Genome-wide mapping of Sox6 binding sites in skeletal muscle reveals both direct and indirect regulation of muscle terminal differentiation by Sox6

<p>Abstract</p> <p>Background</p> <p>Sox6 is a multi-faceted transcription factor involved in the terminal differentiation of many different cell types in vertebrates. It has been suggested that in mice as well as in zebrafish Sox6 plays a role in the terminal differentiation of skeletal muscle by suppressing transcription of slow fiber specific genes. In order to understand how Sox6 coordinately regulates the transcription of multiple fiber type specific genes during muscle development, we have performed ChIP-seq analyses to identify Sox6 target genes in mouse fetal myotubes and generated muscle-specific Sox6 knockout (KO) mice to determine the Sox6 null muscle phenotype in adult mice.</p> <p>Results</p> <p>We have identified 1,066 Sox6 binding sites using mouse fetal myotubes. The Sox6 binding sites were found to be associated with slow fiber-specific, cardiac, and embryonic isoform genes that are expressed in the sarcomere as well as transcription factor genes known to play roles in muscle development. The concurrently performed RNA polymerase II (Pol II) ChIP-seq analysis revealed that 84% of the Sox6 peak-associated genes exhibited little to no binding of Pol II, suggesting that the majority of the Sox6 target genes are transcriptionally inactive. These results indicate that Sox6 directly regulates terminal differentiation of muscle by affecting the expression of sarcomere protein genes as well as indirectly through influencing the expression of transcription factors relevant to muscle development. Gene expression profiling of Sox6 KO skeletal and cardiac muscle revealed a significant increase in the expression of the genes associated with Sox6 binding. In the absence of the Sox6 gene, there was dramatic upregulation of slow fiber-specific, cardiac, and embryonic isoform gene expression in Sox6 KO skeletal muscle and fetal isoform gene expression in Sox6 KO cardiac muscle, thus confirming the role Sox6 plays as a transcriptional suppressor in muscle development.</p> <p>Conclusions</p> <p>Our present data indicate that during development, Sox6 functions as a transcriptional suppressor of fiber type-specific and developmental isoform genes to promote functional specification of muscle which is critical for optimum muscle performance and health.</p

Sox6 Directly Silences Epsilon Globin Expression in Definitive Erythropoiesis

Sox6 is a member of the Sox transcription factor family that is defined by the conserved high mobility group (HMG) DNA binding domain, first described in the testis determining gene, Sry. Previous studies have suggested that Sox6 plays a role in the development of the central nervous system, cartilage, and muscle. In the Sox6-deficient mouse, p(100H), ɛy globin is persistently expressed, and increased numbers of nucleated red cells are present in the fetal circulation. Transfection assays in GM979 (erythroleukemic) cells define a 36–base pair region of the ɛy proximal promoter that is critical for Sox6 mediated repression. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrate that Sox6 acts as a repressor by directly binding to the ɛy promoter. The normal expression of Sox6 in wild-type fetal liver and the ectopic expression of ɛy in p(100H) homozygous fetal liver demonstrate that Sox6 functions in definitive erythropoiesis. The present study shows that Sox6 is required for silencing of ɛy globin in definitive erythropoiesis and suggests a role for Sox6 in erythroid cell maturation. Thus, Sox6 regulation of ɛy globin might provide a novel therapeutical target in the treatment of hemoglobinopathies such as sickle cell anemia and thalassemia

Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells

Abstract Background A sophisticated level of coordinated gene expression is necessary for skeletal muscle fibers to obtain their unique functional identities. We have previously shown that the transcription factor Sox6 plays an essential role in coordinating muscle fiber type differentiation by acting as a transcriptional suppressor of slow fiber-specific genes. Currently, mechanisms regulating the activity of Sox6 in skeletal muscle and how these mechanisms affect the fiber phenotype remain unknown. Methods Yeast two-hybrid screening was used to identify binding partners of Sox6 in muscle. Small interfering RNA (siRNA)-mediated knockdown of one of the Sox6 binding proteins, Trip12, was used to determine its effect on Sox6 activity in C2C12 myotubes using quantitative analysis of fiber type-specific gene expression. Results We found that the E3 ligase Trip12, a HECT domain E3 ubiquitin ligase, recognizes and polyubiquitinates Sox6. Inhibiting Trip12 or the 26S proteasome activity resulted in an increase in Sox6 protein levels in C2C12 myotubes. This control of Sox6 activity in muscle cells via Trip12 ubiquitination has significant phenotypic outcomes. Knockdown of Trip12 in C2C12 myotubes led to upregulation of Sox6 protein levels and concurrently to a decrease in slow fiber-specific Myh7 expression coupled with an increased expression in fast fiber-specific Myh4. Therefore, regulation of Sox6 cellular levels by the ubiquitin-proteasome system can induce identity-changing alterations in the expression of fiber type-specific genes in muscle cells. Conclusions Based on our data, we propose that in skeletal muscle, E3 ligases have a significant role in regulating fiber type-specific gene expression, expanding their importance in muscle beyond their well-established role in atrophy

Transcriptome Dynamics and Potential Roles of Sox6 in the Postnatal Heart.

The postnatal heart undergoes highly coordinated developmental processes culminating in the complex physiologic properties of the adult heart. The molecular mechanisms of postnatal heart development remain largely unexplored despite their important clinical implications. To gain an integrated view of the dynamic changes in gene expression during postnatal heart development at the organ level, time-series transcriptome analyses of the postnatal hearts of neonatal through adult mice (P1, P7, P14, P30, and P60) were performed using a newly developed bioinformatics pipeline. We identified functional gene clusters by principal component analysis with self-organizing map clustering which revealed organized, discrete gene expression patterns corresponding to biological functions associated with the neonatal, juvenile and adult stages of postnatal heart development. Using weighted gene co-expression network analysis with bootstrap inference for each of these functional gene clusters, highly robust hub genes were identified which likely play key roles in regulating expression of co-expressed, functionally linked genes. Additionally, motivated by the role of the transcription factor Sox6 in the functional maturation of skeletal muscle, the role of Sox6 in the postnatal maturation of cardiac muscle was investigated. Differentially expressed transcriptome analyses between Sox6 knockout (KO) and control hearts uncovered significant upregulation of genes involved in cell proliferation at postnatal day 7 (P7) in the Sox6 KO heart. This result was validated by detecting mitotically active cells in the P7 Sox6 KO heart. The current report provides a framework for the complex molecular processes of postnatal heart development, thus enabling systematic dissection of the developmental regression observed in the stressed and failing adult heart

Sox6 regulation of cardiac myocyte development

A mouse mutation (p(100H)/p(100H)) has been identified that is associated with cardioskeletal myopathy, heart block, delayed growth and early postnatal death. The gene that is disrupted in this mutation encodes the transcription factor Sox6. P19CL6 cells were used as an in vitro cardiomyocyte differentiation system and revealed that Sox6 is expressed exclusively when the cells are committed to differentiate to beating cardiac myocytes. We used the yeast two-hybrid system to identify the Prtb (Proline-rich transcript of the brain) protein as a Sox6 interactor, and subsequently confirmed the interaction by co-immunoprecipitation. Prtb expression in P19CL6 cells increased with differentiation to beating cardiomyocytes. Using the P19CL6 cells stably transfected with noggin, an antagonist of BMP (Bone Morphogenic Protein), we found that BMP expression is required for Sox6 expression in cardiomyocyte differentiation. Surprisingly, the expression of the α(1c)-subunit gene of the L-type Ca(2+) channel decreased in P19CL6 cells as they differentiated to beating cardiac cells. Ectopic expression of Sox6 or Prtb alone in P19CL6 cells caused down-regulation of L-type Ca(2+) α(1c) expression, but when Sox6 and Prtb were co-transfected to the cells, L-type Ca(2+) α(1c) remained at basal levels. A similar relationship of Sox6 and L-type Ca(2+) α(1c) expression was seen in vivo (comparing wild-type and p(100H)/p(100H) mutant mice). Thus, Sox6 is within the BMP pathway in cardiac differentiation, interacts with Prtb and may play a critical role in the regulation of a cardiac L-type Ca(2+) channel

Autologous angiogenic therapy with cultured mesenchymal stromal cells in platelet-rich plasma for critical limb ischemia

Introduction: The prevalence of diabetes mellitus is increasing globally, including in Japan. Patients with diabetes often experience microangiopathy and macroangiopathy, which lead to difficult-to-treat foot ulcers and diabetic gangrene. Conventional cellular therapies have limited safety and are invasive. In this study, we investigated the use of cultured autologous mesenchymal stromal cells derived from the bone marrow and grown in platelet-rich plasma as a potential treatment for diabetic complications. Methods: A prospective clinical trial was conducted to assess safety as the primary endpoint and efficacy as the secondary endpoint of the aforementioned therapy in five patients with critical limb ischemia, with or without hemodialysis. Results: Five patients with critical limb ischemia were enrolled between 2016 and 2019, three of whom underwent hemodialysis. Platelet-rich plasma was obtained from 288 ± 39.6 mL of blood/patient, yielding 31.6 ± 1.67 mL of platelet-rich plasma. Bone marrow aspiration yielded 18.4 ± 4.77 mL/patient, and 4.64 ± 1.51 × 107 cells were incubated for 16 ± 2.8 days to obtain 3.26 ± 0.33 × 107 mesenchymal stromal cells. Although several adverse events were observed, none were directly attributed to cell therapy. Clinical severity, as assessed by both the Fontaine stage and Rutherford category, improved significantly following therapy. This improvement was accompanied by enhancements in the 6-min walking distance, dorsal skin perfusion pressure, ankle transcutaneous partial oxygen pressure, and ankle brachial pressure index. Conclusion: Autologous angiogenic therapy with cultured mesenchymal stromal cells derived from the bone marrow and grown in platelet-rich plasma is a safe and feasible, and was expected as a potential treatment for critical limb ischemia