MEF2 Transcription Factors Research Articles

A1.20 HDAC5 regulates CXCL chemokine expression in RA FLS via the transcription factor IRF1

Background and ObjectivesHDAC inhibitors (HDACi) display anti-inflammatory properties in animal and in vitro models of rheumatoid arthritis (RA), as well as initial safety and efficacy in the treatment of systemic...

Comparative Analysis Reveals Distinct and Overlapping Functions of Mef2c and Mef2d during Cardiogenesis in Xenopus laevis

The family of vertebrate Mef2 transcription factors is comprised of four members named Mef2a, Mef2b, Mef2c, and Mef2d. These transcription factors are regulators of the myogenic programs with crucial roles in development of skeletal, cardiac and smooth muscle cells. Mef2a and Mef2c are essential for cardiac development in mice. In Xenopus, mef2c and mef2d but not mef2a were recently shown to be expressed during cardiogenesis. We here investigated the function of Mef2c and Mef2d during Xenopus laevis cardiogenesis. Knocking down either gene by corresponding antisense morpholino oligonucleotides led to profound heart defects including morphological abnormalities, pericardial edema, and brachycardia. Marker gene expression analyses and rescue experiments revealed that (i) both genes are required for proper cardiac gene expression, (ii) Mef2d can compensate for the loss of Mef2c but not vice versa, and (iii) the γ domain of Mef2c is required for early cardiac development. Taken together, our data provide novel insights into the function of Mef2 during cardiogenesis, highlight evolutionary differences between species and might have an impact on attempts of direct reprogramming.

Metabolic and immune integration in aging and age-related disease

Metabolic regulation and immune responses are tightly coupled in numerous contexts. Chronic and acute infections can drive metabolic disruption and lead to wasting of fatty and lean tissues, a phenomenon apparent in many human infections. Furthermore, several metabolic syndromes, particularly obesity and diabetes, have an inflammatory component which is thought to be a driving force behind the pathology associated with these increasingly common diseases [1]. In aging human populations metabolic dysfunction, particularly insulin resistance, and inflammatory disorders become increasingly common; however, the etiology of these age-related diseases remains unclear [2,3]. Identifying the molecular mechanisms that underlie metabolic-immune integration is therefore of critical importance to our understanding of metabolic syndromes, infection-induced pathology and aging. In Drosophila, the metabolic and immune consequences of aging are tightly correlated, as individuals that show increased antimicrobial gene expression also show systemic metabolic dysfunction [4]. The metabolic and immune phenotypes of individuals undergoing normal aging therefore begin to mimic those of animals experiencing infection-induced metabolic patho-physiology. This occurs in the apparent absence of infection, and its proximate cause remains unknown. In recent work, we have identified the transcription factor MEF2 as a critical transcriptional switch between metabolism and immunity [5]. In Drosophila, as in vertebrates, chronic or acute infection results in metabolic disruption. In the adult fat body, a structure analogous to the vertebrate liver and adipose tissue, MEF2 exists in two states with distinct physiological functions. Under normal conditions MEF2 phosphorylated at T20 promotes the expression of key enzymes involved in anabolic metabolism. However, in infected animals T20 phosphorylation is lost and MEF2 associates with the TATA-binding protein, and drives normal expression of multiple antimicrobial peptides. MEF2 therefore acts as a switch between distinct transcriptional states. The integration of anabolic signaling and immune activation in this manner may represent an efficient program allowing an animal to alter energy usage in the face of challenge. Alternatively, it may arise from mechanistic constraints resulting from the need for MEF2 to serve different transcriptional functions under different conditions. In either case, prolonged immune activation forces the animal into metabolic pathophysiology. In the absence of infection, when chronic immune activation is unnecessary and detrimental to the animal, MEF2 is an ideal target for intervention. In the context of animal aging the identification of a single protein which when appropriately controlled switches the organism toward anabolic metabolism and away from immune activation is of particular interest. It is easy to speculate that modulation of the phosphorylation status of MEF2 in adult Drosophila may ameliorate the affects of aging on two major physiological systems, with profound consequences for the aging organism. Identifying the signaling mechanisms that regulate T20 phosphorylation of MEF2 will be a key step toward elucidating upstream inputs that modulate immune-metabolic integration via MEF2. Pursuing this question will reveal the events leading to immune and metabolic dysfunction, be it in the context of infection, metabolic syndromes, or normal aging. Such insights from the Drosophila model may be particularly helpful in narrowing the field of candidate pathways as this work moves into mammalian model systems. Immune and metabolic signaling pathways are highly conserved throughout species; it is therefore sensible to suggest that the molecular mechanisms underlying the integration of these signals may also be conserved across organisms. In mammals, Mef2 family proteins have roles in hematopoiesis, and in B cell and T cell function. In addition, Mef2 family proteins are critical regulators of muscle metabolism [6,7]. However, the potential roles of these proteins as direct activators of innate immune responses and regulators of adipose metabolism remains to be explored in mammalian models. Nevertheless, identification of the Drosophila MEF2 immune-metabolic switch represents a significant advance in our understanding of the integrated physiology of the organism.

Two different vestigial like 4 genes are differentially expressed during Xenopus laevis development.

The vestigial gene (vg) was first characterized in Drosophila and several homologues were identified in vertebrates and called vestigial like 1-4 (vgll1-4). Vgll proteins interact with the transcription factors TEF-1 and MEF-2 through a conserved region called TONDU (TDU). Vgll4s are characterized by two tandem TDU domains which differentiate them from other members of the vestigial family. In Xenopus two genes were identified as vgll4. Our bioinformatic analysis demonstrated that these two genes are paralogues and must be named differently. We designated them as vgll4 and vgll4l. In situ hybridization analysis revealed that the expression of these two genes is rather different. At gastrula stage, both were expressed in the animal pole. However, at neurula stage, vgll4 was mainly expressed in the neural plate and neural folds, while vgll4l prevailed in the neural folds and epidermis. From the advanced neurula stage onward, expression of both genes was strongly enhanced in neural tissues, anterior neural plate, migrating neural crest, optic and otic vesicles. Nevertheless, there were some differences: vgll4 presented somite expression and vgll4l was localized at the skin and notochord. Our results demonstrate that Xenopus has two orthologues of the vgll4 gene, vgll4 and vgll4l with differential expression in Xenopus embryos and they may well have different roles during development.

The Histone Deacetylase HDAC4 Regulates Long-Term Memory in Drosophila

A growing body of research indicates that pharmacological inhibition of histone deacetylases (HDACs) correlates with enhancement of long-term memory and current research is concentrated on determining the roles that individual HDACs play in cognitive function. Here, we investigate the role of HDAC4 in long-term memory formation in Drosophila. We show that overexpression of HDAC4 in the adult mushroom body, an important structure for memory formation, resulted in a specific impairment in long-term courtship memory, but had no affect on short-term memory. Overexpression of an HDAC4 catalytic mutant also abolished LTM, suggesting a mode of action independent of catalytic activity. We found that overexpression of HDAC4 resulted in a redistribution of the transcription factor MEF2 from a relatively uniform distribution through the nucleus into punctate nuclear bodies, where it colocalized with HDAC4. As MEF2 has also been implicated in regulation of long-term memory, these data suggest that the repressive effects of HDAC4 on long-term memory may be through interaction with MEF2. In the same genetic background, we also found that RNAi-mediated knockdown of HDAC4 impairs long-term memory, therefore we demonstrate that HDAC4 is not only a repressor of long-term memory, but also modulates normal memory formation.

MEF2C exon α: Role in gene activation and differentiation

Myocyte enhancer factor 2C (MEF2C) belongs to the MEF2 transcription factors. All products of MEF2 genes have a common amino-terminal DNA binding and dimerization domain. All four vertebrate MEF2 gene transcripts are also alternatively spliced. In the present study we identify two novel MEF2C splice variants, named VP and VP2. These variants are generated by the skipping of exon α. The identified α− variants are ubiquitously expressed, although at very low levels compared to the α+ variants. The existence of MEF2C α− variants gave us the opportunity to study for the first time the function of exon α. Transactivation experiments show that the presence of exon α induces a reduction of transcription levels. Moreover, α− variants are significantly expressed during neuronal cell differentiation, indicating a putative role of these variants in development.

Abstract 115: Nuclear CaMKII is a Histone H3 Kinase that Remodels Chromatin During Cardiac Hypertrophy

Background and Purpose: Calcium/calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous serine/threonine kinase implicated in pathological events such as cardiac hypertrophy. In this study we investigated the role of a specific nuclear isoform of CaMKII in chromatin remodeling and in transcriptional regulation in cardiac muscle. Methods: Comprehensive experimental approaches performed in primary cardiomyocyte cultures were used including chromatin immunoprecipitation assays (ChIP), q-PCR, chromatin remodeling assays, in vitro phosphorylation/transcription assays, production of recombinant adenovirus, siRNA technology, fluorescence microscopy and mass spectrometry. Results: We found that CaMKIIδB targets specific components of chromatin during cardiac hypertrophy and binds to nucleosomes through its association domain in a cooperative model. CaMKIIδB also increased chromatin relaxation, and this action was dependent on its kinase activity. The observation that CaMKIIδB interacts with chromatin suggested to us that histones maybe novel substrates of the kinase in cardiac muscle. To test this hypothesis, we performed in vitro kinase assays and found that histone H3 is a bona fide CaMKIIδB substrate and Ser-10 appears to be a predominant phosphorylation site. Increased histone H3 Ser-10 phosphorylation was observed following hypertrophic stimulation and was not associated with cellular proliferation, whereas depletion of CaMKIIδB significantly reduced histone H3 Ser-10 phosphorylation in primary cardiomyocytes. Interestingly, we found that H3 S10 phosphorylation and recruitment of CaMKIIδB occur at promoters of fetal cardiac genes. To establish the functional link between H3 phosphorylation by CaMKIIδB, chromatin remodeling and transcription activation, we developed an in vitro transcription system and using it we found that CaMKIIδB increased chromatin accessibility and mediated transcription of the Mef2 transcription factor. Conclusion: Taken together, these findings highlight a new role of CaMKIIδB as relevant histone H3 kinase and link for the first time epigenetic changes by CaMKII to cardiac hypertrophy.

Abstract A42: Epigenetic silencing of Brahma, a subunit of the chromatin remodeling complex SWI/SNF

Abstract Both BRM and BAF47 are subunits of the SWI/SNF complex and have increasingly clear roles in cancer development. While BAF47 is inactivated via deletions/mutations, BRM is epigenetically silenced. Central to this silencing of BRM are two polymorphisms in the BRM promoter, which have been found to correlate with BRM loss in both cancer cell lines and primary lung tumors. In addition, the induction of BRM by pan-HDAC inhibitors, lead to the discovery that HDAC3 and HDAC9 are regulators of BRM. As these polymorphism sites are 6-7 base pair AT-rich insertions, they have >90% homology with MEF2, POU, and FOX transcription factors. Knockdown experiments of a number of transcription factors lead to uncovering GATA3, MEF2D, and BRN2 as additional regulators of BRM. Both GATA3 and HDAC9 are highly overexpressed in both BRM-deficient cancer cell lines as well as primary lung tumors. Knocking down of GATA3, MEF2D, or both leads to down regulation of HDAC9 and the up regulation of BRM. To determine if any of these proteins might be binding the BRM promoter we conducted ChIP experiments and found that both MEF2D and HDAC9 bind to the BRM promoter in the areas of both polymorphic sites. Since HDACs remove histone acetylation, we looked to see if several chromatin marks changed with the induction of BRM expression. We found that H3K9 and H3K27 become acetylated when BRM is induced. These data suggest a functional role for HDACs in silencing BRM by removing these chromatin marks. To determine if these polymorphic sites have a direct regulatory function on BRM, we conducted BRM promoter-swap experiments where the BRM promoter (and into exon 2) was linked to a luciferase-IRES-neomycin construct. Through homologous recombination we replaced the endogenous BRM promoter with this BRM reporter containing either none or both BRM polymorphic sites and found that removal of the BRM polymorphisms caused about 7-8 fold induction in luciferase indicating a role of these polymorphic sites in BRM regulation. To determine which proteins bind these polymorphic sites we first conducted EMSA experiments and found that these polymorphic sites, compared to wild-type sequence, yielded a gel shift. To determine which sequence elements of these polymorphisms are involved in this gel shift or protein binding, we sequentially changed each of three identical TTAAAA sequences to GCGCGC. When the center sequence was changed from TTAAAA to GCGCGC, no change in the gel shift was observed; however, when either these outer TTAAAA sequences were changed to GCGCGC, the gel shift was lost. This gel shift was also not observed if the center sequence was doubled in length. These results indicate that the outer sequences are involved in protein binding while the center sequence appears to function as a spacer. To further determine what proteins might actually be binding these polymorphic sites, we conducted DNA affinity purification experiments and compared the binding of 40mer with and without the -741 polymorphic sites. This analysis showed four bands uniquely binding these polymorphic sites. By mass spec, we identified the 100kd band as HDAC9 and the 30-35kd band was identified as the scaffold proteins 14-3-3 gamma, delta, and epsilon. Knockdown of either of the 14-3-3 proteins in BRM-deficient cell lines induced BRM and caused growth arrest in a BRM-dependent manner. As such, the involvement of these 14-3-3 proteins suggests a large regulatory complex is involved in the binding to the polymorphisms affecting the regulation of BRM. As we observed experimentally essentially identical results from BRM-deficient Rhabdoid, cervical, and lung cancer cell lines, the findings suggest a conservation of this epigenetic mechanism across very different cancer types. Citation Format: Bhaskar Kahali, Sarah Gramling, Ken Thompson, Stefanie Marquez, Lu Lu, David Reisman. Epigenetic silencing of Brahma, a subunit of the chromatin remodeling complex SWI/SNF. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A42.

The Transcription Factor Mef2 Links the Drosophila Core Clock to Fas2, Neuronal Morphology, and Circadian Behavior

The transcription factor Mef2 regulates activity-dependent neuronal plasticity and morphology in mammals, and clock neurons are reported to experience activity-dependent circadian remodeling in Drosophila. We show here that Mef2 is required for this daily fasciculation-defasciculation cycle. Moreover, the master circadian transcription complex CLK/CYC directly regulates Mef2 transcription. ChIP-Chip analysis identified numerous Mef2 target genes implicated in neuronal plasticity, including the cell-adhesion gene Fas2. Genetic epistasis experiments support this transcriptional regulatory hierarchy, CLK/CYC- > Mef2- > Fas2, indicate that it influences the circadian fasciculation cycle within pacemaker neurons, and suggest that this cycle also contributes to circadian behavior. Mef2 therefore transmits clock information to machinery involved in neuronal remodeling, which contributes to locomotor activity rhythms.

Nuclear CaMKII enhances histone H3 phosphorylation and remodels chromatin during cardiac hypertrophy.

Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in pathological cardiac hypertrophy, but the mechanisms by which it modulates gene activity in the nucleus to mediate hypertrophic signaling remain unclear. Here, we report that nuclear CaMKII activates cardiac transcription by directly binding to chromatin and regulating the phosphorylation of histone H3 at serine-10. These specific activities are demonstrated both in vitro and in primary neonatal rat cardiomyocytes. Activation of CaMKII signaling by hypertrophic agonists increases H3 phosphorylation in primary cardiac cells and is accompanied by concomitant cellular hypertrophy. Conversely, specific silencing of nuclear CaMKII using RNA interference reduces both H3 phosphorylation and cellular hypertrophy. The hyper-phosphorylation of H3 associated with increased chromatin binding of CaMKII occurs at specific gene loci reactivated during cardiac hypertrophy. Importantly, H3 Ser-10 phosphorylation and CaMKII recruitment are associated with increased chromatin accessibility and are required for chromatin-mediated transcription of the Mef2 transcription factor. Unlike phosphorylation of H3 by other kinases, which regulates cellular proliferation and immediate early gene activation, CaMKII-mediated signaling to H3 is associated with hypertrophic growth. These observations reveal a previously unrecognized function of CaMKII as a kinase signaling to histone H3 and remodeling chromatin. They suggest a new epigenetic mechanism controlling cardiac hypertrophy.

Opposing HDAC4 nuclear fluxes due to phosphorylation by β‐adrenergic activated protein kinase A or by activity or Epac activated CaMKII in skeletal muscle fibres

Class IIa histone deacetylases (HDACs) move between skeletal muscle fibre cytoplasm and nuclei in response to various stimuli, suppressing activity of the exclusively nuclear transcription factor Mef2. Protein kinase A (PKA) phosphorylates class IIa HDACs in cardiac muscle, resulting in HDAC nuclear accumulation, but this has not been examined in skeletal muscle. Using HDAC4-green fluorescent protein (HDAC4-GFP) expressed in isolated skeletal muscle fibres, we now show that activation of PKA by the beta-receptor agonist isoproterenol or dibutyryl (Db) cAMP causes a steady HDAC4-GFP nuclear influx. The beta-receptor blocker propranolol or PKA inhibitor Rp-cAMPS blocks the effects of isoproterenol on the nuclear influx of HDAC4-GFP, and Rp-cAMPS blocks the effects of Db cAMP. The HDAC4-GFP construct having serines 265 and 266 replaced with alanines, HDAC4 (S265/266A)-GFP, did not respond to beta-receptor or PKA activation. Immunoprecipitation results show that HDAC4-GFP is a substrate of PKA, but HDAC4 (S265/266A)-GFP is not, implicating HDAC4 serines 265/266 as the site(s) phosphorylated by PKA. During 10 Hz trains of muscle fibre electrical stimulation, the nuclear efflux rate of HDAC4-GFP, but not of HDAC4 (S265/266)-GFP, was decreased by PKA activation, directly demonstrating antagonism between the effects of fibre stimulation and beta-adrenergic activation of PKA on HDAC4 nuclear fluxes. 8-CPT, a specific activator of Epac, caused nuclear efflux of HDAC4-GFP, opposite to the effect of PKA. Db cAMP increased both phosphorylated PKA and GTP-bound Rap1. Our results demonstrate that the PKA and CaMKII pathways play important opposing roles in skeletal muscle gene expression by oppositely affecting the subcellular localization of HDAC4.

Tissue-specific splicing of a ubiquitously expressed transcription factor is essential for muscle differentiation

Alternate splicing contributes extensively to cellular complexity by generating protein isoforms with divergent functions. However, the role of alternate isoforms in development remains poorly understood. Mef2 transcription factors are essential transducers of cell signaling that modulate differentiation of many cell types. Among Mef2 family members, Mef2D is unique, as it undergoes tissue-specific splicing to generate a muscle-specific isoform. Since the ubiquitously expressed (Mef2Dα1) and muscle-specific (Mef2Dα2) isoforms of Mef2D are both expressed in muscle, we examined the relative contribution of each Mef2D isoform to differentiation. Using both in vitro and in vivo models, we demonstrate that Mef2D isoforms act antagonistically to modulate differentiation. While chromatin immunoprecipitation (ChIP) sequencing analysis shows that the Mef2D isoforms bind an overlapping set of genes, only Mef2Dα2 activates late muscle transcription. Mechanistically, the differential ability of Mef2D isoforms to activate transcription depends on their susceptibility to phosphorylation by protein kinase A (PKA). Phosphorylation of Mef2Dα1 by PKA provokes its association with corepressors. Conversely, exon switching allows Mef2Dα2 to escape this inhibitory phosphorylation, permitting recruitment of Ash2L for transactivation of muscle genes. Thus, our results reveal a novel mechanism in which a tissue-specific alternate splicing event has evolved that permits a ubiquitously expressed transcription factor to escape inhibitory signaling for temporal regulation of gene expression.

JAK-STAT pathway and myogenic differentiation

Myogenic differentiation plays an important role in muscle regeneration and is regulated by two transcription factor families, MRFs and MEF2, which induce differentiation of myoblasts through expression of the muscle-specific gene, myogenin. In addition, many intracellular signaling pathways are also involved in myogenic differentiation, including p38 MAPK, ERK/MAPK and PI3K/AKT. The JAK-STAT pathway is activated by various cytokines and positively or negatively regulates the differentiation of myoblasts. JAK1 plays a notable role in proliferation; whereas, JAK2 and JAK3 function mainly in differentiation. The STATs, molecules downstream of JAK, regulate myogenesis. With JAK1, STAT1 promotes proliferation, while STAT3 has a dual effect on proliferation and differentiation. The JAK-STAT negative regulator, SOCS, is also associated with myogenesis; although, its role is controversial. In this review, we will discuss the role of the JAK-STAT pathway on myogenic differentiation.

The Tumor Suppressor Kinase LKB1 Activates the Downstream Kinases SIK2 and SIK3 to Stimulate Nuclear Export of Class IIa Histone Deacetylases

Histone deacetylases 4 (HDAC4), -5, -7, and -9 form class IIa within the HDAC superfamily and regulate diverse physiological and pathological cellular programs. With conserved motifs for phosphorylation-dependent 14-3-3 binding, these deacetylases serve as novel signal transducers that are able to modulate histone acetylation and gene expression in response to extracellular cues. Here, we report that in a PKA-sensitive manner the tumor suppressor kinase LKB1 acts through salt-inducible kinase 2 (SIK2) and SIK3 to promote nucleocytoplasmic trafficking of class IIa HDACs. Both SIK2 and SIK3 phosphorylate the deacetylases at the conserved motifs and stimulate 14-3-3 binding. SIK2 activates MEF2-dependent transcription and relieves repression of myogenesis by the deacetylases. Distinct from SIK2, SIK3 induces nuclear export of the deacetylases independent of kinase activity and 14-3-3 binding. These findings highlight the difference among members of the SIK family and indicate that LKB1-dependent SIK activation constitutes an important signaling module upstream from class IIa deacetylases for regulating cellular programs controlled by MEF2 and other transcription factors.

Activation of Transcription Factor MEF2D by Bis(3)-cognitin Protects Dopaminergic Neurons and Ameliorates Parkinsonian Motor Defects

Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

The Transcription Factor MEF2 Directs Developmental Visually Driven Functional and Structural Metaplasticity

Natural sensory input shapes both structure and function of developing neurons, but how early experience-driven morphological and physiological plasticity are interrelated remains unclear. Using rapid time-lapse two-photon calcium imaging of network activity and single-neuron growth within the unanesthetized developing brain, we demonstrate that visual stimulation induces coordinated changes to neuronal responses and dendritogenesis. Further, we identify the transcription factor MEF2A/2D as a major regulator of neuronal response to plasticity-inducing stimuli directing both structural and functional changes. Unpatterned sensory stimuli that change plasticity thresholds induce rapid degradation of MEF2A/2D through a classical apoptotic pathway requiring NMDA receptors and caspases-9 and -3/7. Knockdown of MEF2A/2D alone is sufficient to induce a metaplastic shift in threshold of both functional and morphological plasticity. These findings demonstrate how sensory experience acting through altered levels of the transcription factor MEF2 fine-tunes the plasticity thresholds of brain neurons during neural circuit formation.

A “Twist box” Code of p53 Inactivation: Twist box:p53 Interaction Promotes p53 Degradation

Twist proteins have been shown to contribute to cancer development and progression by impinging on different regulatory pathways, but their mechanism of action is poorly defined. By investigating the role of Twist in sarcomas, we found that Twist1 acts as a mechanism alternative to TP53 mutation and MDM2 overexpression to inactivate p53 in mesenchymal tumors. We provide evidence that Twist1 binds p53 C terminus through the Twist box. This interaction hinders key posttranslational modifications of p53 and facilitates itsMDM2-mediated degradation. Our study suggests the existence of a Twist box code of p53 inactivation and provides the proof of principle that targeting the Twist box:p53 interaction might offer additional avenues for cancer treatment.

Dilated cardiomyopathy and mitochondrial dysfunction in Sirt1-deficient mice: A role for Sirt1-Mef2 in adult heart

The deacetylase Sirtuin-1 (Sirt1) is involved in the cardiac hypertrophic responses and cardiac embryo morphogenesis. However, the physiological function of Sirt1 deficiency in the postnatal development of the heart remains to be characterized. The aim of the study was to investigate the relevance of Sirt1 in the development and function of the myocardium. Hearts from Sirt1-deficient mice partially or totally lacking Sirt1 protein activity were analyzed. Loss of Sirt1 activity led to dilated cardiomyopathy in adult hearts, a phenotype accompanied by reduced cardiomyocyte size and the absence of fibrosis. Morphological and functional mitochondrial abnormalities were observed in the adult hearts lacking Sirt1, suggesting that mitochondrial dysfunction contributes to the progression of the observed cardiomyopathy. Moreover, gene expression analyses revealed that mitochondrial genes were the most affected in Sirt1-deficient mice, showing a reduction in their expression. No overt cardiac dilatation was observed in neonates lacking Sirt1 activity, but first signs of mitochondrial alterations were already present. Immunoblot analyses revealed that Sirt1 is highly expressed in the heart after birth, indicating the importance of Sirt1 in the neonatal period. Finally, Sirt1 deficiency affected the acetylation pattern of the myocyte enhancer factor 2 (Mef2) transcription factors, which are critical for normal heart development and mitochondrial integrity. Collectively, our findings indicate that Sirt1 is essential for the maintenance of cardiac mitochondrial integrity and normal postnatal myocardium development.

Zebrafish Mef2ca and Mef2cb are essential for both first and second heart field cardiomyocyte differentiation

Mef2 transcription factors have been strongly linked with early heart development. D-mef2 is required for heart formation in Drosophila, but whether Mef2 is essential for vertebrate cardiomyocyte (CM) differentiation is unclear. In mice, although Mef2c is expressed in all CMs, targeted deletion of Mef2c causes lethal loss of second heart field (SHF) derivatives and failure of cardiac looping, but first heart field CMs can differentiate. Here we examine Mef2 function in early heart development in zebrafish. Two Mef2c genes exist in zebrafish, mef2ca and mef2cb. Both are expressed similarly in the bilateral heart fields but mef2cb is strongly expressed in the heart poles at the primitive heart tube stage. By using fish mutants for mef2ca and mef2cb and antisense morpholinos to knock down either or both Mef2cs, we show that Mef2ca and Mef2cb have essential but redundant roles in myocardial differentiation. Loss of both Mef2ca and Mef2cb function does not interfere with early cardiogenic markers such as nkx2.5, gata4 and hand2 but results in a dramatic loss of expression of sarcomeric genes and myocardial markers such as bmp4, nppa, smyd1b and late nkx2.5 mRNA. Rare residual CMs observed in mef2ca;mef2cb double mutants are ablated by a morpholino capable of knocking down other Mef2s. Mef2cb over-expression activates bmp4 within the cardiogenic region, but no ectopic CMs are formed. Surprisingly, anterior mesoderm and other tissues become skeletal muscle. Mef2ca single mutants have delayed heart development, but form an apparently normal heart. Mef2cb single mutants have a functional heart and are viable adults. Our results show that the key role of Mef2c in myocardial differentiation is conserved throughout the vertebrate heart.

A Direct HDAC4-MAP Kinase Crosstalk Activates Muscle Atrophy Program

Prolonged deficits in neural input activate pathological muscle remodeling, leading to atrophy. In denervated muscle, activation of the atrophy program requires HDAC4, a potent repressor of the master muscle transcription factor MEF2. However, the signaling mechanism that connects HDAC4, a protein deacetylase, to the atrophy machinery remains unknown. Here, we identify the AP1 transcription factor as a critical target of HDAC4 in neurogenic muscle atrophy. In denervated muscle, HDAC4 activates AP1-dependent transcription, whereas AP1 inactivation recapitulates HDAC4 deficiency and blunts the muscle atrophy program. We show that HDAC4 activates AP1 independently of its canonical transcriptional repressor activity. Surprisingly, HDAC4 stimulates AP1 activity by activating the MAP kinase cascade. We present evidence that HDAC4 binds and promotes the deacetylation and activation of a key MAP3 kinase, MEKK2. Our findings establish an HDAC4-MAPK-AP1 signaling axis essential for neurogenic muscle atrophy and uncover a direct crosstalk between acetylation- and phosphorylation-dependent signaling cascades.