well as in motoneurons derived from ALS-iPSCs. Our approach will provide mechanistic insight on the interplay between VCP, PQC and SGs. It will also demonstrate whether/how SG composition and dynamics are affected in ALS and how this correlates with proteostasis alterations, representing a common pathogenic mechanism; finally, our data will provide the molecular basis for the design of new therapeutic strategy
Proline Isomerase Pin1 represses terminal differentiation and Myocyte Enhancer Factor 2C function in Skeletal Muscle Cells
No full textMEF2 (myocyte enhancer factor 2) transcription factors (MEF2A-D) are highly expressed in skeletal muscle cells, they bind to a conserved AT rich DNA sequence through their N-ter MADS and MEF2 domains and activate transcription via their C-ter transcriptional activation domains (TAD), the functional domains of MEF2C are indicated in Figure 1. MEF2 proteins interact with members of the MyoD family of basic helix–loop–helix (bHLH) proteins to establish a unique transcriptional code for skeletal muscle gene activation. Recent studies have revealed multiple signaling systems that stimulate and inhibit myogenesis by altering MEF2 phosphorylation and its association with other transcriptional cofactors. We show that the Pin1 isomerase, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds, interacts with phosphorylated MEF2C in muscle cells. This interaction requires two novel phospho-Ser-Pro motifs in MEF2C: Ser(98) and Ser(110), which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenesis. Furthermore Pin1 modulates the skeletal muscle differentiation program because down-regulation of Pin1 markedly promotes myogenic differentiation. We suggest that Pin1 is a novel regulator of MEF2C function and muscle differentiation, it is expressed in muscle cells and a significant proportion of Pin1 in myotubes but not in myoblasts is excluded from the nucleus. We observed a reduction of phosphorylation of the Ser(98) and Ser(110) Pin1 binding sites in differentiated myocytes. Based on these results we propose a model in which, in proliferating myoblasts, Pin1, upon binding to phosphorylated nuclear MEF2C, leads to decreased levels and transcriptional activity of MEF2C. Upon induction of terminal differentiation, to establish a full activity of MEF2 proteins, a reduced Pin1-MEF2C association is required, possibly due to the relegation of Pin1 to the cytoplasm and to a reduced level of phosphorylation of Ser98 and Ser110
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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