SoxC Proteins Research Articles

SEM-2/SoxC regulates multiple aspects of C. elegans postembryonic mesoderm development.

Development of multicellular organisms requires well-orchestrated interplay between cell-intrinsic transcription factors and cell-cell signaling. One set of highly conserved transcription factors that plays diverse roles in development is the SoxC group. C. elegans contains a sole SoxC protein, SEM-2. SEM-2 is essential for embryonic development, and for specifying the sex myoblast (SM) fate in the postembryonic mesoderm, the M lineage. We have identified a novel partial loss-of-function sem-2 allele that has a proline to serine change in the C-terminal tail of the highly conserved DNA-binding domain. Detailed analyses of mutant animals harboring this point mutation uncovered new functions of SEM-2 in the M lineage. First, SEM-2 functions antagonistically with LET-381, the sole C. elegans FoxF/C forkhead transcription factor, to regulate dorsoventral patterning of the M lineage. Second, in addition to specifying the SM fate, SEM-2 is essential for the proliferation and diversification of the SM lineage. Finally, SEM-2 appears to directly regulate the expression of hlh-8, which encodes a basic helix-loop-helix Twist transcription factor and plays critical roles in proper patterning of the M lineage. Our data, along with previous studies, suggest an evolutionarily conserved relationship between SoxC and Twist proteins. Furthermore, our work identified new interactions in the gene regulatory network (GRN) underlying C. elegans postembryonic development and adds to the general understanding of the structure-function relationship of SoxC proteins.

The transcription factor prospero homeobox protein 1 is a direct target of SoxC proteins during developmental vertebrate neurogenesis.

The high-mobility-group domain containing SoxC transcription factors Sox4 and Sox11 are expressed and required in the vertebrate central nervous system in neuronal precursors and neuroblasts. To identify genes that are widely regulated by SoxC proteins during vertebrate neurogenesis we generated expression profiles from developing mouse brain and chicken neural tube with reduced SoxC expression and found the transcription factor prospero homeobox protein 1 (Prox1) strongly down-regulated under both conditions. This led us to hypothesize that Prox1 expression depends on SoxC proteins in the developing central nervous system of mouse and chicken. By combining luciferase reporter assays and over-expression in the chicken neural tube with invivo and invitro binding studies, we identify the Prox1 gene promoter and two upstream enhancers at -44kb and -40kb relative to the transcription start as regulatory regions that are bound and activated by SoxC proteins. This argues that Prox1 is a direct target gene of SoxC proteins during neurogenesis. Electroporations in the chicken neural tube furthermore show that Prox1 activates a subset of SoxC target genes, whereas it has no effects on others. We propose that the transcriptional control of Prox1 by SoxC proteins may ensure coupling of two types of transcription factors that are both required during early neurogenesis, but have at least in part distinct functions. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Inflammatory Cytokines Stabilize SOXC Transcription Factors to Mediate the Transformation of Fibroblast-Like Synoviocytes in Arthritic Disease.

ObjectiveFibroblast‐like synoviocytes (FLS) produce key synovial fluid and tissue components to ensure joint integrity under healthy conditions, whereas they become cancer‐like and aggressively contribute to joint degeneration in inflammatory arthritis. The aim of this study was to determine whether the SOXC transcription factors SOX4 and SOX11, whose functions are critical in joint development and many cancer types, contribute to FLS activities under normal and inflammatory conditions.MethodsWe inactivated the SOXC genes in FLS from adult mice and studied the effect on joint homeostasis and tumor necrosis factor (TNF)–induced arthritis. We used primary cells and synovial biopsy specimens from arthritis patients to analyze the interactions between inflammatory signals and SOXC proteins.ResultsPostnatal inactivation of the SOXC genes had no major effect on joint integrity in otherwise healthy mice. However, it hampered synovial hyperplasia and joint degeneration in transgenic mice expressing human TNF. These effects were explained by the ability of SOX4/11 to amplify the pathogenic impact of TNF on FLS by increasing their survival and migration. SOXC RNA levels were not changed by TNF and other proinflammatory cytokines, but SOXC proteins were strongly stabilized and able to potentiate the TNF‐induced up‐regulation of genes involved in FLS transformation. Substantiating the relevance of these findings in human disease, SOXC protein levels, but not RNA levels, were significantly higher in inflamed synovium than in noninflamed synovium from arthritis patients.ConclusionSOXC proteins are targets and pivotal mediators of proinflammatory cytokines during FLS transformation in arthritic diseases. Targeting of these proteins could thus improve current strategies to treat arthritic diseases and possibly other inflammatory diseases.

SOX4 regulates gonad morphogenesis and promotes male germ cell differentiation in mice

The group C SOX transcription factors SOX4, −11 and −12 play important and mutually overlapping roles in development of a number of organs. Here, we examined the role of SoxC genes during gonadal development in mice. All three genes were expressed in developing gonads of both sexes, predominantly in somatic cells, with Sox4 being most strongly expressed. Sox4 deficiency resulted in elongation of both ovaries and testes, and an increased number of testis cords. While female germ cells entered meiosis normally, male germ cells showed reduced levels of differentiation markers Nanos2 and Dnmt3l and increased levels of pluripotency genes Cripto and Nanog, suggesting that SOX4 may normally act to restrict the pluripotency period of male germ cells and ensure their proper differentiation. Finally, our data reveal that SOX4 (and, to a lesser extent, SOX11 and −12) repressed transcription of the sex-determining gene Sox9 via an upstream testis-specific enhancer core (TESCO) element in fetal gonads, raising the possibility that SOXC proteins may function as transcriptional repressors in a context-dependent manner.

SOXC Genes and the Control of Skeletogenesis.

The SOXC group of transcription factors, composed of SOX4, SOX11, and SOX12, has evolved to fulfill key functions in cell fate determination. Expressed in many types of progenitor/stem cells, including skeletal progenitors, SOXC proteins potentiate pathways critical for cell survival and differentiation. As skeletogenesis unfolds, SOXC proteins ensure cartilage primordia delineation by amplifying canonical WNT signaling and antagonizing the chondrogenic action of SOX9 in perichondrium and presumptive articular joint cells. They then ensure skeletal elongation by inducing growth plate formation via enabling non-canonical WNT signaling. Human studies have associated SOX4 with bone mineral density and fracture risk in osteoporotic patients, and SOX11 with Coffin-Siris, a syndrome that includes skeletal dysmorphism. Meanwhile, in vitro and mouse studies have suggested important cell-autonomous roles for SOXC proteins in osteoblastogenesis. We here review current knowledge and gaps in understanding of SOXC protein functions, with an emphasis on the skeleton and possible links to osteoporosis.

SOXC Transcription Factors Induce Cartilage Growth Plate Formation in Mouse Embryos by Promoting Noncanonical WNT Signaling.

Growth plates are specialized cartilage structures that ensure the elongation of most skeletal primordia during vertebrate development. They are made by chondrocytes that proliferate in longitudinal columns and then progress in a staggered manner towards prehypertrophic, hypertrophic and terminal maturation. Complex molecular networks control the formation and activity of growth plates, but remain incompletely understood. We investigated here the importance of the SoxC genes, which encode the SOX4, SOX11 and SOX12 transcription factors, in growth plates. We show that the three genes are expressed robustly in perichondrocytes and weakly in growth plate chondrocytes. SoxC(Prx1Cre) mice, which deleted SoxC genes in limb bud skeletogenic mesenchyme, were born with tiny appendicular cartilage primordia because of failure to form growth plates. In contrast, SoxC(Col2Cre) and SoxC(ATC) mice, which deleted SoxC genes primarily in chondrocytes, were born with mild dwarfism and fair growth plates. Chondrocytes in the latter mutants matured normally, but formed irregular columns, proliferated slowly and died ectopically. Asymmetric distribution of VANGL2 was defective in both SoxC(Prx1Cre) and SoxC(ATC) chondrocytes, indicating impairment of planar cell polarity, a noncanonical WNT signaling pathway that controls growth plate chondrocyte alignment, proliferation and survival. Accordingly, SoxC genes were necessary in perichondrocytes for expression of Wnt5a, which encodes a noncanonical WNT ligand required for growth plate formation, and in chondrocytes and perichondrocytes for expression of Fzd3 and Csnk1e, which encode a WNT receptor and casein kinase-1 subunit mediating planar cell polarity, respectively. Reflecting the differential strengths of the SOXC protein transactivation domains, SOX11 was more powerful than SOX4, and SOX12 interfered with the activity of SOX4 and SOX11. Altogether, these findings provide novel insights into the molecular regulation of skeletal growth by proposing that SOXC proteins act cell- and non-cell-autonomously in perichondrocytes and chondrocytes to establish noncanonical WNT signaling crosstalk essential for growth plate induction and control.

Keeping an eye on SOXC proteins.

The formation of a mature, functional eye requires a complex series of cell proliferation, migration, induction among different germinal layers, and cell differentiation. These processes are regulated by extracellular cues such as the Wnt/BMP/Hh/Fgf signaling pathways, as well as cell intrinsic transcription factors that specify cell fate. In this review article, we provide an overview of stages of embryonic eye morphogenesis, extrinsic and intrinsic factors that are required for each stage, and pediatric ocular diseases that are associated with defective eye development. In addition, we focus on recent findings about the roles of the SOXC proteins in regulating vertebrate ocular development and implicating SOXC mutations in human ocular malformations.

SOXC proteins amplify canonical WNT signaling to secure nonchondrocytic fates in skeletogenesis.

Canonical WNT signaling stabilizes β-catenin to determine cell fate in many processes from development onwards. One of its main roles in skeletogenesis is to antagonize the chondrogenic transcription factor SOX9. We here identify the SOXC proteins as potent amplifiers of this pathway. The SOXC genes, i.e., Sox4, Sox11, and Sox12, are coexpressed in skeletogenic mesenchyme, including presumptive joints and perichondrium, but not in cartilage. Their inactivation in mouse embryo limb bud caused massive cartilage fusions, as joint and perichondrium cells underwent chondrogenesis. SOXC proteins govern these cells cell autonomously. They replace SOX9 in the adenomatous polyposis coli-Axin destruction complex and therein inhibit phosphorylation of β-catenin by GSK3. This inhibition, a crucial, limiting step in canonical WNT signaling, thus becomes a constitutive event. The resulting SOXC/canonical WNT-mediated synergistic stabilization of β-catenin contributes to efficient repression of Sox9 in presumptive joint and perichondrium cells and thereby ensures proper delineation and articulation of skeletal primordia. This synergy may determine cell fate in many processes besides skeletogenesis.

Can the 'neuron theory' be complemented by a universal mechanism for generic neuronal differentiation.

With the establishment of the 'neuron theory' at the turn of the twentieth century, this remarkably powerful term was introduced to name a breathtaking diversity of cells unified by a characteristic structural compartmentalization and unique information processing and propagating features. At the beginning of the twenty-first century, developmental, stem cell and reprogramming studies converged to suggest a common mechanism involved in the generation of possibly all vertebrate, and at least a significant number of invertebrate, neurons. Sox and, in particular, SoxB and SoxC proteins as well as basic helix-loop-helix proteins play major roles, even though their precise contributions to progenitor programming, proliferation and differentiation are not fully resolved. In addition to neuronal development, these transcription factors also regulate sensory receptor and endocrine cell development, thus specifying a range of cells with regulatory and communicative functions. To what extent microRNAs contribute to the diversification of these cell types is an upcoming question. Understanding the transcriptional and post-transcriptional regulation of genes coding for cell type-specific cytoskeletal and motor proteins as well as synaptic and ion channel proteins, which mark differences but also similarities between the three communicator cell types, will provide a key to the comprehension of their diversification and the signature of 'generic neuronal' differentiation. Apart from the general scientific significance of a putative universal core instruction for neuronal development, the impact of this line of research for cell replacement therapy and brain tumor treatment will be of considerable interest.

SoxC Transcription Factors Are Required for Neuronal Differentiation in Adult Hippocampal Neurogenesis

Neural stem cells (NSCs) generate new hippocampal dentate granule neurons throughout adulthood. The genetic programs controlling neuronal differentiation of adult NSCs are only poorly understood. Here we show that, in the adult mouse hippocampus, expression of the SoxC transcription factors Sox4 and Sox11 is initiated around the time of neuronal commitment of adult NSCs and is maintained in immature neurons. Overexpression of Sox4 and Sox11 strongly promotes in vitro neurogenesis from adult NSCs, whereas ablation of Sox4/Sox11 prevents in vitro and in vivo neurogenesis from adult NSCs. Moreover, we demonstrate that SoxC transcription factors target the promoters of genes that are induced on neuronal differentiation of adult NSCs. Finally, we show that reprogramming of astroglia into neurons is dependent on the presence of SoxC factors. These data identify SoxC proteins as essential contributors to the genetic network controlling neuronal differentiation in adult neurogenesis and neuronal reprogramming of somatic cells.

Structural Basis for the Oxidation of Protein-bound Sulfur by the Sulfur Cycle Molybdohemo-Enzyme Sulfane Dehydrogenase SoxCD

The sulfur cycle enzyme sulfane dehydrogenase SoxCD is an essential component of the sulfur oxidation (Sox) enzyme system of Paracoccus pantotrophus. SoxCD catalyzes a six-electron oxidation reaction within the Sox cycle. SoxCD is an α(2)β(2) heterotetrameric complex of the molybdenum cofactor-containing SoxC protein and the diheme c-type cytochrome SoxD with the heme domains D(1) and D(2). SoxCD(1) misses the heme-2 domain D(2) and is catalytically as active as SoxCD. The crystal structure of SoxCD(1) was solved at 1.33 Å. The substrate of SoxCD is the outer (sulfane) sulfur of Cys-110-persulfide located at the C-terminal peptide swinging arm of SoxY of the SoxYZ carrier complex. The SoxCD(1) substrate funnel toward the molybdopterin is narrow and partially shielded by side-chain residues of SoxD(1). For access of the sulfane-sulfur of SoxY-Cys-110 persulfide we propose that (i) the blockage by SoxD-Arg-98 is opened via interaction with the C terminus of SoxY and (ii) the C-terminal peptide VTIGGCGG of SoxY provides interactions with the entrance path such that the cysteine-bound persulfide is optimally positioned near the molybdenum atom. The subsequent oxidation reactions of the sulfane-sulfur are initiated by the nucleophilic attack of the persulfide anion on the molybdenum atom that is, in turn, reduced. The close proximity of heme-1 to the molybdopterin allows easy acceptance of the electrons. Because SoxYZ, SoxXA, and SoxB are already structurally characterized, with SoxCD(1) the structures of all key enzymes of the Sox cycle are known with atomic resolution.

TheC. elegansSoxC protein SEM-2 opposes differentiation factors to promote a proliferative blast cell fate in the postembryonic mesoderm

The proper development of multicellular organisms requires precise regulation and coordination of cell fate specification, cell proliferation and differentiation. Abnormal regulation and coordination of these processes could lead to disease, including cancer. We have examined the function of the sole C. elegans SoxC protein, SEM-2, in the M lineage, which produces the postembryonic mesoderm. We found that SEM-2/SoxC is both necessary and sufficient to promote a proliferating blast cell fate, the sex myoblast fate, over a differentiated striated bodywall muscle fate. A number of factors control the specific expression of sem-2 in the sex myoblast precursors and their descendants. This includes direct control of sem-2 expression by a Hox-PBC complex. The crucial nature of the HOX/PBC factors in directly enhancing expression of this proliferative factor in the C. elegans M lineage suggests a possible more general link between Hox-PBC factors and SoxC proteins in regulating cell proliferation.

Organogenesis relies on SoxC transcription factors for the survival of neural and mesenchymal progenitors

During organogenesis, neural and mesenchymal progenitor cells give rise to many cell lineages, but their molecular requirements for self-renewal and lineage decisions are incompletely understood. In this study, we show that their survival critically relies on the redundantly acting SoxC transcription factors Sox4, Sox11 and Sox12. The more SoxC alleles that are deleted in mouse embryos, the more severe and widespread organ hypoplasia is. SoxC triple-null embryos die at midgestation unturned and tiny, with normal patterning and lineage specification, but with massively dying neural and mesenchymal progenitor cells. Specific inactivation of SoxC genes in neural and mesenchymal cells leads to selective apoptosis of these cells, suggesting SoxC cell-autonomous roles. Tead2 functionally interacts with SoxC genes in embryonic development, and is a direct target of SoxC proteins. SoxC genes therefore ensure neural and mesenchymal progenitor cell survival, and function in part by activating this transcriptional mediator of the Hippo signalling pathway.

Sequential requirement of Sox4 and Sox11 during development of the sympathetic nervous system

The highly related transcription factors Sox4 and Sox11 are expressed in the developing sympathetic nervous system. In the mouse, Sox11 appears first, whereas Sox4 is prevalent later. Using mouse mutagenesis and overexpression strategies in chicken, we studied the role of both SoxC proteins in this tissue. Neither Sox4 nor Sox11 predominantly functioned by promoting pan-neuronal or noradrenergic differentiation of sympathetic neurons as might have been expected from studies in neuronal precursors of the central nervous system. The transcriptional network that regulates the differentiation of sympathetic neurons remained intact and expression of noradrenergic markers showed only minor alterations. Instead, Sox11 was required in early sympathetic ganglia for proliferation of tyrosine hydroxylase-expressing cells, whereas Sox4 ensured the survival of these cells at later stages. In the absence of both Sox4 and Sox11, sympathetic ganglia remained hypoplastic throughout embryogenesis because of consecutive proliferation and survival defects. As a consequence, sympathetic ganglia were rudimentary in the adult and sympathetic innervation of target tissues was impaired leading to severe dysautonomia.

Characterization of SoxB2 and SoxC genes in amphioxus (Branchiostoma belcheri): Implications for their evolutionary conservation

Most Sox genes directly affect cell fate determination and differentiation. In this study, we isolated two Sox genes: SoxB2 and SoxC from amphioxus (Branchiostoma belcheri), the closest living invertebrate relative of the vertebrates. Alignments of SoxB2 and SoxC protein sequences and their vertebrate homologs show high conservation of their HMG domains. Phylogenic analysis shows that amphioxus SoxB2 and SoxC fall out of the vertebrate branches, suggesting that vertebrate homologs might arise from gene duplications during evolution. The two genes possess similar spatial and temporal expression patterns during embryogenesis and in adults. They are both maternally inherited. During neurulation, they are expressed in the neural ectoderm and archenterons. In adults, they are expressed not only in the nerve cord, but also in the gut, midgut diverticulum, gill and oocytes. These results suggest that amphioxus SoxB2 and SoxC might co-function and have conserved functions in the nervous system and gonads as their vertebrate homologs.

The three SoxC proteins—Sox4, Sox11 and Sox12—exhibit overlapping expression patterns and molecular properties

The group C of Sry-related high-mobility group (HMG) box (Sox) transcription factors has three members in most vertebrates: Sox4, Sox11 and Sox12. Sox4 and Sox11 have key roles in cardiac, neuronal and other major developmental processes, but their molecular roles in many lineages and the roles of Sox12 remain largely unknown. We show here that the three genes are co-expressed at high levels in neuronal and mesenchymal tissues in the developing mouse, and at variable relative levels in many other tissues. The three proteins have conserved remarkable identity through evolution in the HMG box DNA-binding domain and in the C-terminal 33 residues, and we demonstrate that the latter residues constitute their transactivation domain (TAD). Sox11 activates transcription several times more efficiently than Sox4 and up to one order of magnitude more efficiently than Sox12, owing to a more stable α-helical structure of its TAD. This domain and acidic domains interfere with DNA binding, Sox11 being most affected and Sox4 least affected. The proteins are nevertheless capable of competing with one another in reporter gene transactivation. We conclude that the three SoxC proteins have conserved overlapping expression patterns and molecular properties, and might therefore act in concert to fulfill essential roles in vivo.

Evidence for two pathways of thiosulfate oxidation in Starkeya novella (formerly Thiobacillus novellus).

The pathway of thiosulfate oxidation in the facultatively chemolithotrophic, sulfur-oxidizing bacterium Starkeya novella (formerly Thiobacillus novellus) has not been established beyond doubt. Recently, isolation of the sorAB genes, which encode a soluble sulfite:cytochrome c oxidoreductase, has been reported, indicating that a thiosulfate-oxidizing pathway not involving a multienzyme complex may exist in this organism. Here we report the cloning and sequencing of the soxBCD genes from S. novella, which are closely related to the corresponding genes encoding the thiosulfate-oxidizing multienzyme complex from Paracoccus pantotrophus. These findings suggest two distinct pathways for thiosulfate oxidation in S. novella. The expression of sorAB and soxC in cells grown on thiosulfate- and/or glucose-containing media was studied by Western blot analysis. The results showed that the SorAB protein is synthesized in the presence of thiosulfate irrespective of the presence of glucose. In contrast, the SoxC protein is subject to repression by glucose; the repression, however, appears to be dependent on the relative amounts of glucose and thiosulfate present. The regulatory effects observed for the expression of sorAB are likely to be mediated by an extracytoplasmic function sigma factor encoded by the sigE gene identified upstream of sorAB.