CAPN11: A calpain with high mRNA levels in testis and located on chromosome 6.

Calpains are a superfamily of related proteins, some of which have been shown to function as calcium-dependent cysteine proteases. In mammals, eight different calpains have been identified. We report the identification of a new mammalian calpain gene, CAPN11. The predicted protein possesses the features typical of calpains including potential protease and calcium-binding domains. The CAPN11 mRNA exhibits a highly restricted tissue distribution with highest levels present in testis. Radiation hybrid mapping localized the gene to human chromosome 6, within a region mapped to p12. Phylogenetic analysis suggests that, in mammals, the predicted CAPN11 protein is most closely related to CAPN1 and CAPN2. However, of the calpain sequences available, the predicted CAPN11 sequence exhibits greatest homology to the chicken micro/m calpain. Thus CAPN11 may be the human orthologue of micro/m calpain. The discovery of this new calpain emphasizes the complexity of the calpain family, with members being distinguished on the basis of protease activity, calcium dependence, and tissue expression.     

Targeted suppression of μ‐calpain and caspase 9 expression and its effect on caspase 3 and caspase 7 in satellite cells of Korean Hanwoo cattle

The calpains play an important role in cell death and cell signalling. Caspases catalyse wholesale destruction of cellular proteins which is a major cause of cellular death. The current study looks at the function of μ‐calpain and caspase 9, using RNAi (RNA interference)‐mediated silencing, and to observe the mRNA expression level of caspase genes during satellite cell growth. The satellite cells were treated with siRNA (small interfering RNA) of μ‐calpain and caspase 9 separately. There was reduction of 16 and 24% in CAPN1 (calpain1)‐siRNA2 and CAPN1‐siRNA3 transfected cells respectively, whereas it was 60 and 56% in CAPN1‐siRNA1 and CAPN1‐siRNA4 transfected cells respectively. CAPN1‐siRNA4 and CAPN1‐siRNA1 treated cells showed more reduction in caspase 3 and 7 gene expression. CARD9 (caspase recruitment domain 9)‐siRNA1 and CARD9‐siRNA2‐treated cells showed reduction of 40 and 49% respectively. CARD9‐siRNA1 and CARD9‐siRNA2 showed an increase in caspase 3 gene expression, whereas CARD9‐siRNA2 showed reduction in caspase 7 gene expression. These results suggest a strong cross‐talk between μ‐calpain and the caspase enzyme systems. Suppression of target genes, such as μ‐calpain and caspase 9, might have genuine potential in the treatment of skeletal muscle atrophy.     

Role of calpains in diabetes mellitus: A mini review

Type 2 diabetes mellitus (T2DM) is characterized by defects in haepatic glucose production, insulin action and insulin secretion, which can also lead to a variety of secondary disorders. The disease can lead to death without treatment and it has been predicted that T2DM will affect 215 million people world-wide by 2010. T2DM is a multifactorial condition whose precise genetic causes and biochemical defects have not been fully elucidated but at both levels, calpains appear to play a role. Positional cloning studies mapped T2DM susceptibility to CAPN10, the gene encoding the intracellular cysteine protease, calpain 10. Further studies have shown a number of non-coding polymorphisms in CAPN10 to be functionally associated with T2DM whilst the identification of coding polymorphisms, suggested that mutant calpain 10 proteins may also contribute to the disease. The presence of both calpain 10 and its mRNA have been demonstrated in tissues from several mammalian species whilst calpain 10 appears to be associated with pathways involved in glucose metabolism, insulin secretion and insulin action. It appears that other calpains may also participate in these pathways and here we present an overview of recent studies on calpains and their putative role in T2DM. (Mol Cell Biochem 261: 161–167, 2004)     

Changes in calpain and caspase gene expression at the mRNA level during bovine muscle satellite cell myogenesis and the correlation between the cell model and the muscle tissue

The calpain proteolytic system plays a central role in cell death and cell signaling. Caspases are a family of proteases implicated in apoptosis. The objective of this study was to explore the regulation and change trend of calpains (CAPN1 and CAPN3) and caspases (caspase-3, caspase-7, and caspase-9) expression at the mRNA level in Luxi cattle skeletal muscle satellite cells during proliferation and differentiation into myotubes. We also sought to assess whether there is a relationship between the muscle satellite cell model and skeletal muscle tissue. Satellite cells were isolated from longissimus dorsi muscle from Luxi cattle and cultured in vitro. Immunofluorescence was used to characterize satellite cells. Our study was divided into three groups: stage one, satellite cells proliferated at 50- and 80-% confluence; stage two, satellite cells differentiated at days 1, 3, 5, 7, and 15; stage three, not the satellite cells but the skeletal muscle tissue. Real-time PCR was used to quantify expression of calpains and the caspases at the mRNA level. These data demonstrated that CAPN1, CAPN3, CASP7, Myf5, and MyoG gene expression significantly increased from satellite cell proliferation to differentiation phases (P < 0.05). In contrast, CASP3 and CASP9 gene expression was significantly down-regulated during myogenesis (P < 0.05). Moreover, we put the CAPN1, CAPN3, CASP3, CASP7, CASP9, Myf5, and MyoG together to say that these genes expression had no significant correlation between the satellite cell model and the skeletal muscle tissue (P > 0.05). Here, we conclude that calpains (CAPN1 and CAPN3), caspases (caspase-3, caspase-7, and caspase-9), and Myf5 and MyoG all have important roles in satellite cell myogenesis. However, there is no relationship between the cell model and muscle tissue.     

The calpains: modular designs and functional diversity

The calpain family is named for the calcium dependence of the papain-like, thiol protease activity of the well-studied ubiquitous vertebrate enzymes calpain-1 (μ-calpain) and calpain-2 (m-calpain). Proteins showing sequence relatedness to the catalytic core domains of these enzymes are included in this ancient and diverse eukaryotic protein family. Calpains are examples of highly modular organization, with several varieties of amino-terminal or carboxy-terminal modules flanking a conserved core. Acquisition of the penta-EF-hand module involved in calcium binding (and the formation of heterodimers for some calpains) seems to be a relatively late event in calpain evolution. Several alternative mechanisms for binding calcium and associating with membranes/phospholipids are found throughout the family. The gene family is expanded in mammals, trypanosomes and ciliates, with up to 26 members in Tetrahymena, for example; in striking contrast to this, only a single calpain gene is present in many other protozoa and in plants. The many isoforms of calpain and their multiple splice variants complicate the discussion and analysis of the family, and challenge researchers to ascertain the relationships between calpain gene sequences, protein isoforms and their distinct or overlapping functions. In mammals and plants it is clear that a calpain plays an essential role in development. There is increasing evidence that ubiquitous calpains participate in a variety of signal transduction pathways and function in important cellular processes of life and death. In contrast to relatively promiscuous degradative proteases, calpains cleave only a restricted set of protein substrates and use complex substrate-recognition mechanisms, involving primary and secondary structural features of target proteins. The detailed physiological significance of both proteolytically active calpains and those lacking key catalytic residues requires further study.     

The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants

DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML–CysPc–C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc–C2L domains of land plant calpains form a separate sub‐clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1‐like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1‐3 mutant using CysPc–C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc–C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1‐3 mutant phenotype. In contrast, neither the CysPc–C2L domains from M. viride nor chimeric animal–plant calpains complement this mutant. Co‐evolution analysis identified differences in the interactions between the CysPc–C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1‐3 complementation assay, we show that four conserved amino acid residues of two Ca²⁺‐binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.     

A comparative study of soluble calcium-dependent proteolytic activity in brain

Recent studies have shown that soluble calcium activated proteases (calpains) in brain degrade proteins associated with the cytoskeleton and vary markedly in activity across regions and as a function of development. It was suggested that the observed differences in calpain activity reflect differences in the turnover rate of structural elements. The present study extends this analysis by measuring the properties and activity of calpain in representatives of the five classes of vertebrates with particular emphasis on the mammals. No evidence for proteolysis was found in soluble fractions of fish brains at neutral pH in the presence or absence of added calcium. A substantial calcium-independent proteolytic activity was found in amphibian brains—the effects of a variety of protease inhibitors indicated that it is also a neurtral thiol (cysteine) protease. Reptilian brains exhibited both calcium-independent and calcium-dependent proteolytic activity. Virtually all proteolytic activity in birds (5 species) and mammals (9 species) measured at neutral pH was calcium-dependent. The endogenous substrates for the calcium activated proteases were very similar in several species of birds and mammals as were the effects of a variety of protease inhibitors. However, the activity of the enzyme, expressed per mg of soluble protein, was highly and negatively correlated with brain size in the mammals. The allometric expression for this relationship was similar to that found for the density of neurons in cerebral cortex as a function of absolute brain size. These results indicate that soluble proteolytic enzymes in brain are differentially expressed among classes of vertebrates and suggest that the turnover of cytoskeletal elements in birds and mammals differs in important ways from that found in fish and amphibians. The results obtained for mammals raise the possibility of a relationship between brain size and the rate at which structural elements are broken down and replaced in this vertebrate class.     

Calpain Expression and Activity during Lens Fiber Cell Differentiation

In animal models, the dysregulated activity of calcium-activated proteases, calpains, contributes directly to cataract formation. However, the physiological role of calpains in the healthy lens is not well defined. In this study, we examined the expression pattern of calpains in the mouse lens. Real time PCR and Western blotting data indicated that calpain 1, 2, 3, and 7 were expressed in lens fiber cells. Using controlled lysis, depth-dependent expression profiles for each calpain were obtained. These indicated that, unlike calpain 1, 2, and 7, which were most abundant in cells near the lens surface, calpain 3 expression was strongest in the deep cortical region of the lens. We detected calpain activities in vitro and showed that calpains were active in vivo by microinjecting fluorogenic calpain substrates into cortical fiber cells. To identify endogenous calpain substrates, membrane/cytoskeleton preparations were treated with recombinant calpain, and cleaved products were identified by two-dimensional difference electrophoresis/mass spectrometry. Among the calpain substrates identified by this approach was αII-spectrin. An antibody that specifically recognized calpain-cleaved spectrin was used to demonstrate that spectrin is cleaved in vivo, late in fiber cell differentiation, at or about the time that lens organelles are degraded. The generation of the calpain-specific spectrin cleavage product was not observed in lens tissue from calpain 3-null mice, indicating that calpain 3 is uniquely activated during lens fiber differentiation. Our data suggest a role for calpains in the remodeling of the membrane cytoskeleton that occurs with fiber cell maturation.Calpains comprise a family of cysteine proteases named for the calcium dependence of the founder members of the family, the ubiquitously expressed enzymes, calpain 1 (μ-calpain) and calpain 2 (m-calpain). The calpain family includes more than a dozen members with sequence relatedness to the catalytic subunits of calpain 1 and 2. Calpains have a modular domain architecture. By convention, the family is subdivided into classical and nonclassical calpains, according to the presence or absence, respectively, of a calcium-binding penta-EF-hand module in domain IV of the protein (1). Classical calpains include calpain 1, 2, 3, 8, 9, and 11. Nonclassical calpains include calpain 5, 6, 7, 10, 12, 13, and 14.Transgenic and gene knock-out approaches in mice have demonstrated an essential role for calpains during embryonic development. Knock-out of the small regulatory subunit (Capn4) results in embryonic lethality (2, 3). Similarly, inactivation of the Capn2 gene blocks development between the morula and blastocyst stage (4). In humans, mutations in CAPN3 underlie limb-girdle muscular dystrophy-2A, and polymorphisms in CAPN10 may predispose to type 2 diabetes mellitus (5, 6).Even under conditions of calcium overload, where calpains are presumably activated maximally, only a subset (<5%) of cellular proteins are hydrolyzed (7). Calpains typically cleave their substrates at a limited number of sites to generate large polypeptide fragments that, in many cases, retain bioactivity. Thus, under physiological conditions, calpains probably participate in the regulation of protein function rather than in non-specific protein degradation.More than 100 proteins have been shown to serve as calpain substrates in vitro, including cytoskeletal proteins (8), signal transduction molecules (9), ion channels (10), and receptors (11). In vivo, calpains are believed to function in myoblast fusion (12), long term potentiation (13), and cellular mobility (14). Unregulated calpain activity, secondary to intracellular calcium overload, is associated with several pathological conditions, including Alzheimer disease (15), animal models of cataract (16), myocardial (17), and cerebral ischemia (18).In addition to their domain structure, calpains are often classified according to their tissue expression patterns. Calpain 1, 2, and 10 are widely expressed in mammalian tissues, but other members of the calpain family show tissue-specific expression patterns. Calpain 8, for example, is a stomach-specific calpain (19), whereas expression of calpain 9 is restricted to tissues of the digestive tract (20). The expression of calpain 3 was originally thought to be limited to skeletal muscle (21), but splice variants of calpain 3 have since been detected in a range of tissues. At least 12 isoforms of calpain 3 have been described in rodents (22), of which several are expressed in the mammalian eye, including Lp82 (lens), Cn94 (cornea), and Rt88 (retina) (23).Calpains have been studied intensively in the ocular lens because of their suspected involvement in lens opacification (cataract). Calpain-mediated proteolysis of lens crystallin proteins causes increased light scatter (24). Unregulated activation of calpains is observed in rodent cataract models (25), where calpain-mediated degradation of crystallin proteins (26) and cytoskeletal elements (27) is commonly observed. Calpain inhibitors are effective in delaying or preventing cataract in vitro (28, 29) and in vivo (30, 31).It is likely, however, that calpains have important physiological roles in the lens beyond their involvement in tissue pathology. Terminal differentiation of lens fiber cells involves a series of profound morphological and biochemical transformations. For example, differentiating lens fiber cells undergo an enormous (>100-fold) increase in cell length, accompanied by extensive remodeling of the plasma membrane system (32). Early in the differentiation process, fusion pores are established between cells, as neighboring fibers are incorporated into the lens syncytium (33). A later stage of fiber cell differentiation involves the dissolution of all intracellular organelles, a process that is thought to eliminate light-scattering particles from the light path and contribute to the transparency of the tissue (34). Any or all of these phenomena might require the developmentally regulated activation of calpains. This is consistent with our previous observation that in calpain 3 knock-out mice the transition zone is altered, suggesting a change in the differentiation program (35).In the current study, therefore, we examined the depth-dependent expression pattern and activity of calpains in the mouse lens. Fluorogenic substrates were microinjected into the intact lens to visualize calpain activity directly, and proteomic approaches were used to identify endogenous calpain substrates. The cleavage pattern of one of these, αII-spectrin, was examined in detail. Immunocytochemical and immunoblot analysis with wild type and calpain 3-null lenses indicated that αII-spectrin is a specific calpain 3 substrate in maturing lens fiber cells. Together, the data suggest that calpains are activated relatively late in fiber cell differentiation and may contribute to the remodeling of the membrane cytoskeleton that accompanies fiber cell maturation.     

Calpain对细胞骨架蛋白tau降解作用的研究(英文)

Calpain是钙依赖性中性蛋白酶 ,根据其对钙敏感性的不同 ,可分为m 和 μ calpain两型 .分别用不同浓度CaCl2 溶液孵育Wistar大鼠脑皮质匀浆液 ,并用蛋白质印迹和定量图像分析技术检测不同亚型calpain对tau蛋白的降解作用 .研究发现 :在 3 7℃用 1mmol/LCa2 孵育底物 15min ,可见tau蛋白明显降解 ,并在分子质量为 2 9ku处出现tau蛋白降解片段 ;当Ca2 浓度为 5mmol/L时 ,tau蛋白几乎全部被降解 ;这种tau蛋白降解可被calpain特异性抑制剂完全逆转 .进一步的研究发现 ,分别用 μ calpain抑制剂 (0 0 5μmol/Lcalpastatin) ,m calpain抑制剂 (10 0 μmol/LcalpaininhibitorⅣ )或总calpain抑制剂 (552 μmol/Lcalpeptin)与 1mmol/LCa2 共同孵育Wistar大鼠脑皮质匀浆液 ,Ca2 激活的tau蛋白降解分别被抑制8 6% ,92 5%和 97 8% .结果表明一定浓度的Ca2 可同时激活 μ calpain和m calpain ,这两种亚型calpain均参与降解tau蛋白 ,但m calpain的作用比 μ calpain更强     

Characterization of Ca2+-dependent neutral protease (calpain) from human blood flukes, Schistosoma mansoni

Calcium-dependent, neutral cysteine-proteases (calpain) were purified from human blood flukes, Schistosoma mansoni. The electrophoretic mobilities, Western blot analyses and high specificity to peptide inhibitors confirmed the presence of both calpain I and II in the purified preparation. The schistosome calpains were localized in the surface syncytial epithelium and underlying musculature. Using peptide inhibitors, calpain was shown to function as a mediator of the surface membrane synthetic process. Since there was also no immunological cross-reactivity between vertebrate and schistosome calpains using antibodies affinity-purified from native and recombinant schistosome calpains, this protease may be usefully investigated as forming the basis of a molecular vaccine against schistosomiasis.     

Characterization of insect cytosolic juvenile hormone binding protein gene: Highly homology with vertebrate glyoxalase domain containing protein 4

Cytosolic juvenile hormone binding protein (cJHBP) is a carrier of juvenile hormone (JH) in insects, however knowledge about its evolution and expression remains extremely limited. In this study, a gene encoding for cJHBP was isolated from the Chinese oak silkmoth Antheraea pernyi. A database search showed that the homologous sequences were present in several animal species including nematodes, insects, tunicates, fish, and mammals. The A. pernyi cJHBP had 54–85% identity with its homolog from other insects, and 58–62% identity with vertebrate glyoxalase domain containing protein 4 (Glod-4). Phylogenetic analysis supported the hypothesis that insect cJHBP shares a common ancestor with vertebrate Glod-4. RT-PCR detection showed that the cJHBP gene was expressed throughout the developmental stages and in all tested tissues of A. pernyi, which agreed with the data from Bombyx mori cJHBP and Homo sapiens Glod-4. These data suggest that insect cJHBP may play a similar function as vertebrate Glod-4.     

Mutations in CAPN1 Cause Autosomal-Recessive Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a genetically and clinically heterogeneous disease characterized by spasticity and weakness of the lower limbs with or without additional neurological symptoms. Although more than 70 genes and genetic loci have been implicated in HSP, many families remain genetically undiagnosed, suggesting that other genetic causes of HSP are still to be identified. HSP can be inherited in an autosomal-dominant, autosomal-recessive, or X-linked manner. In the current study, we performed whole-exome sequencing to analyze a total of nine affected individuals in three families with autosomal-recessive HSP. Rare homozygous and compound-heterozygous nonsense, missense, frameshift, and splice-site mutations in CAPN1 were identified in all affected individuals, and sequencing in additional family members confirmed the segregation of these mutations with the disease (spastic paraplegia 76 [SPG76]). CAPN1 encodes calpain 1, a protease that is widely present in the CNS. Calpain 1 is involved in synaptic plasticity, synaptic restructuring, and axon maturation and maintenance. Three models of calpain 1 deficiency were further studied. In Caenorhabditis elegans, loss of calpain 1 function resulted in neuronal and axonal dysfunction and degeneration. Similarly, loss-of-function of the Drosophila melanogaster ortholog calpain B caused locomotor defects and axonal anomalies. Knockdown of calpain 1a, a CAPN1 ortholog in Danio rerio, resulted in abnormal branchiomotor neuron migration and disorganized acetylated-tubulin axonal networks in the brain. The identification of mutations in CAPN1 in HSP expands our understanding of the disease causes and potential mechanisms.     

BmCalpains are involved in autophagy and apoptosis during metamorphosis and after starvation in Bombyx mori

Apoptosis and autophagy play crucial roles during Bombyx mori metamorphosis and in response to various adverse conditions, including starvation. Recently, calpain, one of the major intracellular proteases, has been reported to be involved in apoptosis and autophagy in mammals. BmATG5 and BmATG6 have been identified to mediate apoptosis following autophagy induced by 20‐hydroxyecdysone and starvation in B. mori. However, B. mori calpains and their functions remain unclear. In this study, phylogenetic analysis of calpains from B. mori, Drosophila melanogaster and Homo sapiens were performed and the results showed distinct close relationships of BmCalpain‐A/B with DmCalpain‐A/B, BmCalpain‐C with DmCalpain‐C, and BmCalpain‐7 with HsCalpain‐7. Then, the expression profiles of BmCalpains were analyzed by quantitative real‐time polymerase chain reaction, and results showed that expression of BmCalpain‐A/B, BmCalpain‐C and BmCalpain‐7 was significantly increased during B. mori metamorphosis and induced in the fat body and midgut of starved larvae, which is consistent with the expression profiles of BmAtg5, BmAtg6 and BmCaspase‐1. Moreover, the apoptosis‐associated cleavage of BmATG6 in Bm‐12 cells was significantly enhanced when BmCalpain‐A/B and BmCalpain‐7 were induced by starvation, and was partially inhibited by the inhibitor of either calpain or caspase, but completely inhibited when both types of inhibitors were applied together. Our results indicated that BmCalpains, including BmCalpain‐A/B, ‐C and ‐7, may be involved in autophagy and apoptosis during B. mori metamorphosis and after starvation, and may also contribute to the apoptosis‐associated cleavage of BmATG6.     

Calpains — An elaborate proteolytic system

Calpain is an intracellular Ca²⁺-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02). Recent expansion of sequence data across the species definitively shows that calpain has been present throughout evolution; calpains are found in almost all eukaryotes and some bacteria, but not in archaebacteria. Fifteen genes within the human genome encode a calpain-like protease domain. Interestingly, some human calpains, particularly those with non-classical domain structures, are very similar to calpain homologs identified in evolutionarily distant organisms. Three-dimensional structural analyses have helped to identify calpain's unique mechanism of activation; the calpain protease domain comprises two core domains that fuse to form a functional protease only when bound to Ca²⁺via well-conserved amino acids. This finding highlights the mechanistic characteristics shared by the numerous calpain homologs, despite the fact that they have divergent domain structures. In other words, calpains function through the same mechanism but are regulated independently. This article reviews the recent progress in calpain research, focusing on those studies that have helped to elucidate its mechanism of action. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Flow on the right side of the gastrocoel roof plate is dispensable for symmetry breakage in the frog Xenopus laevis

Leftward flow of extracellular fluid breaks the bilateral symmetry of most vertebrate embryos, manifested by the ensuing asymmetric induction of Nodal signaling in the left lateral plate mesoderm (LPM). Flow is generated by rotational beating of polarized monocilia at the posterior notochord (PNC; mammals), Kupffer's vesicle (KV; teleost fish) and the gastrocoel roof plate (GRP; amphibians). To manipulate flow in a defined way we cloned dynein heavy chain genes dnah5, 9 and 11 in Xenopus. dnah9 expression was closely related to motile cilia from neurulation onwards. Morphant tadpoles showed impaired epidermal ciliary beating. Leftward flow at the GRP was absent, resulting in embryos with loss of asymmetric marker gene expression. Remarkably, unilateral knockdown on the right side of the GRP did not affect laterality, while left-sided ablation of flow abolished marker gene expression. Thus, flow was required exclusively on the left side of the GRP to break symmetry in the frog. Our data suggest that the substrate of flow is generated within the GRP and not at its margin, disqualifying Nodal as a candidate morphogen.     

Calcium-dependent proteinases of some invertebrates and fish

In comparative-evolutionary aspect, the experimental data are considered about activity, biochemical properties, and peculiarities of structural organization of proteins of the calpain family in some invertebrates and fish. Peculiarities of calpain-like proteins of invertebrates—the predecessors of calpains of higher animals are revealed. By the example of the studied taxa, there is traced complication of the structural organization and mechanisms of control of the calpain activities, which reflects stages of molecular evolution of the protein family.     

Conserved regulatory modules in the Sox9 testis-specific enhancer predict roles for SOX,TCF/LEF,Forkhead, DMRT,and GATA proteins in vertebrate sex determination

While the primary sex determining switch varies between vertebrate species, a key downstream event in testicular development, namely the male-specific up-regulation of Sox9, is conserved. To date, only two sex determining switch genes have been identified, Sry in mammals and the Dmrt1-related gene Dmy (Dmrt1bY) in the medaka fish Oryzias latipes. In mice, Sox9 expression is evidently up-regulated by SRY and maintained by SOX9 both of which directly activate the core 1.3 kb testis-specific enhancer of Sox9 (TESCO). How Sox9 expression is up-regulated and maintained in species without Sry (i.e. non-mammalian species) is not understood. In this study, we have undertaken an in-depth comparative genomics approach and show that TESCO contains an evolutionarily conserved region (ECR) of 180 bp which is present in marsupials, monotremes, birds, reptiles and amphibians. The ECR contains highly conserved modules that predict regulatory roles for SOX, TCF/LEF, Forkhead, DMRT, and GATA proteins in vertebrate sex determination/differentiation. Our data suggest that tetrapods share common aspects of Sox9 regulation in the testis, despite having different sex determining switch mechanisms. They also suggest that Sox9 autoregulation is an ancient mechanism shared by all tetrapods, raising the possibility that in mammals, SRY evolved by mimicking this regulation. The validation of ECR regulatory sequences conserved from human to frogs will provide new insights into vertebrate sex determination.