Generation of both an shRNA-resistant MEF2A over expression construct and a dominant negative construct in adenovirus for rescue and knockout experiments in muscle

The Myocyte Enhancer Factor-2, or MEF2, transcription factor family is necessary for the differentiation and regeneration of both skeletal and cardiac muscle tissue. The transcription factors in this family are responsible for the activation of many muscle specific growth factor-induced and differentiation genes. There are four individual isoforms of MEF2; MEF2A, -B, -C, and –D, and the roles of these individual transcription factors are not completely understood. Knockdowns of these individual isoforms revealed that a MEF2A knockdown mouse model displays severe myofibrillar defects in cardiac muscle. This knockdown also has shown that MEF2A is required for myogenesis in vitro, where the other 3 isoforms, -B, -C, and –D, are not necessary for this process. One method of knocking down MEF2A to study its roles further is through the use of short hairpin RNAs (shRNA). The purpose of my research was two-fold. First, in order to test the specificity of this shRNA method, an shRNA-resistant MEF2A over expression construct in an adenoviral vector was created to perform rescue experiments. Second, to compare individual MEF2 isoform knockouts to a complete knockout of the entire MEF2 family, a dominant negative construct was created in an adenoviral vector. In both cases, a pShuttle-CMV adenoviral vector was used. The results of this experiment can be used to further investigate the roles of MEF2A in both regeneration and differentiation of skeletal and cardiac muscle tissue

THE REPRESSION OF MEF2 TRANSCRIPTION FACTORS EXERTED BY CLASS IIA HDACS AND THEIR DEGRADATION STIMULATED BY CDK4 DETERMINE THE ACQUISITION OF HALLMARKS OF TRANSFORMATION IN FIBROBLASTS.

MEF2 transcription factors (TFs) are well known regulators of differenziative and adaptive responses, with predominant roles in muscular, cerebral and immune districts. However, literature concerning the contribution of MEF2 TFs in processes of transformation and oncogenesis is scattered and contradictory; class IIa HDACs (HDAC4, HDAC5, HDAC7, HDAC9) are well-established repressors of MEF2 activity and increasing numbers of selective class IIa HDACs inhibitors are under preclinical screening for various diseases, including cancer. However, a clear demonstration of the oncogenic functions of these proteins is still missing. The aim of this work was to clarify the possible involvement of the HDAC-MEF2 axis in carcinogenesis using as a model different mesenchymal cell lines with varying degrees of immortalization. Here, we incontrovertibly demonstrate a pro-oncogenic role of a nuclear resident form of HDAC4/HDAC7 in NIH-3T3 and BALB/c fibroblasts. Through a DNA microarray experiment we identified the signature of HDAC4 and, as expected, among the genes directly repressed by HDAC4 many are MEF2 targets. We demonstrated that most of the transforming potential of HDAC4 is due to the repression of MEF2 transcriptional activity and that the MEF2-HDAC axis is particularly active in Soft-tissue Sarcomas; in these tumors the binding between HDAC4 and MEF2 could be an effective therapeutic target, as proved by us in vitro. We also demonstrated that the repression of MEF2 activity could also be exerted by common oncogenes, such as RAS and AKT, which act independently from class IIa HDACs by inducing a decrease in the half-life of MEF2C and MEF2D proteins. We reported that MEF2C/D are subjected to a cyclic degradation during cell-cycle with peaks of dysregulation concomitant with S phase entry. The signal that controls the cyclic degradation of MEF2 is the phosphorylation by CDK4/CyclinD1 on two serine residues, conserved among the MEF2 family members, except for MEF2B and a transcriptional variant expressed in skeletal muscles. As a consequence of this phosphorylation, MEF2C/D are bound by the E3-ligase SKP2 that mediates their poly-ubiquitylation and degradation in the proteasome. The cyclic degradation of MEF2 proteins is required for the correct progression of the cell-cycle, as any interference in this degradation process causes an arrest in G1 because of MEF2-mediated transcription of p21/CDKN1A; on the contrary, any increase in MEF2 degradation causes an aberrant progression in the cell-cycle, a common feature of cancer cells. In summary, we demonstrated that in fibroblasts MEF2 activity could be alternatively repressed by class IIa HDACs or through a cell-cycle based degradation process; in both the cases MEF2 repression results in an increase in cell proliferation and in the acquisition of hallmarks of transformation

Global MEF2 Target Gene Analysis in Skeletal and Cardiac Muscle

A loss of muscle mass or function occurs in many genetic and acquired pathologies such as heart disease, sarcopenia and cachexia which are predominantly found among the rapidly increasing elderly population. Developing effective treatments relies on understanding the genetic networks that control these disease pathways. Transcription factors occupy an essential position as regulators of gene expression. Myocyte enhancer factor 2 (MEF2) is an important transcription factor in striated muscle development in the embryo, skeletal muscle maintenance in the adult and cardiomyocyte survival and hypertrophy in the progression to heart failure. We sought to identify common MEF2 target genes in these two types of striated muscles using chromatin immunoprecipitation and next generation sequencing (ChIP-seq) and transcriptome profiling (RNA-seq). Using a cell culture model of skeletal muscle (C2C12) and primary cardiomyocytes we found 294 common MEF2A binding sites within both cell types. Individually MEF2A was recruited to approximately 2700 and 1600 DNA sequences in skeletal and cardiac muscle, respectively. Two genes were chosen for further study: DUSP6 and Hspb7. DUSP6, an ERK1/2 specific phosphatase, was negatively regulated by MEF2 in a p38MAPK dependent manner in striated muscle. Furthermore siRNA mediated gene silencing showed that MEF2D in particular was responsible for repressing DUSP6 during C2C12 myoblast differentiation. Using a p38 pharmacological inhibitor (SB 203580) we observed that MEF2D must be phosphorylated by p38 to repress DUSP6. This established a unique model whereby MAPK signaling results in repression of a MAPK phosphatase. The second MEF2 target gene studied was Hspb7, a small heat shock protein that is highly expressed in striated muscle. Using a combination of bioinformatic and biochemical analysis we found that AP-1 can inhibit Hspb7 transcription, in contrast to MEF2 which activates it. Additionally, the glucocorticoid receptor (GR) regulates Hspb7 in a manner dependent on the presence of MEF2. We also demonstrate an in vivo role for Hspb7 in autophagy which has significant implications in skeletal muscle wasting. Overall we found that MEF2A regulates distinct gene networks in skeletal and cardiac muscle, yet important shared target genes such as DUSP6 and Hspb7 also illustrate that MEF2A regulates some common gene programs that are critical to striated muscle health

HDAC4: studying the pro-oncogenic role in human immortalized fibroblasts

In vitro transformation of primary human fibroblasts has been commonly used to understand the specific steps required to generate neoplastic cells following the ordered introduction of cellular and viral oncogenes and/or the down-modulation of tumor suppressor genes. In this thesis I have applied this strategy to explore the pro-oncogenic function of class IIa HDACs. Class IIa histone deacetylases deregulation can contribute to cancer development and progression in different ways. However their real involvement in tumor biology is still debated. To clarify this issue I have investigated the role of HDAC4, a representative member of this class, in human immortalized foreskin fibroblast (BJ/hTERT). I have demonstrated that HDAC4 negatively influences the isolation of clones after retroviral infection. This effect is MEF2-independent and is in part due to the activation of an apoptotic response. Through the generation of BJ/hTERT cells expressing BCL-xL, a Bcl-2 family member characterized by a pro-survival function, it was possible to isolate clones expressing HDAC4 mutated in the 14-3-3 binding sites, suggesting that HDAC4 deregulation can elicit apoptosis. Isolated clones were characterized, and alterations in the cell cycle profile were not observed. However strong repressive forms of HDAC4 were also subject to intense proteolytic degradation. The apoptotic response and the proteasome-mediated degradation were also described using a doxycycline-inducible system. In this case the nuclear resilient mutants of HDAC4 render BJ/hTERT cells more susceptible to apoptosis only when triggered by DNA damage and protein synthesis inhibition, but not by proteasome inhibitors or oxidative stress. In addition all the nuclear resident mutants evidenced a higher rate of proteasomal degradation. Finally, ectopically expressing in BJ/hTERT cells, a form of HDAC4 mutated only in NES sequence (HDAC4-L/A), allowed the isolation of clones characterized by a MEF2-repressed phenotype. This mutation causes the accumulation of the deacetylase in the nuclear compartment, without interfering with 14-3-3 binding. This result suggests a possible implication of these adaptor proteins in the HDAC4 anti-proliferative activity. In parallel murine fibroblast expressing the HDAC4-L/A mutant acquire the ability to growth in an anchorage-independent manner. Overall this thesis sheds some light on the HDAC4 potential of eliciting oncogenic conversion also in human cells managed also by the 14-3-3 bindin

Alternative splicing of MEF2C pre-mRNA controls its activity in normal myogenesis and promotes tumorigenicity in rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Many cellular disruptions contribute to the progression of this pediatric cancer, including aberrant alternative splicing. The MEF2 family of transcription factors regulates many developmental programs, including myogenesis. MEF2 gene transcripts are subject to alternate splicing to generate protein isoforms with divergent functions. We found that MEF2Cα1 was the ubiquitously expressed isoform that exhibited no myogenic activity and that MEF2Cα2, the muscle-specific MEF2C isoform, was required for efficient differentiation. We showed that exon α in MEF2C was aberrantly alternatively spliced in RMS cells, with the ratio of α2/α1 highly down-regulated in RMS cells compared with normal myoblasts. Compared with MEF2Cα2, MEF2Cα1 interacted more strongly with and recruited HDAC5 to myogenic gene promoters to repress muscle-specific genes. Overexpression of the MEF2Cα2 isoform in RMS cells increased myogenic activity and promoted differentiation in RMS cells. We also identified a serine protein kinase, SRPK3, that was down-regulated in RMS cells and found that expression of SRPK3 promoted the splicing of the MEF2Cα2 isoform and induced differentiation. Restoration of either MEF2Cα2 or SPRK3 inhibited both proliferation and anchorage-independent growth of RMS cells. Together, our findings indicate that the alternative splicing of MEF2C plays an important role in normal myogenesis and RMS development. An improved understanding of alternative splicing events in RMS cells will potentially reveal novel therapeutic targets for RMS treatment

RNA microarray analysis in prenatal mouse cochlea reveals novel IGF-I target genes: implication of MEF2 and FOXM1 transcription factors

Background: Insulin-like growth factor-I (IGF-I) provides pivotal cell survival and differentiation signals during inner ear development throughout evolution. Homozygous mutations of human IGF1 cause syndromic sensorineural deafness, decreased intrauterine and postnatal growth rates, and mental retardation. In the mouse, deficits in IGF-I result in profound hearing loss associated with reduced survival, differentiation and maturation of auditory neurons. Nevertheless, little is known about the molecular basis of IGF-I activity in hearing and deafness. Methodology/Principal Findings: A combination of quantitative RT-PCR, subcellular fractionation and Western blotting, along with in situ hybridization studies show IGF-I and its high affinity receptor to be strongly expressed in the embryonic and postnatal mouse cochlea. The expression of both proteins decreases after birth and in the cochlea of E18.5 embryonic Igf1(-/-) null mice, the balance of the main IGF related signalling pathways is altered, with lower activation of Akt and ERK1/2 and stronger activation of p38 kinase. By comparing the Igf1(-/-) and Igf1(+/+) transcriptomes in E18.5 mouse cochleae using RNA microchips and validating their results, we demonstrate the up-regulation of the FoxM1 transcription factor and the misexpression of the neural progenitor transcription factors Six6 and Mash1 associated with the loss of IGF-I. Parallel, in silico promoter analysis of the genes modulated in conjunction with the loss of IGF-I revealed the possible involvement of MEF2 in cochlear development. E18.5 Igf1(+/+) mouse auditory ganglion neurons showed intense MEF2A and MEF2D nuclear staining and MEF2A was also evident in the organ of Corti. At P15, MEF2A and MEF2D expression were shown in neurons and sensory cells. In the absence of IGF-I, nuclear levels of MEF2 were diminished, indicating less transcriptional MEF2 activity. By contrast, there was an increase in the nuclear accumulation of FoxM1 and a corresponding decrease in the nuclear cyclin-dependent kinase inhibitor p27(Kip1). Conclusions/Significance: We have defined the spatiotemporal expression of elements involved in IGF signalling during inner ear development and reveal novel regulatory mechanisms that are modulated by IGF-I in promoting sensory cell and neural survival and differentiation. These data will help us to understand the molecular bases of human sensorineural deafness associated to deficits in IGF-I

MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia

Regulation and Function of MEF2 in Cardiomyocytes

Regular formation of the mammalian heart needs precise spatial and temporal transcriptional regulation of gene programs in ardiomyocytes. Cardiac transcription factors are defined, in this context, as essential transcriptional activators that are expressed predominantly in the myocardium which regulate the expression of the cardiac genes encoding structural proteins of cardiomyocytes. Unsurprisingly, disruptions in this elaborate transcriptional machinery can lead to severe cardiac abnormalities including hypertension, cardiomyopathy, and congenital heart disease. In this field, Myocyte Enhancer Factor 2 (MEF2) transcription factor is considered one of only a few core cardiac transcription factors that play important roles in cardiac development, survival, contractility, and in postnatal adaption to a wide array of physiological and pathological signals. MEF2 functions as a transcriptional switch by potently activating or repressing transcription through interaction with a variety of co-factors which serve as positive and negative regulators of transcription. The interaction of MEF2 with its co-factors is controlled by a multitude of signaling pathways that result in post-translational modification of MEF2 and in the subsequent MEF2-dependent repression or activation of target gene transcription. Our project studied regulation and function of MEF2A in cardiomyocytes. We hypothesized that the combinatorial interactions between transcription factors and promoter elements that are required for the regulation of cardiac gene expression may operate in pathological cardiac remodeling and hypertrophy. Therefore, studying and characterizing the regulation of proteins which bind to MEF2A in cardiomyocytes may unravel the underlying dysregulation of the cardiac transcriptome in the pathogenesis of cardiovascular disease and heart failure. In this project, HL-1 cardiomyocytes have been chosen as a model of study. An agonist (Isoproterenol) was used to mimic cardiomyocytes hypertrophy in HL-1 cells. Isoproterenol activates adrenergic signaling which can trigger many mechanisms in the heart contributing to the hypertrophic phenotype. We developed two different methods to capture MEF2A interacting partners (interactome), including immunoprecipitation (IP) of endogenous MEF2A and IP of Flag-MEF2A proteins in normal and hypertrophy conditions. Our optimization will allow characterization of MEF2A interactome partners through state of the art quantitative proteomics approaches. In previous research, transcriptome analysis (RNA-seq) from left ventricular RNA samples and MEF2A depleted cardiomyocytes identified some genes, including kf2, junb, alas2 and rarres2 which may have implications for cardiac hypertrophy. Our ChIP-qPCR data indicated that MEF2A is recruited to the rarres2 promoter in primary cardiomyocytes. Thus, rarres2 is a novel MEF2A target gene and further, it will be interesting to uncover functions of MEF2A interactome partners on rarres2 gene regulation in cardiac diseases. A study has indicated that klf2, junB, alas2, and rarres2 may have a role in promoting cardiomyocyte hypertrophy in cultured HL-1 cells and primary neonatal rat cardiomyocytes. Taken together, this project developed the methods to study the characterization of MEF2A interactome in cardiomyocytes. Additionally, we showed the capacity of some MEF2 target genes, including rarres2 to promote cardiomyocyte hypertrophy

Analysis of Protein Arginine Methyltransferase Function during Myogenic Gene Transcription: A Dissertation

Skeletal muscle differentiation requires synergy between tissue-specific transcription factors, chromatin remodeling enzymes and the general transcription machinery. Here we demonstrate that two distinct protein arginine methyltransferases are required to complete the differentiation program. Prmt5 is a type II methyltransferase, symmetrically dimethylates histones H3 and H4 and has been shown to play a role in transcriptional repression. An additional member of the Prmt family, Carm1 is a type I methyltransferase, and asymmetrically methylates histone H3 and its substrate proteins. MyoD regulates the activation of the early class of skeletal muscle genes, which includes myogenin. Prmt5 was bound to and dimethylates H3R8 at the myogenin promoter in a differentiation-dependent fashion. When proteins levels of Prmt5 were reduced by antisense, disappearance of H3R8 dimethylation and Prmt5 binding was observed. Furthermore, binding of Brg1 to regulatory sequences of the myogenin promoter was abolished. All subsequent events relying on Brg1 function, such as chromatin remodeling and stable binding by muscle specific transcription factors such as MyoD, were eliminated. Robust association of Prmt5 and dimethylation of H3R8 at myogenin promoter sequences was observed in mouse satellite cells, the precursors of mature myofibers. Prmt5 binding and histone modification were observed to a lesser degree in mature myofibers. Therefore, these results indicate that Prmt5 is required for dimethylating histone at the myogenin locus during skeletal muscle differentiation in order to facilitate the binding of Brg1, the ATPase subunit of the chromatin remodeling complex SWI/SNF. Further exploration of the role of Prmt5 during the activation of the late class of muscle genes revealed that though Prmt5 is associated with and dimethylates histones at the regulatory elements of late muscle genes in tissue and in culture, it was dispensable for late gene activation. Previous reports had indicated that Carm1 was involved during late gene activation. We observed that Carm1 was bound to and responsible for dimethylating histones at late muscle gene promoters in tissue and in culture. In contrast to Prmt5, a complete knockout of Carm1 resulted in abrogation of late muscle gene activation. Furthermore, loss of Carm1 binding and dimethylated histones resulted in a disappearance of Brg1 binding and chromatin remodeling at late muscle gene loci. Time course chromatin immunoprecipitations revealed that Carm1 binding and histone dimethylation occurred concurrently with the onset of late gene activation. In vitro binding assays revealed that an interaction between Carm1, myogenin and Mef2D exists. These results demonstrate that Carm1 is recruited to the regulatory sequences of late muscle genes via its interaction with either myogenin or Mef2D and is responsible for dimethylates histones in order to facilitate the binding of Brg1. Therefore, these results indicate that during skeletal muscle differentiation, distinct roles exist for these Prmts such that Prmt5 is required for activation of early genes while Carm1 is essential for late gene induction