[Show abstract][Hide abstract] ABSTRACT: Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c.
[Show abstract][Hide abstract] ABSTRACT: The DUX4 gene, encoded within D4Z4 repeats on human chromosome 4q35, has recently emerged as a key factor in the pathogenic mechanisms underlying Facioscapulohumeral muscular dystrophy (FSHD). This recognition prompted development of animal models expressing the DUX4 open reading frame (ORF) alone or embedded within D4Z4 repeats. In the first published model, we used adeno-associated viral vectors (AAV) and strong viral control elements (CMV promoter, SV40 poly A) to demonstrate that the DUX4 cDNA caused dose-dependent toxicity in mouse muscles. As a follow-up, we designed a second generation of DUX4-expressing AAV vectors to more faithfully genocopy the FSHD-permissive D4Z4 repeat region located at 4q35. This new vector (called AAV.D4Z4.V5.pLAM) contained the D4Z4/DUX4 promoter region, a V5 epitope-tagged DUX4 ORF, and the natural 3' untranslated region (pLAM) harboring two small introns, DUX4 exons 2 and 3, and the non-canonical poly A signal required for stabilizing DUX4 mRNA in FSHD. AAV.D4Z4.V5.pLAM failed to recapitulate the robust pathology of our first generation vectors following delivery to mouse muscle. We found that the DUX4.V5 junction sequence created an unexpected splice donor in the pre-mRNA that was preferentially utilized to remove the V5 coding sequence and DUX4 stop codon, yielding non-functional DUX4 protein with 55 additional residues on its carboxyl-terminus. Importantly, we further found that aberrant splicing could occur in any expression construct containing a functional splice acceptor and sequences resembling minimal splice donors. Our findings represent an interesting case study with respect to AAV.D4Z4.V5.pLAM, but more broadly serve as a note of caution for designing constructs containing V5 epitope tags and/or transgenes with downstream introns and exons.
[Show abstract][Hide abstract] ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder with a 1 in 7500 incidence. Symptoms typically arise in young adulthood and most patients show clinical features before age 30. FSHD is characterized by progressive wasting and weakness of facial and shoulder-girdle muscles, although no consistent pattern of penetrance or severity exists. A hallmark characteristic of FSHD is asymmetrical muscle weakness. There are also non-muscular features including retinal vasculopathy and high frequency hearing loss. The current model of pathogenesis in FSHD involves mis-expression of the myotoxic DUX4 gene, but developing animal models has proven difficult. At previous meetings, we first described our transgenic reporter mice containing a putative DUX4 promoter cloned upstream of GFP. We generated three separate lines of DUX4 promoter-GFP mice to identify DUX4 expression patterns and the involvement of selected muscles in FSHD. In short, we found the DUX4 promoter directed GFP expression in the face and limbs of newborn and adult mice, as well as multiple cell types in the retina. Essentially all other organs were GFP negative with a few exceptions including the pancreas and brain. Closer analysis of GFP positive tissues has revealed extensive expression in both myogenic and neuronal cell types. Strikingly, all lines also showed variable penetrance and asymmetrical expression in all GFP-positive tissues, even within individual litters. We have recently created another transgenic line expressing a GFP-CRE fusion protein from the same DUX4 promoter. Importantly we see similar expression patterns to the original mice and are now using them to create an inducible DUX4 transgenic mouse. We have also created analogous AAV vectors to induce localized DUX4 expression at physiologically relevant levels. These models will aid in deciphering molecular mechanisms and developing therapeutic interventions for FSHD.
No preview · Article · Oct 2014 · Neuromuscular Disorders
[Show abstract][Hide abstract] ABSTRACT: Recent progress suggests gene therapy may one day be an option for treating some forms of limb girdle muscular dystrophy (LGMD). Nevertheless, approaches targeting LGMD have so far focused on gene replacement strategies for recessive forms of the disease. In contrast, no attempts have been made to develop molecular therapies for any of the eight dominantly inherited forms of LGMD. Importantly, the emergence of RNA interference (RNAi) therapeutics in the last decade provided new tools to combat dominantly inherited LGMDs with molecular therapy. In this study, we describe the first RNAi-based, preclinical gene therapy approach for silencing a gene associated with dominant LGMD. To do this, we developed adeno-associated viral vectors (AAV6) carrying designed therapeutic microRNAs targeting mutant myotilin (MYOT), which is the underlying cause of LGMD type 1A (LGMD1A). Our best MYOT-targeted microRNA vector (called miMYOT) significantly reduced mutant myotilin mRNA and soluble protein expression in muscles of LGMD1A mice (the TgT57I model) both 3 and 9 months after delivery, demonstrating short- and long-term silencing effects. This MYOT gene silencing subsequently decreased deposition of MYOT-seeded intramuscular protein aggregates, which is the hallmark feature of LGMD1A. Histological improvements were accompanied by significant functional correction, as miMYOT-treated animals showed increased muscle weight and improved specific force in the gastrocnemius, which is one of the most severely affected muscles in TgT57I mice and patients with dominant myotilin mutations. These promising results in a preclinical model of LGMD1A support the further development of RNAi-based molecular therapy as a prospective treatment for LGMD1A. Furthermore, this study sets a foundation that may be refined and adapted to treat other dominant LGMD and related disorders.
[Show abstract][Hide abstract] ABSTRACT: Facioscapulophumeral muscular dystrophy is a common form of muscular dystrophy with significant social and economic impact on affected patients. The pathological features are relatively nonspecific and frequently nondescript, although inflammatory cell infiltrates are a common feature. Muscle biopsy seldom adds to the diagnosis, which is usually made on the basis of the striking clinical features, including typically asymmetrical weakness and atrophy of the facial, shoulder fixator, and humeral compartment muscles. Ankle dorsiflexion and pelvic girdle weakness are also frequently present. Genetic confirmation requires Southern blot analysis, which in most patients reveals an integral deletion of a heterochromatic repeat element (D4Z4) in the subtelomere of chromosome 4q. Recent work confirms that this deletion – or alternately, in rare cases, altered epigenetic regulation of the repeat array – results in the expression of the DUX4 gene, encoding a transcription factor. The molecular pathways by which DUX4 overexpression results in myopathy is the subject of intense research, and may hold keys to future therapeutics.
[Show abstract][Hide abstract] ABSTRACT: Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×10(10) -1 × 10(11) particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 10(9) AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hrGFP as a reporter gene in mouse muscle. These results may impact the design of future preclinical gene therapy studies targeting muscles and non-muscle tissues alike.Molecular Therapy - Nucleic Acids (2013) 2, e86; doi:10.1038/mtna.2013.16; published online 16 April 2013.
[Show abstract][Hide abstract] ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disorder that most commonly affects specific muscles of the face, shoulder girdle, and limbs and has no effective treatment. The relative lack of focus on targeted FSHD therapies existed because the pathogenic mechanisms underlying the disease were not well understood. The field may have reached a tipping point recently with the emergence of a new FSHD pathogenesis model involving mis-expression of the pro-apoptotic DUX4 transcription factor. DUX4 belongs to the homeodomain family of proteins that bind a common AT-rich core motif. Two previous studies reported DUX4 binding sites using ChIP-seq and EMSA methodology. However, in our preliminary work using gel shifts we found that the DUX4 protein was capable of binding numerous homeodomain sites, suggesting the protein may be “sticky”. We therefore set out to better characterize DUX4 binding sequences and their relative affinities with respect to other double homeodomain proteins. To do this, we developed a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method using human DUX4 protein. SELEX provides an unbiased method to determine binding motifs, and has been used to ascertain novel binding sites within genomes. Briefly, this method involved incubating purified full-length DUX4 protein with a randomized, 27-mer double-stranded oligonucleotide pool, washing unbound sequences, and PCR-amplifying eluted, DUX4-enriched binding sites. High-throughput Illumina sequencing in enriched samples was used to identify DUX4 binding motifs. We are now using bioinformatic analysis to search for these DUX4 binding sites in promoters of genes changed in FSHD patient samples and mouse muscles expressing our previously described AAV·DUX4 vectors. This study is an important early step in identifying DUX4-modulated target genes in muscle, and may pinpoint relevant pathways for targeted FSHD therapeutics, downstream of DUX4.
No preview · Article · Oct 2012 · Neuromuscular Disorders
[Show abstract][Hide abstract] ABSTRACT: Limb-girdle muscular dystrophy refers to a group of 23 disorders characterized by progressive wasting and weakness of shoulder and hip girdle muscles. The onset and progression of LGMD varies among individuals and genetic subtypes. Pre-clinical studies support that gene therapy is a promising treatment approach for the LGMDs, and importantly one such strategy (for LGMD2D) was recently translated to human clinical trials. Despite the positive direction of LGMD-targeted gene therapies, all strategies to date focused on gene replacement approaches for recessive forms, while treatments for dominant LGMDs have been largely unexplored. This lack of focus on gene therapy for dominant LGMDs arose primarily because these disorders require disease gene knockdown, and the molecular tools to feasibly accomplish this did not exist until recently, with the emergence of RNA interference (RNAi). We hypothesized that patients with dominantly inherited LGMD would benefit from RNAi-mediated reduction of the pathogenic alleles underlying their disease. In this study, we developed the first RNAi-based, pre-clinical treatment for LGMD1A, caused by dominant mutation in one allele of the myotilin (MYOT) gene. To do this, we engineered and delivered MYOT-targeted artificial microRNA (miMYOT) vectors to knockdown mutant MYOT in muscles of an LGMD1A mouse model. Three months after treatment, miMYOT vectors significantly reduced soluble mutant MYOT protein to undetectable levels, and the protein aggregates that are characteristic of LGMD1A were either absent or very small in treated muscles. This reduction was associated with significantly improved muscle mass and whole muscle strength in LGMD1A mice. We are now assessing body wide improvements of miMYOT treatment following global vascular delivery. This work is an important first step toward translating targeted RNAi gene therapy approaches for LGMD1A, and our method could be adapted to impact a large class of dominant muscle disorders.
No preview · Article · Oct 2012 · Neuromuscular Disorders
[Show abstract][Hide abstract] ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disorder with a one in 7500 incidence. Symptoms typically arise in young adulthood and most patients show clinical features before age 30. FSHD is most commonly characterized by progressive wasting and weakness of facial and shoulder-girdle muscles, although no consistent pattern of penetrance or severity exists, even within affected families. The hallmark characteristic of FSHD is asymmetrical muscle weakness. There are also non-muscular features including retinal vasculopathy and high frequency hearing loss. The pathogenic mechanisms underlying FSHD have been difficult to discern, and scientists in the field have spent most of the last two decades attempting to decipher FSHD pathogenesis. Several recent breakthroughs now support a model in which mis-expression of the myotoxic DUX4 gene is a primary pathogenic event underlying FSHD. We recently showed that DUX4 is generally toxic to muscles, and we therefore hypothesized that there was a direct correlation between DUX4 expression patterns and the involvement of only selected muscles (and ear and retinal pathologies) in FSHD. In short, we proposed that if DUX4 over-expression is indeed the underlying pathogenic event in FSHD, it must be preferentially expressed in FSHD-affected regions. To test this hypothesis, we developed transgenic reporter mice containing a putative DUX4 promoter cloned upstream of GFP. We generated three separate lines of DUX4 promoter-GFP mice. We found the DUX4 promoter directed GFP expression in the face and limbs of newborn and adult mice, as well as the retina and inner ear. Essentially all other organs were GFP negative. Strikingly, all lines showed asymmetrical expression and variable penetrance, even within individual litters. We conclude that our mice faithfully recapitulate expected DUX4 expression patterns in regions of FSHD pathology, and further support the role of DUX4 as pathogenic insult in FSHD.
No preview · Article · Oct 2012 · Neuromuscular Disorders
[Show abstract][Hide abstract] ABSTRACT: Limb Girdle Muscular Dystrophy (LGMD) refers to a group of 25 genetic diseases linked by common clinical features, including wasting of muscles supporting the pelvic and shoulder girdles. Cardiac involvement may also occur. Like other muscular dystrophies, LGMDs are currently incurable, but prospective gene replacement therapies targeting recessive forms have shown promise in pre-clinical and clinical studies. In contrast, little attention has been paid to developing gene therapy approaches for dominant forms of LGMD, which would likely benefit from disease gene silencing. Despite the lack of focus to date on developing gene therapies for dominant LGMDs, the field is not starting at square one, since translational studies on recessive LGMDs provided a framework that can be applied to treating dominant forms of the disease. In this manuscript, we discuss the prospects of treating dominantly inherited forms of LGMD with gene silencing approaches.
No preview · Article · Aug 2012 · Current Gene Therapy
[Show abstract][Hide abstract] ABSTRACT: Paired-like homeodomain transcription factor 1 (PITX1) was specifically up-regulated in patients with facioscapulohumeral muscular dystrophy (FSHD) by comparing the genome-wide mRNA expression profiles of 12 neuromuscular disorders. In addition, it is the only known direct transcriptional target of the double homeobox protein 4 (DUX4) of which aberrant expression has been shown to be the cause of FSHD. To test the hypothesis that up-regulation of PITX1 contributes to the skeletal muscle atrophy seen in patients with FSHD, we generated a tet-repressible muscle-specific Pitx1 transgenic mouse model in which expression of PITX1 in skeletal muscle can be controlled by oral administration of doxycycline. After PITX1 was over-expressed in the skeletal muscle for 5 weeks, the mice exhibited significant loss of body weight and muscle mass, decreased muscle strength, and reduction of muscle fiber diameters. Among the muscles examined, the tibialis anterior, gastrocnemius, quadricep, bicep, tricep and deltoid showed significant reduction of muscle mass, while the soleus, masseter and diaphragm muscles were not affected. The most prominent pathological change was the development of atrophic muscle fibers with mild necrosis and inflammatory infiltration. The affected myofibers stained heavily with NADH-TR with the strongest staining in angular-shaped atrophic fibers. Some of the atrophic fibers were also positive for embryonic myosin heavy chain using immunohistochemistry. Immunoblotting showed that the p53 was up-regulated in the muscles over-expressing PITX1. The results suggest that the up-regulation of PITX1 followed by activation of p53-dependent pathways may play a major role in the muscle atrophy developed in the mouse model.
[Show abstract][Hide abstract] ABSTRACT: No treatment exists for facioscapulohumeral muscular dystrophy (FSHD), one of the most common inherited muscle diseases. Although FSHD can be debilitating, little effort has been made to develop targeted therapies. This lack of focus on targeted FSHD therapy perpetuated because the genes and pathways involved in the disorder were not understood. Now, more than 2 decades after efforts to decipher the root cause of FSHD began, this barrier to translation is finally lowering. Specifically, several recent studies support an FSHD pathogenesis model involving overexpression of the myopathic DUX4 gene. DUX4 inhibition has therefore emerged as a promising therapeutic strategy for FSHD. In this study, we tested a preclinical RNA interference (RNAi)-based DUX4 gene silencing approach as a prospective treatment for FSHD. We found that adeno-associated viral (AAV) vector-delivered therapeutic microRNAs corrected DUX4-associated myopathy in mouse muscle. These results provide proof-of-principle for RNAi therapy of FSHD through DUX4 inhibition.
[Show abstract][Hide abstract] ABSTRACT: RNA interference (RNAi) is a conserved gene silencing mechanism mediated by small inhibitory microRNAs (miRNAs).Promoter-driven miRNA expression vectors have emerged as important tools for delivering natural or artificially designed miRNAs to eukaryotic cells and organisms. Such systems can be used to query the normal or pathogenic functions of natural miRNAs or messenger RNAs, or to therapeutically silence disease genes.
As with any molecular cloning procedure, building miRNA-based expression constructs requires a time investment and some molecular biology skills. To improve efficiency and accelerate the construction process, we developed a method to rapidly generate miRNA expression vectors using recombinases instead of more traditional cut-and-paste molecular cloning techniques. In addition to streamlining the construction process, our cloning strategy provides vectors with added versatility. In our system, miRNAs can be constitutively expressed from the U6 promoter, or inducibly expressed by Cre recombinase. We also engineered a built-in mechanism to destroy the vector with Flp recombinase, if desired. Finally, to further simplify the construction process, we developed a software package that automates the prediction and design of optimal miRNA sequences using our system.
We designed and tested a modular system to rapidly clone miRNA expression cassettes. Our strategy reduces the hands-on time required to successfully generate effective constructs, and can be implemented in labs with minimal molecular cloning expertise. This versatile system provides options that permit constitutive or inducible miRNA expression, depending upon the needs of the end user. As such, it has utility for basic or translational applications.
[Show abstract][Hide abstract] ABSTRACT: Muscular dystrophies, and other diseases of muscle, arise from recessive and dominant gene mutations. Gene replacement strategies may be beneficial for the former, while gene silencing approaches may provide treatment for the latter. In the last two decades, muscle-directed gene therapies were primarily focused on treating recessive disorders. This disparity at least partly arose because feasible mechanisms to silence dominant disease genes lagged behind gene replacement strategies. With the discovery of RNA interference (RNAi) and its subsequent development as a promising new gene silencing tool, the landscape has changed. In this study, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo. We tested the potential of adeno-associated viral (AAV)-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1), to correct myopathic features in mice expressing toxic levels of human FRG1 (FRG1(-high) mice). We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1(-high) mice, thereby demonstrating therapeutic promise for treatment of dominantly inherited myopathies using RNAi. This approach potentially applies to as many as 29 different gene mutations responsible for myopathies inherited as dominant disorders.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) have emerged as important modulators of eukaryotic gene expression through a process called RNA interference
(RNAi). Over the last several years, a large amount of work has focused on understanding how miRNAs are expressed and processed
to a biologically functional form. This knowledge has enabled the development of RNAi as a molecular tool for investigating
basic biological questions or as a therapeutic technique. Artificial miRNA shuttle vectors can be engineered to mimic natural
miRNAs and subsequently used to suppress any gene of interest. Here, we describe a simple method to build and functionally
validate artificial miRNA shuttles.
Key wordsRNAi–MicroRNA–miRNA, siRNA–Inhibitory RNA–Gene silencing–Gene therapy
[Show abstract][Hide abstract] ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is associated with D4Z4 repeat contraction on human chromosome 4q35. This genetic lesion does not result in complete loss or mutation of any gene. Consequently, the pathogenic mechanisms underlying FSHD have been difficult to discern. In leading FSHD pathogenesis models, D4Z4 contractions are proposed to cause epigenetic changes, which ultimately increase expression of genes with myopathic potential. Although no gene has been conclusively linked to FSHD development, recent evidence supports a role for the D4Z4-encoded DUX4 gene in FSHD. In this study, our objective was to test the in vivo myopathic potential of DUX4.
We delivered DUX4 to zebrafish and mouse muscle by transposon-mediated transgenesis and adeno-associated viral vectors, respectively.
Overexpression of DUX4, which encodes a transcription factor, caused abnormalities associated with muscular dystrophy in zebrafish and mice. This toxicity required DNA binding, because a DUX4 DNA binding domain mutant produced no abnormalities. Importantly, we found the myopathic effects of DUX4 were p53 dependent, as p53 inhibition mitigated DUX4 toxicity in vitro, and muscles from p53 null mice were resistant to DUX4-induced damage.
Our work demonstrates the myopathic potential of DUX4 in animal muscle. Considering previous studies showed DUX4 was elevated in FSHD patient muscles, our data support the hypothesis that DUX4 overexpression contributes to FSHD development. Moreover, we provide a p53-dependent mechanism for DUX4 toxicity that is consistent with previous studies showing p53 pathway activation in FSHD muscles. Our work justifies further investigation of DUX4 and the p53 pathway in FSHD pathogenesis.
No preview · Article · Mar 2011 · Annals of Neurology
[Show abstract][Hide abstract] ABSTRACT: Protein phosphatase 2A (PP2A) is one of the most abundantly expressed serine/threonine protein phosphatases. A large body of evidence suggests that PP2A is a tumor suppressor and plays critical roles in regulating apoptosis. PP2A is a heterotrimeric protein complex. Its substrate specificity, localization, and activity are regulated by regulatory subunits
of PP2A. A recent study has demonstrated that single nucleotide polymorphism in B56ϵ (PPP2R5E), a B56 family regulatory subunit of PP2A, is associated with human soft tissue sarcoma. This raises the possibility that B56ϵ is involved in tumorigenesis and plays important roles in regulating apoptosis. However, this hypothesis has not been tested
experimentally. Our previous studies revealed that B56ϵ regulates a number of developmental signaling pathways during early embryonic patterning. Here we report novel functions
of B56ϵ in regulating apoptosis. We provide evidence that B56ϵ has both anti- and pro-apoptotic functions. B56ϵ suppresses p53-independent apoptosis during neural development, but triggers p53-dependent apoptosis. Mechanistically, B56ϵ regulates the p53-dependent apoptotic pathway solely through controlling the stability of p53 protein. In addition to its function in regulating apoptosis, we show that B56ϵ undergoes proteolytic cleavage. The cleavage of B56ϵ is mediated by caspase-3 and occurs on the carboxyl side of an evolutionarily conserved N-terminal “DKXD” motif. These results demonstrate that B56ϵ, a substrate of caspase-3, is an essential regulator of apoptosis. So far, we have identified an alternative translation isoform and a caspase cleavage
product of B56ϵ. The significance of post-transcriptional regulation of B56ϵ is discussed.
Preview · Article · Nov 2010 · Journal of Biological Chemistry