Nobuko Hagiwara

Sox6 is required for normal fiber type differentiation of fetal skeletal muscle in mice

Sox6, a member of the Sox family of transcription factors, is highly expressed in skeletal muscle. Despite its abundant expression, the role of Sox6 in muscle development is not well understood. We hypothesize that, in fetal muscle, Sox6 functions as a repressor of slow fiber type‐specific genes. In the wild‐type mouse, differentiation of fast and slow fibers becomes apparent during late fetal stages (after approximately embryonic day 16). However, in the Sox6 null‐p100H mutant mouse, all fetal muscle fibers maintain slow fiber characteristics, as evidenced by expression of the slow myosin heavy chain MyHC‐β. Knockdown of Sox6 expression in wild‐type myotubes results in a significant increase in MyHC‐β expression, supporting our hypothesis. Analysis of the MyHC‐β promoter revealed a Sox consensus sequence that likely functions as a negative cis‐regulatory element. Together, our results suggest that Sox6 plays a critical role in the fiber type differentiation of fetal skeletal muscle. Developmental Dynamics 236:2062–2076, 2007.

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, p100H, 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 p100H 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.

Slow and fast fiber isoform gene expression is systematically altered in skeletal muscle of the Sox6 mutant, p100H

We have previously demonstrated that p100H mutant mice, which lack a functional Sox6 gene, exhibit skeletal and cardiac muscle degeneration and develop cardiac conduction abnormalities soon after birth. To understand the role of Sox6 in skeletal muscle development, we identified muscle‐specific genes differentially expressed between wild‐type and p100H mutant skeletal muscles and investigated their temporal expression in the mutant muscle. We found that, in the mutant skeletal muscle, slow fiber and cardiac isoform genes are expressed at significantly higher levels, whereas fast fiber isoform genes are expressed at significantly lower levels than wild‐type. Onset of this aberrant fiber type‐specific gene expression in the mutant coincides with the beginning of the secondary myotube formation, at embryonic day 15–16 in mice. Together with our earlier report, demonstrating early postnatal muscle defects in the Sox6 null‐p100H mutant, the present results suggest that Sox6 likely plays an important role in muscle development. Developmental Dynamics 234:301–311, 2005.

Cloning, characterization and chromosome mapping of the human SOX6 gene.

The Sox gene family encodes an important group of transcription factors harboring the conserved high-mobility group (HMG) box originally identified in the mouse and human testis determining gene Sry. We have cloned and sequenced SOX6, a member of the human Sox gene family. SOX6 cDNAs isolated from a human myoblast cDNA library show 94.3% amino acid identity to mouse Sox6 throughout the gene, and 100% identity in the critical HMG box and coiled-coil domains. The human SOX6 gene was localized to chromosome 11p15.2-11p15.3 in a region of shared synteny with distal mouse chromosome 7. An analysis of the genomic structure of the human SOX6 gene revealed 16 exons. We identified three SOX6 cDNAs that are generated by alternative splicing. Northern blot analysis revealed that SOX6 is expressed in a wide variety of tissues, most abundantly in skeletal muscle, suggesting an important role for SOX6 in muscle. Mice homozygous for a null mutation of Sox6 (p(100H)) die suddenly within the first 2 weeks after birth, most likely from cardiac conduction defects (Hagiwara et al., 2000). Thus, there is a possibility that human SOX6 is similarly involved in an, as yet, unidentified human cardiac disorder.

Nonneuronal expression of the GABAA β3 subunit gene is required for normal palate development in mice

Cleft palate is one of the most common birth defects in humans, in which both genetic and environmental factors are involved. In mice, loss of the GABA(A) receptor beta3 subunit gene (Gabrb3) or the targeted mutagenesis of the GABA synthetic enzyme (Gad1) leads to cleft palate. These observations indicate that a GABAergic system is important in normal palate development. To determine what cell types, neuronal or nonneuronal, are critical for GABA signaling in palate development, we used the neuron-specific enolase promoter to express the beta3 subunit in Gabrb3 mutant mice. Expression of this construct was able to rescue the neurological phenotype, but not the cleft palate phenotype. Combined with the previous observation demonstrating that ubiquitous expression of the beta3 subunit rescued the cleft palate phenotype, a nonneuronal GABAergic system is implicated in palate development. Using immunohistochemistry, we detected GABA in the developing palate, initially in the nasal aspect of palatal epithelium of the vertical shelves; later in the medial edge epithelium of the horizontally oriented palatal shelves and in the epithelial seam during fusion. Based on these observations, we propose that GABA, synthesized by the palatal epithelium, acts as a signaling molecule during orientation and fusion of the palate shelves.

Sox6, jack of all trades: a versatile regulatory protein in vertebrate development.

Approximately 20,000 genes are encoded in our genome, one tenth of which are thought to be transcription factors. Considering the complexity and variety of cell types generated during development, many transcription factors likely play multiple roles. Uncovering the versatile roles of Sox6 in vertebrate development sheds some light on how an organism efficiently utilizes the limited resources of transcription factors. The structure of the Sox6 gene itself may dictate its functional versatility. First, Sox6 contains no known regulatory domains; instead, it utilizes various cofactors. Second, Sox6 has a long 3′‐UTR that contains multiple microRNA targets, thus its protein level is duly adjusted by cell type–specific microRNAs. Just combining these two characteristics alone makes Sox6 extremely versatile. To date, Sox6 has been reported to regulate differentiation of tissues of mesoderm, ectoderm, and endoderm origins, making Sox6 a truly multifaceted transcription factor. Developmental Dynamics 240:1311–1321, 2011.

Regulation of heat shock protein 60 and 72 expression in the failing heart

Heart failure, a progressive, fatal disease of the heart muscle, is a state of chronic inflammation and injury. Heat shock protein (HSP) 72, a ubiquitous protective protein that is well-established as cardioprotective, is not increased in heart failure. In contrast, HSP60 levels are doubled in the failing heart. We hypothesized that HSF-1 is not activated in heart failure and that the increased expression of HSP60 was driven by NFkappaB activation. To test this hypothesis, we measured levels of heat shock factor (HSF) -1 and -2, the transcription factors controlling HSP expression, which were increased in heart failure. There was no increased phosphorylation of serine 230 or serine 303/307 in HSF-1, which are thought to regulate its activity; EMSA showed no increase in HSF binding activity with heart failure. Nonetheless, mRNA was increased for HSP60, but not HSP72. In contrast to HSF, NFkappaB activity was increased in heart failure. HSP60, but not HSP72, contained NFkappaB binding elements. ChIP assay demonstrated increased binding of NFkappaB to both of the NFkappaB binding elements in the heart failure HSP60 gene. TNFalpha treatment was used to test the role of NFkappaB activation in HSP60 expression in a cardiac cell line. TNFalpha increased HSP60 expression, and this could be prevented by pretreatment with siRNA inhibiting p65 expression. In conclusion, HSP72 is not increased in heart failure because HSF activity is not changed; increased expression of HSP60 may be driven by NFkappaB activation.

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

BackgroundSox6 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.ResultsWe 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.ConclusionsOur 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.

Mutations in Grxcr1 Are The Basis for Inner Ear Dysfunction in the Pirouette Mouse

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.

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

BackgroundA 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.MethodsYeast 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.ResultsWe 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.ConclusionsBased 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.