Sox Protein Research Articles

Lessons from the Pros

Virology Human cells contain tens of thousands of protein-encoding genes that are transcribed into a much larger numbers of mRNAs. Viruses, on the other hand, are far simpler, carrying with them only a handful of genes and proteins, yet they have developed countless ways of hijacking specific host cellular functions for their own benefit. Discovering these survival mechanisms often offers fascinating insights into normal host cell biology. Eukaryotes use sophisticated ways of regulating gene expression, including polyadenylating the 3′ end of mRNAs to enhance their stability for eventual translation into protein, and possibly also to promote mRNA degradation. Some herpesviruses, including Kaposi's sarcoma–associated herpesvirus (KSHV), manipulate host cells by promoting the widespread destruction of cellular mRNAs. KSHV achieves this via its Sox protein, and Lee and Glaunsinger show that human cells expressing the viral Sox protein contain mRNAs with unusually long poly(A) tails (hyperadenylation), which was mediated by the host enzyme poly(A) polymerase II. This effect of viral Sox was linked to its ability to accelerate mRNA turnover, suggesting that the virus induces host mRNA degradation by modulating poly(A) length. PLoS Biol . 7 , e1000107 (2009).

Sox3: A transcription factor for Cyp19 expression in the frog Rana rugosa

Cyp19 is expressed at a high level in the gonad of the female tadpole of the frog Rana rugosa during sex determination. To identify sequence elements important for expression of Cyp19, we isolated a genomic clone (∼ 40 kbp) carrying R. rugosa Cyp19 and analyzed the nucleotide sequence of the 5′-flanking region to search for potential transcription factor binding sites. Sox (SRY-related HMG box) protein and Sf1 binding sites were found in the ovary-specific promoter region of Cyp19. Because Sox3 is located on the sex chromosome in R. rugosa, we conducted the luciferase reporter assay in Xenopus A6 cells using the promoter region. Sox3 drove the reporter gene in the cells, but Sf1 did not. When sequential deletion of the 2.7 kbp Cyp19-promoter region was undertaken, a fragment spanning nucleotides − 191 to + 48 was sufficient to drive the transcription of the reporter gene. In site-directed mutagenesis, the binding site at − 57 in the region was critical for Sox3 responsiveness. Sox3 lacking the HMG box had no ability to promote Cyp19 transcription. In addition, a chromatin immunoprecipitation (ChIP) assay showed that DNA fragments were enriched 8-fold, as determined by real-time PCR, when chromatin was immunoprecipitated with the anti-His antibody against His-tagged Sox3. The results, taken together, suggest that Sox3 activates Cyp19 transcription by its direct binding to the binding site of the Cyp19 promoter region. Sox3 appears to be a factor that directs indifferent gonads to develop into an ovary in R. rugosa.

Aberrant Herpesvirus-Induced Polyadenylation Correlates With Cellular Messenger RNA Destruction

Author SummaryDuring viral infection, many essential cellular functions are targets for viral manipulation, yet aside from RNA interference, surprisingly few examples of viruses disrupting RNA turnover have been documented. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that induces widespread cellular messenger RNA destabilization during lytic infection. The viral protein SOX is a critical effector of this phenotype, yet it lacks ribonuclease activity, so presumably it targets cellular factors governing RNA stability. Here, we show that SOX stimulates host mRNA destruction via a unique mechanism involving polyadenylation. During SOX expression, newly formed messages have longer than normal poly(A) tails, leading to their retention in the nucleus. Coincident with this hyperadenylation, poly(A) binding protein (PABPC) is relocalized from the cytoplasm to the nucleus. PABPC has prominent roles in translation, messenger RNA stabilization, and quality control in the cytoplasm; we find its nuclear relocalization by SOX correlates with enhanced mRNA turnover in the cytoplasm. Thus, KSHV appears to have evolved distinct polyadenylation-linked mechanisms to target both new messages in the nucleus and preexisting cytoplasmic messages for destruction, thereby effectively inhibiting cellular gene expression.

Having it both ways: Sox protein function between conservation and innovation.

Transcription factors of the Sox family arose around the advent of multicellularity in animals, arguing that their ability to regulate the expression of extracellular matrix, cell adhesion and signaling molecules may have been instrumental in the generation of metazoans. In particular, during vertebrate evolution, the Sox family experienced a phase of expansion that led to the appearance of groups of highly homologous Sox proteins and the division of existing Sox protein functions among group members. It furthermore allowed Sox transcription factors to acquire numerous novel functions. These past events of subfunctionalization and neofunctionalization can still be recognized today in all groups of the Sox family. They have led to partial functional redundancies, but also to interesting species-specific variations in the developmental roles of Sox proteins as shown here for the SoxB and the SoxE groups.

MammalianSox15Gene: Promoter Analysis and Implications for Placental Evolution

Sox15 belongs to the Sox (Sry-type HMG box) protein family, which is involved in placental development and muscle regeneration. Previously, we showed that the Sox15 gene is highly expressed in the trophoblast giant cells of the mouse placenta. To elucidate the molecular mechanisms of the tissue-dependent transcription of the gene, we isolated approximately 2.2 kb of the 5'-flanking sequence upstream of the transcription initiation site and used it to construct luciferase reporter plasmids. A variety of cell lines, including trophoblast stem (TS) cells, placenta-derived Rcho-1 cells, and myoblast C2C12 cells, required the same 5'-flanking sequence, from -109 to -8, for basal promoter activity. In contrast, the sequences from -297 to -149 and from -148 to -110 were required for cell-type-specific promoter activity in myoblast-derived C2C12 cells and placenta-derived Rcho-1 and TS cells, respectively. These results suggest that the region from -297 to -8 of the Sox15 gene contains three distinct cis-elements that respectively control placenta-specific, myoblast-specific, and common basal expression. We also searched for Sox15 ortholog(s) in the genome databases of various vertebrate species. The results indicated that the three regulatory promoter sequences of the Sox15 genes were conserved among eutherian mammals during vertebrate evolution. Interestingly, the marsupial opossum gene that is closest to Sox15 appeared to be a pseudogene. These findings indicate that Sox15 may have been involved in placental evolution.

Partners in Crime

See related article, pages 12–15 Vertebrates have evolved 2 types of blood vessels—arteries and veins—that function to supply oxygen and nutrients and remove cellular waste. To efficiently accomplish these specialized functions, arteries and veins have developed distinct morphological and molecular differences. As a result, the endothelial cells that compose arteries and veins are vastly different in their biochemical and cellular properties. During development, the nascent endothelial cells generated from hemangioblasts or angioblasts1,2 undergo tightly regulated specification and differentiation processes to adopt either the arterial or venous endothelial fate. Differentiated endothelial cells migrate and aggregate to form either arteries or veins according to their adopted fate.3 Dysregulation of these regulated events often results in devastating consequences. For instance, failure to segregate arterial and venous endothelial cells causes potentially fatal clinical conditions such as arteriovenous malformation, hereditary hemorrhagic telangiectasia, and cerebral cavernous malformation.4 Significant progress has been made in understanding how nascent endothelial cells adopt the arterial fate, resulting in identification of several key signaling molecules and their intracellular transducers involved in the specification of arterial endothelial cells.5 However, transcription factors that function …

Modulation ofCaenorhabditis elegansTranscription Factor Activity by HIM-8 and the Related Zinc-Finger ZIM Proteins

The previously reported negative regulatory activity of HIM-8 on the Sox protein EGL-13 is shared by the HIM-8-related ZIM proteins. Furthermore, mutation of HIM-8 can modulate the effects of substitution mutations in the DNA-binding domains of at least four other transcription factors, suggesting broad regulatory activity by HIM-8.

169 INVITED Recent research in opioid treated cancer pain

Investigations into pigment cell biology have relied on the ability to culture both murine and human melanocytes, numerous melanoma cell lines and more recently, murine and human melanoblasts. Melanoblast culture requires medium supplemented with a range of growth factors including Stem Cell Factor, Endothelin-3 and Fibroblast Growth Factor-2, withdrawal of which causes the cells to differentiate into melanocytes. Using the human melanoblast culture system, we have now examined the expression and/or DNA binding activity of several transcription factors implicated in melanocytic development and differentiation. Of these, the POU domain factor BRN2 and the SOX family member SOX10 are both highly expressed in unpigmented melanocyte precursors but are down-regulated upon differentiation. In contrast, the expression levels of the previously described MITF and PAX3 transcription factors remain relatively constant during the melanoblast–melanocyte transition. Moreover, BRN2 ablated melanoma cells lack expression of SOX10 and MITF but retain PAX3. A novel finding implicates a second SOX protein, SOX9, as a potential melanogenic transcriptional regulator, as its expression level is increased following the down-regulation of BRN2 and SOX10 in differentiated melanoblasts. Our results suggest that a complex network of transcription factor interactions requiring proper temporal coordination is necessary for acquisition and maintenance of the melanocytic phenotype.

Host Transcript Accumulation during Lytic KSHV Infection Reveals Several Classes of Host Responses

Lytic infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with an extensive shutoff of host gene expression, mediated chiefly by accelerated mRNA turnover due to expression of the viral SOX protein. We have previously identified a small number of host mRNAs that can escape SOX-mediated degradation. Here we present a detailed, transcriptome-wide analysis of host shutoff, with careful microarray normalization to allow rigorous determination of the magnitude and extent of transcript loss. We find that the extent of transcript reduction represents a continuum of susceptibilities of transcripts to virus-mediated shutoff. Our results affirm that the levels of over 75% of host transcripts are substantially reduced during lytic infection, but also show that another ∼20% of cellular mRNAs declines only slightly (less than 2-fold) during the course of infection. Approximately 2% of examined cellular genes are strongly upregulated during lytic infection, most likely due to transcriptional induction of mRNAs that display intrinsic SOX-resistance.

Long-Term Erythropoietin Therapy Does Not Affect Metalloproteinases and Their Inhibitor Levels, Oxidative Stress and Inflammation in Hemodialyzed Patients

Background: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play an important role in the atherosclerosis. Recombinant human erythropoietin (EPO) has become widely used to treat anemic hemodialyzed (HD) patients; however, an increased mortality has been reported for HD patients with cardiovascular disease when randomly assigned to normal hematocrit by EPO. Therefore, we conducted a study examining the effect of EPO on MMPs/TIMPs system, oxidative stress and inflammation in these patients. Methods: Assessment of MMP-2, MMP-9, TIMP-1 and TIMP-2 were performed in 20 stable HD patients and 15 healthy controls. Additionally, the effects of EPO on malondialdehyde (MDA) – a marker of SOX and C-reactive protein (CRP) levels – as a marker of inflammation were also investigated. Of the 20 patients, 10 were receiving EPO therapy [HD-EPO(+)] for 12 months or more and 10 were not receiving EPO therapy [HD-EPO(–)]. Both groups were not receiving iron supplementation. Results: All parameters, with the exception of MMP-9, were lower in the healthy subjects compared with the HD subjects, irrespective of EPO administration. There was no difference in MMPs/TIMPs system, MDA and C-reactive levels between HD-EPO(+) and HD-EPO(–) patients. Conclusion:Erythropoietin therapy did not influence MMPs/TIMPs system, inflammation, or SOX in a low-risk HD patient population, in the absence of concomitant iron supplementation and mean Hg levels within target.

The genetic programme of pancreatic β‐cells: basic science for the development of β‐cell therapy

The European Molecular Biology Organization/Federation of European Biochemical Societies Workshop on Programming Pancreatic β‐Cells took place in El Perello, Spain, between 18 and 22 October 2006, and was organized by J. Ferrer, F. X. Real and P. L. Herrera. The meeting was also sponsored by Novo Nordisk and the Juvenile Diabetes Research Foundation. ![][1] β‐cells are located in the pancreatic islets of Langerhans and produce insulin in response to a rise in blood glucose; the destruction or dysfunction of β‐cells results in diabetes. Although insulin therapy has proved useful for the treatment of this disease, it is hoped that β‐cell‐replacement therapy will be even more effective; however, there is a limited supply of donor β‐cells. Alternative sources of β‐cells for implantation are therefore required and their activation in situ needs to be controlled. Possible methods to recreate the development, regeneration and growth of pancreatic β‐cells can be learned from naturally occurring processes, such as embryonic β‐cell development and adaptation of the β‐cell mass in adult life to obesity or other stress conditions. During this workshop, scientists from diverse research fields were brought together to foster discussions on the basic mechanisms of pancreatic development, pancreatic regeneration and growth, transdifferentiation and programmed embryonic stem (ES)‐cell differentiation. In recent years research has amassed an impressive set of data describing the mechanisms that control endoderm development, the subsequent morphogenesis of the pancreas and the differentiation of pancreatic cells (Murtaugh & Melton, 2003; Collombat et al , 2006; Jensen, 2004). Concurrently, research has also progressed on the transcriptional regulation involved. The number of known transcription factors involved in β‐cell development continues to increase. M. Sander (Irvine, CA, USA) has investigated the expression of SRY‐box‐containing (Sox) protein family members during pancreatic development, and found that Sox9 is expressed in a subset of progenitor cells. Conditional inactivation … [1]: /embed/graphic-1.gif

Maternal expression and function of the Drosophila sox gene Dichaete during oogenesis

Members of the Sox family of DNA-binding HMG domain proteins have been shown to regulate gene transcription in a wide range of developmental processes, including sex determination, neurogenesis, and chondrogenesis. However, little is known about their potential functions in developing germline tissues. In Drosophila, the Sox protein Dichaete (a.k.a., Fish-hook) is a member of the SoxB subgroup whose HMG domain shares strong sequence similarity to that of vertebrate Sox2. Dichaete exhibits dynamic expression in embryonic and larval stages and has pleiotropic functions in a variety of tissues. In this study, we extend analyses of Dichaete function and show that expression of Dichaete protein is detected in the developing oocyte during early to mid stages of oogenesis. Strikingly, Dichaete exhibits cytoplasmic distribution and is not detected in the oocyte nucleus. Germline mosaic analyses revealed that the Dichaete gene has maternal functions that influence dorsal/ventral patterning of the egg chamber. Dichaete mutant eggs exhibit defects in formation of the dorsal appendages, differentiation of dorsal/anterior follicle cells, and mislocalization of Gurken protein and gurken mRNA. Dichaete protein was shown to possess RNA-binding capabilities, suggesting a direct post-transcriptional role in regulating RNA functions.

Replacement of the Sox10 transcription factor by Sox8 reveals incomplete functional equivalence

Sox8 and Sox10 are two closely related transcription factors of the Sox protein family with overlapping expression patterns during development. They are believed to perform very similar functions because several developmental processes, including enteric nervous system development and oligodendrocyte differentiation, are regulated by both Sox proteins. To analyze the extent of functional equivalence between the two Sox proteins, we employed targeted mutagenesis to replace Sox10 with Sox8 in the mouse. In mice that expressed Sox8 instead of Sox10, Sox10 deficiency was phenotypically rescued to different extents in affected tissues. Whereas development of glial cells and neurons in the sensory and sympathetic parts of the peripheral nervous system was almost normal when Sox10 was replaced by Sox8, melanocyte development was as defective as in Sox10-deficient mice. The ability of Sox8 to rescue the defects in enteric nervous system development and oligodendrocyte differentiation of Sox10-deficient mice was limited. We conclude that the extent of functional equivalence depends on the tissue and that, despite their relatedness, Sox8 and Sox10 have more unique functions than previously appreciated.

C. elegans HIM-8 functions outside of meiosis to antagonize EGL-13 Sox protein function

C. elegans HIM-8 functions outside of meiosis to antagonize EGL-13 Sox protein function

Sox15 enhances trophoblast giant cell differentiation induced by Hand1 in mouse placenta

Some members of the Sry-type HMG box (Sox) protein family play important roles in embryogenesis as transcription factors. Here, we report that Sox15 transcripts were much more abundant in mouse placenta than in the fetus, the yolk sac, or several adult tissues. In situ hybridization analysis of the mouse E8.0 conceptus indicated that Sox15 mRNA was predominantly expressed in the trophoblast giant cells of the placenta. We also observed that the amount of Sox15 mRNA dramatically increased during the differentiation of mouse trophoblast stem cells. Ectopic expression of Sox15 in Rat choriocarcinoma cells enhanced the giant cell differentiation induced by a bHLH transcription factor, Hand1. Binding experiments in cotransfected 293 T cells and in vitro revealed that Sox15 interacted with Hand1. We next examined the effects of this interaction on the transcriptional activity of Hand1 and Sox15 using the luciferase reporter assay. Overexpression of Hand1 repressed the Sox15-driven reporter expression, but Sox15 enhanced the Hand1-driven transcription. This enhancement required both the Hand1-binding region and the transactivation domain of Sox15. These results may suggest that the increased transcriptional activity of Hand1 caused by Sox15 might promote the transcription of the target gene resulting in the trophoblast giant cell differentiation in the mouse placenta.

C. elegans HIM-8 functions outside of meiosis to antagonize EGL-13 Sox protein function

egl-13 encodes a Sox domain protein that is required for proper uterine seam cell development in Caenorhabditis elegans. We demonstrate that mutations of the C2H2 zinc fingers encoded by the him-8 (high incidence of males) gene partially suppress the egg-laying and connection-of-gonad morphology defects caused by incompletely penetrant alleles of egl-13. him-8 alleles have previously characterized recessive effects on recombination and segregation of the X chromosome during meiosis due to failure of X chromosome homolog pairing and subsequent synapsis. However, we show that him-8 alleles are semi-dominant suppressors of egl-13, and the semi-dominant effect is due to haplo-insufficiency of the him-8 locus. Thus, we conclude that the wild-type him-8 gene product acts antagonistically to EGL-13. Null alleles of egl-13 cannot be suppressed, suggesting that this antagonistic interaction most likely occurs either upstream of or in parallel with EGL-13. Moreover, we conclude that suppression of egl-13 is due to a meiosis-independent function of him-8 because suppression is observed in mutants that have severely reduced meiotic germ cell populations and suppression does not depend on the function of him-8 in the maternal germ line. We also show that the chromosomal context of egl-13 seems important in the him-8 suppression mechanism. Interactions between these genes can give insight into function of Sox family members, which are important in many aspects of metazoan development, and into functions of him-8 outside of meiosis.

Differential Roles for Sox15 and Sox2 in Transcriptional Control in Mouse Embryonic Stem Cells

Sox family transcription factors play essential roles in cell differentiation, development, and sex determination. Sox2 was previously thought to be the sole Sox protein expressed in mouse embryonic stem (ES) cells. Sox2 associates with Oct3/4 to maintain self-renewal of ES cells. In the current study, digital differential display identified transcripts for an additional Sox family member, Sox15, enriched in mouse ES cells. Reverse transcription-PCR confirmed that Sox15 expression is highest in undifferentiated ES cells and repressed upon differentiation. Sox15 is expressed at low levels in several tissues, including testis and muscle. In vitro studies showed that Sox15, like Sox2, associated with Oct3/4 on DNA sequences containing the octamer motif and Sox-binding site. Gel mobility shift assays and SELEX analyses showed that Sox15 binds similar DNA sequences as Sox2 but with weaker affinity. In contrast to the early embryonic lethality observed in Sox2-null mice, Sox15-null ES cells and mice were grossly normal. DNA microarray analyses revealed that Otx2, Ctgf, Ebaf, and Hrc are dysregulated in Sox15-null ES cells, however. Chromatin immunoprecipitation showed that Sox15, but not Sox2, bound to a Sox consensus binding site within the Hrc gene. Taken together, these data demonstrate differential roles for Sox15 and Sox2 in transcriptional control in mouse ES cells.

The Exonuclease and Host Shutoff Functions of the SOX Protein of Kaposi's Sarcoma-Associated Herpesvirus Are Genetically Separable

The Kaposi's sarcoma-associated herpesvirus (KSHV) SOX protein, encoded by ORF37, promotes shutoff of host cell gene expression during lytic viral replication by dramatically impairing mRNA accumulation. SOX is the KSHV homolog of the alkaline exonuclease of other herpesviruses, which has been shown to function as a DNase involved in processing and packaging the viral genome. Although the exonuclease activity of these proteins is widely conserved across all herpesviruses, the host shutoff activity observed for KSHV SOX is not. We show here that SOX expression sharply reduces the half-life of target mRNAs. Extensive mutational analysis reveals that the DNase and host shutoff activities of SOX are genetically separable. Lesions affecting the DNase activity cluster in conserved regions of the protein, but residues critical for mRNA degradation are not conserved across the viral family. Additionally, we present evidence suggesting that the two different functions of SOX occur within distinct cellular compartments-DNase activity in the nucleus and host shutoff activity in the cytoplasm.

Co-expression of SOX9 and SOX10 during melanocytic differentiation in vitro

Investigations into pigment cell biology have relied on the ability to culture both murine and human melanocytes, numerous melanoma cell lines and more recently, murine and human melanoblasts. Melanoblast culture requires medium supplemented with a range of growth factors including Stem Cell Factor, Endothelin-3 and Fibroblast Growth Factor-2, withdrawal of which causes the cells to differentiate into melanocytes. Using the human melanoblast culture system, we have now examined the expression and/or DNA binding activity of several transcription factors implicated in melanocytic development and differentiation. Of these, the POU domain factor BRN2 and the SOX family member SOX10 are both highly expressed in unpigmented melanocyte precursors but are down-regulated upon differentiation. In contrast, the expression levels of the previously described MITF and PAX3 transcription factors remain relatively constant during the melanoblast–melanocyte transition. Moreover, BRN2 ablated melanoma cells lack expression of SOX10 and MITF but retain PAX3. A novel finding implicates a second SOX protein, SOX9, as a potential melanogenic transcriptional regulator, as its expression level is increased following the down-regulation of BRN2 and SOX10 in differentiated melanoblasts. Our results suggest that a complex network of transcription factor interactions requiring proper temporal coordination is necessary for acquisition and maintenance of the melanocytic phenotype.

Circulating β-chemokines and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 system in hemodialyzed patients – Role of oxidative stress

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), β-chemokines, increased oxidative stress (SOX) and inflammation have been implicated as important factors in atherosclerosis and vascular remodeling. We hypothesized the possible roles of β-chemokines [monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory proteins (MIP-1α, MIP-1β) and regulated upon activation, normal T-cell expressed and secreted (RANTES)] as regulators of the metabolism of the vascular extracellular matrix in conditions of increased SOX in hemodialysis (HD) patients. We compared pre-dialysis levels of MMP-9/TIMP-1 system, β-chemokines, Cu/Zn superoxide dismutase (Cu/Zn SOD) as a marker of SOX and C-reactive protein (CRP) as a marker of inflammation in HD patients with and without cardiovascular disease (CVD) to those of controls. HD patients, particularly those with CVD, showed a significant increase in values of Cu/Zn SOD, CRP, TIMP-1, TIMP-1/MMP-9 ratio, MCP-1 and MIP-1β, whereas RANTES levels were lower than in the controls. The levels of MIP-1α as well as MMP-9 in all HD groups were similar to the controls. The positive correlations were observed between the MMP-9/TIMP-1 system and β-chemokines, SOX and inflammation in whole HD group and in the subgroup with CVD. Multivariate analysis showed that the duration of dialysis followed by Cu/Zn SOD, MIP-1α and β levels were the significant positive predictors of TIMP-1. In conclusion, our data show that MMP-9/TIMP-1 system and β-chemokines could cooperate in conditions of elevated SOX, which ultimately predisposes hemodialysis patients to accelerated atherosclerosis.