<p>Technologies for engineering new functions into proteins are advancing biological research, biotechnology, and medicine at an astounding rate. Building on fundamental research of natural protein structure and function, scientists are identifying new protein domains with previously undescribed properties and engineering new proteins with expanded functionalities. Such tools are enabling the precise study of fundamental aspects of cellular behavior and the development of a new class of gene therapies that manipulate the expression of endogenous genes. The applications of these gene regulation technologies include but are not limited to controlling cell fate decisions, reprogramming cell lineage commitment, monitoring cellular states, and stimulating expression of therapeutic factors. </p><p>While the field has come a long way in the past 20 years, there are still many limitations. Historically, gene therapy and gene replacement therapies have relied on over-expression of natural transcription factors that activate specific endogenous gene networks. However, natural transcription factors are often inadequate for generating efficient, fast, and homogenous cellular responses. Furthermore, most natural transcription factors have complex structures and functions that are difficult to improve or alter by rational design. This thesis presents three novel and widely applicable methods for engineering transcription factors for programming cell fate decisions in primary human cells. MyoD is the master transcription factor defining the myogenic lineage. Expression of MyoD in certain non-myogenic lineages induces a coordinated change in differentiation state. We use MyoD as a model for developing our protein engineering techniques because myogenesis is a well-studied pathway that is characterized by an easily detected change in phenotype from mono-nucleated to multinucleated cells. Furthermore, efficient generation of myocytes in vitro presents an attractive patient-specific method by which to treat muscle-wasting diseases such as muscular dystrophy.</p><p>We first demonstrate that we can improve the ability of MyoD to convert human dermal fibroblasts and human adipose-derived stem cells into myocyte-like cells. By fusing potent modular activation domains to the MyoD protein, we increased myogenic gene expression, myofiber formation, cell fusion, and global reprogramming of the myogenic gene network. The engineered MyoD transcription factor induced myogenisis in a little as ten days, a process that takes three or more weeks with the natural MyoD protein. </p><p>While increasing the potency of transcriptional activation is one mechanism by which to improve transcription factor function, there are many other possible routes such as increasing DNA-binding affinity, increasing protein stability, altering interactions with co-factors, or inducing post-translational modifications. Endogenous regulatory pathways are complex, and it is difficult to predict specific amino acid changes that will produce the desired outcome. Therefore, we designed and implemented a high-throughput directed evolution system in mammalian cells that allowed us to enrich for MyoD variants that are successful at inducing expression of the myogenic gene network. Directed evolution presents a well-established and currently unexplored approach for uncovering amino acid substitutions that improve the intrinsic properties of transcription factors themselves without any prior knowledge. After ten rounds of selection, we identified amino acid substitutions in MyoD that increase expression of a subset of myogenic gene markers in primary human cells.</p><p>Rather than guide cell fate decisions by expressing an exogenous factor, it may be beneficial to activate expression of the endogenous gene locus. In comparison to delivering the transcription factor cDNA, expression from the endogenous locus may induce chromatin remodeling and activation of positive feedback loops to stimulate autologous expression more quickly. Recent discoveries of the principles of protein-DNA interactions in various species and systems has guided the development of methods for engineering designer enzymes that can be targeted to any DNA target site. We make use of the RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system to induce expression of the endogenous MyoD gene in human induced pluripotent stem cells (iPSCs). Through complementary base pairing, chimeric guide RNAs (gRNAs) direct a Cas9 transcriptional activator to a target DNA sequence, leading to endogenous gene expression. A current limitation of CRISPR/Cas9-based gene regulation is the potency of transcriptional activation and delivery of the CRISPR/Cas9 components. To address these limitations, we first developed a platform to express Cas9 and up to four gRNAs from a single lentiviral vector. We then optimized the gRNAs and Cas9 transcriptional activator to induce endogenous MyoD expression and differentiate iPSCs into myocyte-like cells. </p><p>In summary, the objective of this work is to develop protein engineering techniques to improve both natural and synthetic transcription factor function for programming cell fate decisions in primary human cells. While we focus on myogenesis, each method can be easily adapted to other transcription factors and gene networks. Engineered transcription factors that induce fast and efficient remodeling of gene networks have widespread applications in the fields of biotechnology and regenerative medicine. Continuing to develop these tools for modulating gene expression will lead to an expanded number of disease models and eventually the efficient generation of patient-specific cellular therapies.</p> / Dissertation

Il existe de nombreuses similitudes au niveau des mécanismes génétiques et moléculaires qui contrôlent les différentes étapes de la myogenèse entre la drosophile et les vertébrés. Afin de mettre en évidence de nouveaux gènes impliqués dans ce processus, nous avons sélectionné des gènes conservés au cours de l’évolution afin de tester leur rôle dans la myogenèse. Des gènes candidats conservés entre le poisson zèbre et la drosophile et exprimés dans des compartiments musculaires ont été sélectionnés in silico à partir des bases de données du poisson-zèbre (Zfin) et de la drosophile (BDGP). Ainsi, 120 gènes ont été mis en évidence, dont plus de la moitié jouerait un rôle dans le métabolisme, et sur les 23 testés par ARNi, 20 donnent des phénotypes musculaires suite à la diminution de leur expression. Les défauts musculaires observés ont permis de replacer le rôle putatif de ces 20 gènes dans le processus myogénique, montrant l’efficacité de cette approche. L’analyse fonctionnelle du gène Pglym78 impliqué dans la glycolyse a ensuite été réalisée. Ce gène est exprimé spécifiquement dans les muscles somatiques et son atténuation donne des défauts de différenciation musculaire caractérisés par un blocage de la fusion des myoblastes et la formation de muscles plus fins. L’ensemble des autres gènes de la glycolyse s’exprime de la même façon et leur inhibition donne aussi des problèmes de différenciation. Ainsi, il existerait au moment de la différenciation musculaire un switch métabolique se traduisant par une augmentation de la glycolyse, similaire à celui mis en évidence dans les cellules cancéreuses, pouvant contribuer à former l’ATP ainsi que les molécules nécessaires pour la synthèse des protéines, le tout permettant la croissance musculaire. Enfin, l’inhibition de la voie insuline, connue pour stimuler la glycolyse mais également la croissance musculaire, diminue l’activité glycolytique et donne des phénotypes similaires à ceux observés lorsque l’on bloque la glycolyse. Nos résultats mettent en évidence l’existence d’un switch métabolique vers la glycolyse, médié au moins en partie par la voie insuline afin de permettre l’augmentation de la synthèse de biomasse dans le muscle, nécessaire à la poursuite de sa différenciation. Ce travail révèle ainsi l’existence d’un lien entre le métabolisme et le développement musculaires. / A large number of genes involved in myogenesis has been described, but several gaps in comprehension of mechanisms giving rise to functional muscles are still remaining. To fill in these gaps, we selected conserved uncharacterized genes expressed in muscular compartments in drosophila and zebrafish and tested their functions by RNAi knockdown. We found that most of the candidate genes have a role in different steps of embryonic myogenesis in drosophila and interestingly more than a half of them are involved in metabolism. One of these candidates, Pglym78, encodes a glycolytic enzyme and gives rise to late muscle differentiation defects after knockdown in drosophila. Glycolysis is a major metabolic process providing energy and components for biomass synthesis to rapidly growing/proliferating cells such as cancer cells but its role in embryonic development remains unknown. Here we show that starting from midembryogenesis, drosophila Pglym78 and almost all the glycolytic genes display muscle specific expression and that, consistent with this, an important increase in glycolytic activity appears since embryonic stage 14, suggesting that glycolysis can play a role in late steps of myogenesis. This possibility is supported by the fact that attenuation of Pglym78 and other glycolytic genes results in affected muscle differentiation. As shown in Pglm78 knockdown embryos these phenotypes are due to myoblasts fusion arrest and formation of significantly smaller muscle fibres.In order to understand how glycolysis controls myogenesis, we analysed the insulin pathway known to control glycolytic activity and to positively regulate muscle growth by stimulating protein synthesis. Interestingly, inhibition of insulin pathway in differentiating embryonic drosophila muscles leads to the reduced activity of PyK and to phenotypes that are reminiscent of those of glycolytic genes such as fusion arrest and formation of smaller fibres. Thus, our data reveal that metabolic switch to glycolysis positively regulated by insulin pathway is required to support increased biomass synthesis in syncytial muscle cells, revealing direct link between metabolism and development.

Embryonic stem cells (ESCs) represent a promising source of cells for cell replacement therapy in the context of muscle diseases; however, before ESC-based cell therapy can be translated to the clinic, we must learn to modulate cell-fate decisions in order to maximize the yield of myocytes from this systems. In order to gain a better understanding of the myogenic cell fate, we sought to define the molecular mechanisms underlying the specification and differentiation of ESCs into cardiac and skeletal muscle. More specifically, the central hypothesis of the thesis is that myogenic signalling cascades modulate cell fate via regulation of transcription factors. Retinoic acid (RA) is known to promote skeletal myogenesis, however the molecular basis for this remains unknown. We showed that RA expands the premyogenic progenitor population in mouse stem cells by directly activating pro-myogenic transcription factors such as Pax3 and Meox1. RA also acts indirectly by activating the pro-myogenic Wnt signalling cascade while simultaneously inhibiting the anti-myogenic influence of BMP4. This ultimately resulted in a significant enhancement of skeletal myogenesis. Furthermore, we showed that this effect was conserved in human embryonic stem cells, with implications for directed differentiation and cell therapy. The regulation of cardiomyogenesis by the Wnt pathway was also investigated. We identified a novel interaction between the cardiomyogenic transcription factor Nkx2.5 and the myosin phosphatase (MP) enzyme complex. Interaction with MP resulted in exclusion of Nkx2.5 from the nucleus and inhibition of its transcriptional activity. Finally, we showed that this interaction was modulated by phosphorylation of the Mypt1 subunit of MP by ROCK, downstream of Wnt3a. Treatment of differentiating mouse ESCs with Wnt3a resulted in exclusion of Nkx2.5 from the nucleus and a subsequent failure to undergo terminal differentiation into cardiomyocytes. This likely represents part of the molecular basis for Wnt-mediated inhibition of terminal differentiation of cardiomyocytes. Taken together, our results provide novel insight into the relationship between myogenic signalling cascades and downstream transcription factors and into how they function together to orchestrate the myogenic cell fate in stem cells.

A complex network of transcription factors, which are regulated by signalling molecules, is responsible in coordinating the formation of differentiated skeletal and cardiac myocytes from undifferentiated stem cells. The present study aims to understand and compare the transcriptional regulation of skeletal and/or cardiac muscle development in the absence of Sox7 or in the presence of a collagen-based matrix in P19 embyonal carcinoma (EC) and mouse embryonic stem (ES) cells. First, knock-down of Sox7 , by shRNA, in muscle inducing conditions (+DMSO) and in the absence of RA (-RA), decreased muscle progenitor transcription factor and myogenic regulatory factor (MRF) levels, suggesting that Sox7 is necessary for myogenesis. However, knock-down of Sox7 in the presence of RA (+RA) and DMSO increased expression of muscle progenitor markers and MRFs, suggesting that Sox7 is inhibitory for myogenesis +RA. Furthermore, Sox7 overexpression enhanced myogenesis -RA, but inhibited myogenesis and enhanced neurogenesis +RA. These results suggest an important interplay between RA signalling and Sox7 function during P19 differentiation. Second, Q-PCR analysis showed that compared to the mouse ES cells differentiated on the regular TC plates, differentiation on the collagen matrices had a higher expression of skeletal and cardiac precursors, MRFs and terminal differentiation markers. Collagen alone enhanced myotube formation. The enhanced collagen matrix, containing the oligosaccharide sialyl LewisX (sLeX), specifically enhanced cardiomyogenesis. These studies have added to our understanding of the transcriptional regulation of premyogenic mesoderm factors and the role of Sox7 in this process. In addition these studies provide a vision for possible use of biomaterials in directed differentiation of stem cells for the purpose of cell therapy.

Members of the Mef2 transcription factor family are extensively studied within the muscle field for their ability to cooperate with the myogenic regulatory factors MyoD and myogenin during muscle differentiation. Although it is known that Mef2 pre-mRNAs undergo alternative splicing, the different splice forms have not been functionally annotated. In this thesis, my studies aimed to characterize three Mef2D splice forms: MEF2Dα'β, MEF2Dαβ, MEF2Dαø. Our results show that MEF2D splice forms can be differentially phosphorylated by p38 MAPK and PKA in vitro. Gene expression analysis using cell lines over-expressing each Mef2D splice form suggests that they can differentially activate desmin, myosin heavy chain and myogenin expression. Mass spectrometry analyses from our pull-down assays reveal known and novel MEF2D binding partners. Our work suggests that Mef2D splice forms have overlapping but distinct roles and provides new insight into the importance of Mef2D alternative splicing during skeletal myogenesis.

A complex network of transcription factors, which are regulated by signalling molecules, is responsible in coordinating the formation of differentiated skeletal and cardiac myocytes from undifferentiated stem cells. The present study aims to understand and compare the transcriptional regulation of skeletal and/or cardiac muscle development in the absence of Sox7 or in the presence of a collagen-based matrix in P19 embyonal carcinoma (EC) and mouse embryonic stem (ES) cells. First, knock-down of Sox7 , by shRNA, in muscle inducing conditions (+DMSO) and in the absence of RA (-RA), decreased muscle progenitor transcription factor and myogenic regulatory factor (MRF) levels, suggesting that Sox7 is necessary for myogenesis. However, knock-down of Sox7 in the presence of RA (+RA) and DMSO increased expression of muscle progenitor markers and MRFs, suggesting that Sox7 is inhibitory for myogenesis +RA. Furthermore, Sox7 overexpression enhanced myogenesis -RA, but inhibited myogenesis and enhanced neurogenesis +RA. These results suggest an important interplay between RA signalling and Sox7 function during P19 differentiation. Second, Q-PCR analysis showed that compared to the mouse ES cells differentiated on the regular TC plates, differentiation on the collagen matrices had a higher expression of skeletal and cardiac precursors, MRFs and terminal differentiation markers. Collagen alone enhanced myotube formation. The enhanced collagen matrix, containing the oligosaccharide sialyl LewisX (sLeX), specifically enhanced cardiomyogenesis. These studies have added to our understanding of the transcriptional regulation of premyogenic mesoderm factors and the role of Sox7 in this process. In addition these studies provide a vision for possible use of biomaterials in directed differentiation of stem cells for the purpose of cell therapy.

No description available.

The disruption of the survival motor neuron (SMN1) gene leads to the children’s genetic disease spinal muscular atrophy (SMA). SMA is characterized by the degeneration of α-motor neurons and skeletal muscle atrophy. Although SMA is primarily considered a motor neuron disease, the involvement of muscle in its pathophysiology has not been ruled out. To gain a better understanding of the involvement of skeletal muscle pathophysiology in SMA, we have developed three aims: to identify cell-specific Smn-interacting proteins, to characterize postnatal skeletal muscle development in mouse models of SMA, and to assess the functional capacity of muscles from SMA model mice. We have used tandem affinity purification to discover Smn interacting partners in disease relevant cell types. We have identified novel cell-specific Smn interacting proteins of which we have validated myosin regulatory light chain as a muscle-specific Smn associated protein in vivo. We have taken advantage of two different mouse models of SMA, the severe Smn-/-;SMN2 mouse and the less severe Smn2B/- mouse, to study the postnatal development of skeletal muscle. Primary myoblasts from Smn2B/- mice demonstrate delayed myotube fusion and aberrant expression of the myogenic program. In addition, the expression of myogenic proteins was delayed in muscles from severe Smn-/-;SMN2 and less severe Smn2B/- SMA model mice. Muscle denervation and degeneration, however, are not the cause of the aberrant myogenic program. At the functional level, we demonstrate a significant decrease in force production in pre-symptomatic Smn-/-;SMN2 and Smn2B/- mice indicating that muscle weakness is an early event in these mice. Immunoblot analyses from hindlimb skeletal muscle samples revealed aberrant levels of developmentally regulated proteins important for muscle function, which may impact muscle force production in skeletal muscle of SMA model mice. The present study demonstrates early and profound intrinsic muscle weakness and aberrant expression of muscle proteins in mouse models of SMA, thus demonstrating how muscle defects can contribute to the disease phenotype independently of and in addition to that caused by motor neuron pathology.

A complex network of transcription factors, which are regulated by signalling molecules, is responsible in coordinating the formation of differentiated skeletal and cardiac myocytes from undifferentiated stem cells. The present study aims to understand and compare the transcriptional regulation of skeletal and/or cardiac muscle development in the absence of Sox7 or in the presence of a collagen-based matrix in P19 embyonal carcinoma (EC) and mouse embryonic stem (ES) cells. First, knock-down of Sox7 , by shRNA, in muscle inducing conditions (+DMSO) and in the absence of RA (-RA), decreased muscle progenitor transcription factor and myogenic regulatory factor (MRF) levels, suggesting that Sox7 is necessary for myogenesis. However, knock-down of Sox7 in the presence of RA (+RA) and DMSO increased expression of muscle progenitor markers and MRFs, suggesting that Sox7 is inhibitory for myogenesis +RA. Furthermore, Sox7 overexpression enhanced myogenesis -RA, but inhibited myogenesis and enhanced neurogenesis +RA. These results suggest an important interplay between RA signalling and Sox7 function during P19 differentiation. Second, Q-PCR analysis showed that compared to the mouse ES cells differentiated on the regular TC plates, differentiation on the collagen matrices had a higher expression of skeletal and cardiac precursors, MRFs and terminal differentiation markers. Collagen alone enhanced myotube formation. The enhanced collagen matrix, containing the oligosaccharide sialyl LewisX (sLeX), specifically enhanced cardiomyogenesis. These studies have added to our understanding of the transcriptional regulation of premyogenic mesoderm factors and the role of Sox7 in this process. In addition these studies provide a vision for possible use of biomaterials in directed differentiation of stem cells for the purpose of cell therapy.

Prader-Willi syndrome (PWS) is a recurrent microdeletion syndrome characterized by severe obesity, hyperphagia, hypotonia, and developmental delay, and is caused by the loss of expression of four protein-coding genes and set of small nucleolar RNAs on chromosome 15. NDN, encoding the protein necdin, is one of these genes, and a large body of literature supports the theory that necdin is important for the differentiation and survival of neurons. Given that necdin is also abundant in developing muscle and that hypotonia is a cardinal feature of PWS, I hypothesize that necdin promotes normal skeletal muscle development. I provide two lines of evidence demonstrating that loss of necdin impairs muscle development in mice. First, necdin interacts with the inhibitor of muscle differentiation EID-1 to relieve inhibition of MyoD-dependent transcription by sequestering this protein in the cytoplasm in over-expression assays. Unexpectedly, the presence of necdin increases EID-1 protein abundance in transfected cells and endogenous EID-1 is less abundant in Ndn-null embryonic mouse tissue compared to controls. Finally, conversion from MyoD+ to Myosin Heavy Chain+ cells is impaired in limb bud cultures from Ndn-null embryos, consistent with the hypothesis that loss of necdin impairs muscle differentiation by failing to relieve EID-1-dependent transcriptional inhibition. Second, loss of necdin impairs polarization of muscle progenitors in vitro and in vivo due to failed activation of the actin-myosin cytoskeleton, and reduces the proportional area of forelimb extensor muscles in Ndn-null mice at birth. This conclusion is supported by defective centrosome re-orientation due to impaired nuclear rearward movement and failed Cdc42 activation in Ndn-null mouse embryonic fibroblasts (MEFs), impaired myosin activation in Ndn-null MEFs and cortical neurons, and excessive branching and failure of hippocampal neurons to polarize with respect to a growth factor. Additionally, PWS patient fibroblasts display centrosome re-orientation defects and impaired myosin activation identical to Ndn-null MEFs, indicating that loss of necdin produces a similar phenotype in both mice and humans. These results provide strong evidence that necdin is critical for both the migration and primary differentiation of skeletal muscle, and validates the Ndn-null mouse as a model for hypotonia in PWS.