Sox9 Promoter Research Articles

RBPJκ-Dependent Signaling Is Essential for Long-Term Maintenance of Neural Stem Cells in the Adult Hippocampus

The generation of new neurons from neural stem cells in the adult hippocampal dentate gyrus contributes to learning and mood regulation. To sustain hippocampal neurogenesis throughout life, maintenance of the neural stem cell pool has to be tightly controlled. We found that the Notch/RBPJκ-signaling pathway is highly active in neural stem cells of the adult mouse hippocampus. Conditional inactivation of RBPJκ in neural stem cells in vivo resulted in increased neuronal differentiation of neural stem cells in the adult hippocampus at an early time point and depletion of the Sox2-positive neural stem cell pool and suppression of hippocampal neurogenesis at a later time point. Moreover, RBPJκ-deficient neural stem cells displayed impaired self-renewal in vitro and loss of expression of the transcription factor Sox2. Interestingly, we found that Notch signaling increases Sox2 promoter activity and Sox2 expression in adult neural stem cells. In addition, activated Notch and RBPJκ were highly enriched on the Sox2 promoter in adult hippocampal neural stem cells, thus identifying Sox2 as a direct target of Notch/RBPJκ signaling. Finally, we found that overexpression of Sox2 can rescue the self-renewal defect in RBPJκ-deficient neural stem cells. These results identify RBPJκ-dependent pathways as essential regulators of adult neural stem cell maintenance and suggest that the actions of RBPJκ are, at least in part, mediated by control of Sox2 expression.

Abstract 3911: Dysegulation of Sox9-dependent gene expression leads to enhancement of angiogenesis and metastases in pancreatic cancer

Abstract Background: The understanding of invasion, angiogenesis and metastasis is essential for the development of new targeted molecular therapy against pancreatic cancer. The aim of this study is to analyse the regulation of differentially expressed genes in highly proangiogenic and prometastatic compared to low metastatic and low angiogenic pancreatic cancer cells Materials and methods: The transcriptome of the low metastatic pancreatic cancer cell line and its highly angiogenic and metastatic derivate L3.6pl have been compared under the normoxic and hypoxic conditions using Affymetrix HGU133plus 2.0 arrays. The hierarchic cluster analysis has been performed using SOTA algorithm. Promoters of the differentially expressed genes have been analysed using oPOSSUM 2 program. Affymetrix data have been validated via qRT-PCR and via ELISA. Chromosomal DNA containing promoters of the relevant genes has been amplified via PCR and sequenced. Results: The Affymetrix analysis revealed clusters of differentially regulated genes with regard to the cell line and the oxygen level. While VEGF expression was upregulated in FG cells under hypoxic condition, it was constitutively upregulated already under normoxic conditions in L3.6pl and no further stimulation was observed under hypoxic conditions. This regulation pattern was similar in a cluster of genes that share a promoter sequence coding for a binding site for the transcription factor Sox9. Sox9 itself shows a similar regulation, leading to the assumption that expression changes of Sox9 are involved in the different hypoxic gene expressions. Promoter analysis of Sox9 in FG and L3.6pl cells showed mutations in the promoter region in L3.6pl, while the VEGF promoter was unchanged. Further analysis has shown that this mutation is not relevant for biological function. Refined analysis revealed differential Sox9 promoter methylation as reason for observed phenomenon. Conclusions: Proangiogenic genes such as VEGF are upregulated in the more aggressive cell line L3.6pl, compared to FG. We identified a gene cluster that is constitutively upregulated in L3.6pl, independent of hypoxia. These genes are controlled by the transcription factor Sox9. A Sox9 promoter differential methylation may be the reason for the escape from hypoxic regulation in L3.6pl cells and thus may contribute to the higher angiogenic and metastatic potential. Taken together, Sox9 may be an interesting new diagnostic and therapeutic target in pancreatic cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3911.

Male fetal germ cell differentiation involves complex repression of the regulatory network controlling pluripotency

Mammalian germ cells are derived from the pluripotent epiblast and share features with pluripotent stem cells, including the expression of key genes that regulate developmental potency. The core genes Oct4, Sox2, and Nanog that regulate pluripotency in stem cells also perform important roles in regulating germ cell development and potentially in occurrence of germ line tumors in humans. Despite this, our understanding of the regulation of these genes during germ cell development remains limited. In this study we examine the regulation of pluripotency in the mouse fetal germ line. We show that male-specific methylation occurs in key functional elements of the Nanog and Sox2 promoters, and these genes are suppressed during early male germ cell differentiation. Furthermore, Oct4 translation is suppressed post-transcriptionally as germ cells differentiate down the male lineage and enter mitotic arrest. Combined, our data strongly support the conclusion that repression of the core machinery regulating pluripotency is a robust and early event involved in the differentiation of the male germ cell lineage. We hypothesize that active repression of pluripotency is required for fetal male germ cell differentiation and that failure of this mechanism may render germ cells susceptible to tumor formation.

Abstract 4224: Sox2 control breast cancer stem-like cells in a breast carcinoma cell line

Abstract Many solid tumors, including breast cancer, exhibit a functional hierarchy of cells of which only a small subpopulation of stem-like cells can give rise to the differentiated cells that make the bulk of the tumor. Recently, a method for the culture of undifferentiated human mammary epithelial cells in non-adherent “mammospheres” has been described. Mammospheres are enriched in stem/progenitor cells giving rise to all three mammary cell types. Although gene profiling was used to compare sphere cells to differentiated cells, no specific markers were described. Three main transcription factors: Oct4, Sox2 and Nanog, have probed responsible for maintaining the undifferentiated state of stem cells, particularly embryonic stem cells. Ectopic introduction of these factors into differentiated cells is capable of reprogramming these cells into embryonic stem-like cells (called iPS, induced pluripotent stem cells). It has been shown recently that one of the main regulators of oncogenic transformation, the p53 tumor supressor, controls the induction of pluripotency, thus linking the process of reprogramming with the process of tumorigenesis. By characterizing factors that control mammosphere formation in the breast carcinoma cell line MCF7 we have identified Sox2 as a gene that is up-regulated upon sphere formation. Besides, ectopic expression of Sox2 is capable of inducing augmented sphere formation, suggesting that expression of Sox2 is a key regulator of breast cancer stem cell phenotype. Up-regulation of Sox2 expression is transcriptionally controlled at the level of the RR1 Sox2 promoter enhancer which is also involved in regulation of Sox2 expression in pluripotent stem cell lines. The recent observation that the tumor suppressor p53 pathway acts as a barrier for induced pluripotency reprogramming has lead to the speculation that cancer stem cells may arise through an altered reprogramming-like mechanism. Our data suggests that Sox2 expression may be such an event. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4224.

Early growth response protein 1 acts as an activator of SOX18 promoter

Sex-determining region Y box 18 (Sox18/SOX18) gene is an important regulator of vascular development playing a role in endothelial cell specification or differentiation, angiogenesis and atherogenesis. The aim of this study was to perform comprehensive functional characterization of the human SOX18 promoter, including determination of transcription start point (tsp) and identification of control elements involved in the regulation of SOX18 gene expression, with an emphasis on angiogenesis-related transcription factors. Analyses were performed in HeLa cells, representing a tumor cell line, and in EA.hy926 cells used as an endothelial model system. We have determined unique tsp of SOX18 gene, located 172 nucleotides upstream from ATG codon. Further, we have shown that SOX18 promoter region, -726 to -89 bp relative to tsp, contains positive cis-regulatory element(s) that stimulates SOX18 promoter activity, while region -89 to+166 represents the minimal promoter. Within this region we have recognized the presence of essential element(s), positioned from -89 to +29, which harbors cluster of three putative early growth response 1 (EGR1) binding sites. By in vitro binding assays and functional analyses we have shown that these three putative binding sites are functionally relevant and sufficient for EGR1-induced SOX18 transcription. Mutations of these binding sites significantly impaired activity of the SOX18 promoter, particularly in EA.hy926 cells, indicating the importance of these regulatory elements for SOX18 promoter activity in endothelial setting. By data presented in this study, we have established SOX18 as a novel target gene regulated by EGR1 transcription factor, thus providing the first functional link between two transcription factors previously shown to be involved in the control of angiogenesis.

Epigenetic Regulation in Reproductive Medicine and Gynecologic Cancers

Epigenetic Regulation in Reproductive Medicine and Gynecologic Cancers

Hypermethylation of SOX2 Promoter in Endometrial Carcinogenesis

This paper aimed at investigating the expression and methylation profiles of SOX2, a gene coding for the stem cell-related transcription factor SOX2, in endometrial carcinomas. By methylation-specific polymerase chain reaction (MS-PCR), the methylation status of SOX2 promoter region in 72 endometrial carcinomas and 12 normal endometrial samples was examined. Methylated allele was found in 37.5% (27/72) of endometrial carcinomas but only in 8.3% (1/12) of normal endometrial, significantly more frequent in cancers (P = .0472). SOX2 mRNA level was significantly reduced in endometrial carcinoma compared with nonneoplastic endometrium (P = .045). A significant correlation between SOX2 mRNA expression and hypermethylation of SOX2 was found (P = .024). Hypermethylation of SOX2 tended to be more frequently found in type II serous or clear cell adenocarcinoma. SOX2 methylation was also significantly correlated with shorter survival of patients (P = .046). In conclusion, epigenetic mechanisms may play a crucial role on the transcriptional regulation of SOX2 and loss of SOX2 expression may be related to endometrial carcinogenesis.

PBX1 and MEIS1 up-regulate SOX3 gene expression by direct interaction with a consensus binding site within the basal promoter region

Sox3/SOX3 [SRY (sex determining region Y)-box 3] is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. We have previously reported characterization of the SOX3 promoter and demonstrated that the general transcription factors NF-Y (nuclear factor-Y), Sp1 (specificity protein 1) and USF (upstream stimulatory factor) are involved in transcriptional regulation of SOX3 promoter activity. In the present study we provide the first evidence that the TALE (three-amino-acid loop extension) transcription factors PBX1 (pre-B-cell leukaemia homeobox 1) and MEIS1 (myeloid ecotropic viral integration site 1 homologue) participate in regulating human SOX3 gene expression in NT2/D1 cells by direct interaction with the consensus PBX/MEIS-binding site, which is conserved in all mammalian orthologue promoters analysed. PBX1 is present in the protein complex formed at this site with nuclear proteins from uninduced cells, whereas both PBX1 and MEIS1 proteins were detected in the complex created with extract from RA (retinoic acid)-induced NT2/D1 cells. By functional analysis we also showed that mutations of the PBX1/MEIS1-binding sites resulted in profound reduction of SOX3 promoter responsiveness to RA. Finally, we demonstrated that overexpressed PBX1 and MEIS1 increased endogenous SOX3 protein expression in both uninduced and RA-induced NT2/D1 cells. With the results of the present study, for the first time, we have established a functional link between the TALE proteins, PBX1 and MEIS1, and expression of the human SOX3 gene. This link is of particular interest since both TALE family members and members of the SOX superfamily are recognized as important developmental regulators.

Abstract 5280: Comparing Epigenetic Modifications of Adult Progenitor Cells

Cell therapy is a promising option for treatment of cardiovascular diseases. Recent studies demonstrate that pluripotency can be induced in somatic cells by a combination of genes such as Oct4, Klf4, c-Myc and Sox2. To address whether adult progenitor cells might express some of the pluripotency genes and therefore might be more easily reprogrammable, we analyzed expression and accessibility of the promoters of pluripotency genes in adult progenitor cells. Oct4 mRNA was expressed at low level (0.2–0.5% compared to human embryonic stem cells (ESC)) in peripheral blood derived early endothelial progenitor cells (EPC), bone marrow-derived mesenchymal stem cells (MSC) and CD34+ cells, whereas neuronal stem cells (NSC) did not express Oct4. Klf4 mRNA was highly expressed in EPC, MSC and CD34+ cells but not in NSC. Interestingly, Sox2 was selectively expressed in NSC (71,65±13,01%) but was low in EPC, MSC and CD34+ (< 4% of hESC). To investigate whether epigenetic mechanisms account for the differential expression of the pluripotency genes, we analyzed promoter DNA methylation by bisulfite sequencing with 2 different primers. The Oct4 promoter showed high incidence of methylated CpG islands in Region A (EPC:82%; MSC:87%; CD34+ 71% compared to ESC: 5 %). Interestingly, the DNA methylation of the Nanog promoter in Region B was low in EPC (37%), MSC (8%), and ESC (9 %) in contrast to CD34+ cells (97%). At the level of histones, chromatine immunoprecipitation experiments demonstrated that the active marks, acetylated-histone H3 (H3Ac) and trimethylation of H3 lysine 4 (H3K4m3) were increased at the Nanog promoter in EPC and MSC. The Sox2 promoter showed a bivalent modification with active H3K4me3 and repressive H3K27m3 marks in EPC, MSC and CD34+. The Klf4 promoter exhibits high levels of active H3Ac and H3K4m3 but no repressive modification in EPC, MSC and CD34+ cells. These data indicate that adult progenitor cells express variable levels of the pluripotency genes. Modification of the epigenetic histone code, e.g. by histone demethylases, might facilitate the induction of pluripotency by remodeling the accessibility of the promoter regions. This may provide an attractive tool to reprogram adult progenitor cells to generate patient-specific stem cells.

TIP39/parathyroid hormone type 2 receptor signaling is a potent inhibitor of chondrocyte proliferation and differentiation

Tuberoinfundibular peptide of 39 residues (TIP39) is a member of the parathyroid hormone (PTH) family of peptide hormones that exerts its function by interacting with the PTH type 2 receptor (PTH2R). Presently, no known function has been attributed to this signaling pathway in the developing skeleton. We observed that TIP39 and PTH2R were present in the newborn mouse growth plate, with the receptor localizing in the resting zone whereas ligand expression was restricted exclusively in prehypertrophic and hypertrophic chondrocytes. By 8 wk of life, PTH2R, and to a lesser degree TIP39, immunoreactivity was present in articular chondrocytes. We therefore sought to investigate the role of TIP39/PTH2R signaling in chondrocytes by generating stably transfected CFK2 chondrocytic cells overexpressing PTH2R (CFK2R). TIP39 treatment of CFK2R clones in culture inhibited their proliferation by restricting cells at the G(0)/G(1) phase of the cell cycle, coupled with decreased expression and activity of cyclin-dependent kinases Cdk2 and Cdk4, while p21, an inhibitor of Cdks, was upregulated. In addition, TIP39 treatment decreased expression of differentiation markers in these cells associated with marked alterations in extracellular matrix and metalloproteinase expression. Transcription of Sox9, the master regulator of cartilage differentiation, was reduced in TIP39-treated CFK2R clones. Moreover, Sox9 promoter activity, as measured by luciferase reporter assay, was markedly diminished after TIP39 treatment. In summary, our results show that TIP39/PTH2R signaling inhibits proliferation and alters differentiation of chondrocytes by modulating SOX9 expression, thereby substantiating the functional significance of this signaling pathway in chondrocyte biology.

Sox2-positive dermal papilla cells specify hair follicle type in mammalian epidermis

The dermal papilla comprises the specialised mesenchymal cells at the base of the hair follicle. Communication between dermal papilla cells and the overlying epithelium is essential for differentiation of the hair follicle lineages. We report that Sox2 is expressed in all dermal papillae at E16.5, but from E18.5 onwards expression is confined to a subset of dermal papillae. In postnatal skin, Sox2 is only expressed in the dermal papillae of guard/awl/auchene follicles, whereas CD133 is expressed both in guard/awl/auchene and in zigzag dermal papillae. Using transgenic mice that express GFP under the control of the Sox2 promoter, we isolated Sox2(+) (GFP(+)) CD133(+) cells and compared them with Sox2(-) (GFP(-)) CD133(+) dermal papilla cells. In addition to the 'core' dermal papilla gene signature, each subpopulation expressed distinct sets of genes. GFP(+) CD133(+) cells had upregulated Wnt, FGF and BMP pathways and expressed neural crest markers. In GFP(-) CD133(+) cells, the hedgehog, IGF, Notch and integrin pathways were prominent. In skin reconstitution assays, hair follicles failed to form when dermis was depleted of both GFP(+) CD133(+) and GFP(-) CD133(+) cells. In the absence of GFP(+) CD133(+) cells, awl/auchene hairs failed to form and only zigzag hairs were found. We have thus demonstrated a previously unrecognised heterogeneity in dermal papilla cells and shown that Sox2-positive cells specify particular hair follicle types.

Helicobacter pyloriinduces gastric mucosal intestinal metaplasia through the inhibition of interleukin-4-mediated HMG box protein Sox2 expression

Helicobacter pylori is a major cause of the transdifferentiation into intestinal metaplasia that may develop gastric cancer. However, the molecular pathogenesis of this transdifferentiation is poorly understood. A SRY-related HMG box protein Sox2 is an essential transcription factor of organ development in brain, lung, and stomach. Our aim of this study was to investigate the mechanism responsible for regulation of Sox2 in host Th1-dominant response to H. pylori. Sox2 protein was immunohistochemically expressed in both human oxyntic and pyloric glands with H. pylori infection, but not in intestinal metaplasia. Western immunoblotting of gastric epithelial cell lines showed that IL-4, a Th2-related cytokine, dose dependently enhanced Sox2 expression among H. pylori infection-mediated cytokines. Small changes of Sox2 expression were observed after each treatment with IFN-gamma, IL-1beta, or TNF-alpha. IL-4-mediated Sox2 induction was suppressed by the inhibition of STAT6 activation with STAT6 RNA interference, and electrophoretic mobility shift assay indicated that activation of the Sox2 promoter by IL-4 occurred through the action of STAT6. Furthermore, H. pylori and IFN-gamma inhibited the phosphorylation of STAT6, resulting in the suppression of IL-4-mediated Sox2 expression. Immunohistochemical analyses showed significantly the suppressed STAT6 activity in H. pylori-infected human gastric mucosa. Additionally, downregulation of Sox2 by knockdown experiments led to intestinal phenotype with expressions of Cdx2 and MUC2. These results suggest that H. pylori and IFN-gamma interfere with the differentiation into oxyntic and pyloric glands by the downregulation of Sox2 on IL-4/STAT6 signaling, which may contribute to the transdifferentiation into intestinal metaplasia.

CARM1 is Required in Embryonic Stem Cells to Maintain Pluripotency and Resist Differentiation

Histone H3 methylation at R17 and R26 recently emerged as a novel epigenetic mechanism regulating pluripotency in mouse embryos. Blastomeres of four-cell embryos with high H3 methylation at these sites show unrestricted potential, whereas those with lower levels cannot support development when aggregated in chimeras of like cells. Increasing histone H3 methylation, through expression of coactivator-associated-protein-arginine-methyltransferase 1 (CARM1) in embryos, elevates expression of key pluripotency genes and directs cells to the pluripotent inner cell mass. We demonstrate CARM1 is also required for the self-renewal and pluripotency of embryonic stem (ES) cells. In ES cells, CARM1 depletion downregulates pluripotency genes leading to their differentiation. CARM1 associates with Oct4/Pou5f1 and Sox2 promoters that display detectable levels of R17/26 histone H3 methylation. In CARM1 overexpressing ES cells, histone H3 arginine methylation is also at the Nanog promoter to which CARM1 now associates. Such cells express Nanog at elevated levels and delay their response to differentiation signals. Thus, like in four-cell embryo blastomeres, histone H3 arginine methylation by CARM1 in ES cells allows epigenetic modulation of pluripotency.

Regulation of CDX2 expression in esophageal adenocarcinoma

Reflux of acidic gastric contents and bile acids into the lower esophagus has been identified to have a central role in esophageal malignancy and is reported to upregulate caudal-related homologue 2 (CDX2), a regulatory gene involved in embryonic development and axial patterning of the alimentary tract. The aim of this study was to characterize the expression of CDX2 in a well-defined series of human esophageal tissues, comprising reflux-induced esophagitis, premalignant Barrett esophagus (BE), and primary esophageal adenocarcinoma (EADC). To explore potential molecular regulatory mechanisms, we also studied the expression of beta-catenin, SOX9, and CDX2 promoter methylation in esophageal tissues, in addition to the effect of bile acids and nitric oxide (NO) on CDX2 expression in the normal human esophageal cell line Het1A. Relative to matched normal esophageal epithelia, CDX2 was overexpressed in esophagitis (37% for RNA; cytoplasmic immunoreactivity in 48% of tissues), a high proportion (91%) of BE tissues, and in EADC (57% for RNA; cell nuclear immunopositivity in 80%). An association with beta-catenin expression was seen, but not with SOX9 or CDX2 promoter methylation. In Het1A cells, CDX2 was upregulated following exposure to bile acids and NO, alone and in combination. These results further implicate CDX2 and beta-catenin in the molecular pathogenesis of human EADC. The observed synergistic effect of NO on the efficacy of bile acid-induction of CDX2 suggests a novel role for NO in modulating the development of the Barrett phenotype and esophageal adenocarcinogenesis.

C017 Expression of proteins p16INK4a, p53 and Bmi-1 in hematopoietic stem cells of patients with MDS. The role of cellular senescence

Increasing evidences indicated that Long noncoding RNAs (lncRNAs) played pivotal roles during tumorigenesis in multiple types of cancers, including breast cancer. This study aimed to investigate the role and function of long noncoding RNA PVT1 in the progression of breast cancer and explore the transcription factor which contributes to the regulation of PVT1 in breast cancer. Our results indicated that the expression of PVT1 was significantly upregulated in breast cancer tissues, compared with adjacent normal tissues (ANTs). And the increasing expression of PVT1 was associated with clinical stage, lymph nodes metastasis, and overall survival in breast cancer patients. Using computational screening, a transcriptional factor binding site was found between SOX2 and PVT1 promoter and the interaction between each other was further verified by chromatin immunoprecipitation (ChIP) analysis. In addition, ectopically overexpression of SOX2 can enhance breast cancer cell proliferation and invasion, while knockdown of SOX2 or PVT1 can severely attenuate this effect both in epithelial and mesenchymal types of breast cancer cells. Further evidences confirmed that overexpression of SOX2 can promote breast cancer cell EMT process; while silencing SOX2 or PVT1 expression can undermine this effect. These data suggest that elevated expression of SOX2 can activate lncRNA PVT1 expression promoted breast cancer tumorigenesis and progression. PVT1 may be a prognostic predictive biomarker for breast cancer, and the interaction of PVT1-SOX2 could be a therapeutic target in breast cancer.

Role of the sodium‐dependent phosphate co‐transporter PiT‐1 in mouse development and vascular calcification

Vascular calcification is correlated with morbidity and mortality in coronary artery disease, diabetes and chronic kidney disease. In vitro, elevated phosphate levels induced smooth muscle cell calcification in a PiT‐dependent manner. To determine the in vivo role of PiT‐1, a mouse conditional allele was generated in which loxP sites flanked exons 3 and 4 of the PiT‐1 gene. Recombination occurred in the presence of Cre driven by the ubiquitous Sox‐2 promoter, and the resulting PiT‐1Δ allele would produce a nonfunctional protein with a shift in the reading frame and production of a stop codon at amino acid 121. Wild‐type and PiT‐1Δ/+ mice were produced at expected Mendelian ratios, but no PiT‐1Δ/Δ mice were born. Examination of early development revealed that the PiT‐1Δ/Δ embryos were arresting at embryonic stage 10.5 and most were resorbed by E13.5. Affected embryos displayed cardiac defects and an under‐vascularized yolk sac as compared to siblings. Semi‐quantitative RT‐PCR analysis confirmed that PiT‐1Δ/Δ embryos expressed a truncated form of mRNA, but only embryos harboring at least one wild‐type allele expressed wild‐type mRNA. Thus, we have successfully generated conditional and null PiT‐1 alleles and confirmed that PiT‐1 is required for normal mouse embryonic development.

Transcriptional induction of SOX9 by NF-κB family member RelA in chondrogenic cells

Although SOX9 is a key molecule for chondrogenic differentiation, little is known about the upstream signal. The present study attempted to identify transcription factors to induce SOX9 expression and examined the mechanism. Sequences of about 1 kb of 5'-end flanking regions were compared between human and mouse SOX9 genes. In vivo localization was examined by immunohistochemistry in the limb cartilage of fetal mice. Promoter activities of the SOX9, SOX6, and type II collagen (COL2A1) genes were determined in human non-chondrogenic HeLa cells and mouse chondrogenic ATDC5 cells transfected with a luciferase-reporter gene containing the promoter fragments. Protein-DNA binding was examined by electrophoretic mobility shift and chromatin immunoprecipitation assays. The chondrogenic differentiation was assessed by endogenous SOX9, SOX6, and COL2A1 mRNA levels, and by Alcian blue staining and alkaline phosphatase activity. Among transcription factors whose binding motifs were identified in the highly-conserved regions between human and mouse SOX9 promoters, a nuclear factor kappa B (NF-kappaB) member RelA strongly activated the promoter activity. RelA and SOX9 were co-localized in the limb cartilage. Deletion, mutagenesis, and tandem-repeat analyses identified the core region responsive to RelA at the NF-kappaB binding motif to be around -250bp of the human SOX9 promoter, and this was confirmed to show specific binding to RelA. RelA induced the chondrogenic differentiation parameters in HeLa and ATDC5 cells. We have identified RelA as a transcriptional factor for SOX9 induction and chondrogenic differentiation via binding to an NF-kappaB binding motif in the SOX9 promoter.

Conditional Inactivation of TACE by a Sox9 Promoter Leads to Osteoporosis and Increased Granulopoiesis via Dysregulation of IL-17 and G-CSF

The TNF-alpha converting enzyme (TACE/ADAM17) is involved in the proteolytic release of the ectodomain of diverse cell surface proteins with critical roles in development, immunity, and hematopoiesis. As the perinatal lethality of TACE-deficient mice has prevented an analysis of the roles of TACE in adult animals, we generated mice in which floxed Tace alleles were deleted by Cre recombinase driven by a Sox9 promoter. These mutant mice survived up to 9-10 mo, but exhibited severe growth retardation as well as skin defects and infertility. The analysis of the skeletal system revealed shorter long bones and prominent bone loss, characterized by an increase in osteoclast and osteoblast activity. In addition, these mice exhibited hypercellularity in the bone marrow and extramedullary hematopoiesis in the spleen and liver. Flow cytometric analysis of the bone marrow cells showed a sharp increase in granulopoiesis and in the population of c-Kit-1(+) Sca-1(+) lineage(-) cells, and a decrease in lymphopoiesis. Moreover, we found that serum levels of IL-17 and G-CSF were significantly elevated compared with control littermates. These findings indicate that TACE is associated with a regulation of IL-17 and G-CSF expression in vivo, and that the dysregulation in G-CSF production is causally related to both the osteoporosis-like phenotype and the defects in the hematopoietic system.

SWI/SNF-Brg1 Regulates Self-Renewal and Occupies Core Pluripotency-Related Genes in Embryonic Stem Cells

The SWI/SNF-Brg1 chromatin remodeling protein plays critical roles in cell-cycle control and differentiation through regulation of gene expression. Loss of Brg1 in mice results in early embryonic lethality, and recent studies have implicated a role for Brg1 in somatic stem cell self-renewal and differentiation. However, little is known about Brg1 function in preimplantation embryos and embryonic stem (ES) cells. Here we report that Brg1 is required for ES cell self-renewal and pluripotency. RNA interference-mediated knockdown of Brg1 in blastocysts caused aberrant expression of Oct4 and Nanog. In ES cells, knockdown of Brg1 resulted in phenotypic changes indicative of differentiation, downregulation of self-renewal and pluripotency genes (e.g., Oct4, Sox2, Sall4, Rest), and upregulation of differentiation genes. Using genome-wide promoter analysis (chromatin immunoprecipitation) we found that Brg1 occupied the promoters of key pluripotency-related genes, including Oct4, Sox2, Nanog, Sall4, Rest, and Polycomb group (PcG) proteins. Moreover, Brg1 co-occupied a subset of Oct4, Sox2, Nanog, and PcG protein target genes. These results demonstrate an important role for Brg1 in regulating self-renewal and pluripotency in ES cells.

002. REGULATION OF PLURIPOTENCY AND CELL CYCLE IN FETAL GERM CELLS

The germ cell lineage is unique in that it must ensure that the genome retains the complete developmental potential (totipotency) that supports development in the following generation. This is achieved through a number of mechanisms that prevent the early germ cell lineage from somatic differentiation and promote the capactity for functional totipotency. Part of this process involves the retained germ line expression of key genes that regulate pluripotency in embryonic stem cells, embryonic germ cells and some embryonal carcinoma cells, the stem cells of testicular tumours. Despite this, germ cells are not intrinsically pluripotent and must differentiate along the male or female pathways, a process which requires commitment of the bi-potential primordial germ cells to the spermatogenic (male) pathway and their entry into mitotic arrest, or to the oogenic pathway (females) and entry into meiosis. This involves robust regulation of regulatory networks controlling pluripotency, cell cycle and sex specific differentiation. Our work aims to further understand the mechanisms controlling differentiation, pluripotency and cell cycle in early male and female germ cells. Our data shows that mitotic arrest of male germ cells involves strict regulation of the G1-S phase check-point through the retinoblastoma protein. In addition, suppression of pluripotency in differentiating male germ cells involves post-transcriptional regulation of OCT4, transcriptional regulation of Sox2 and Nanog and methylation of the Sox2 and Nanog promoters. Further understanding of these processes promises to lead to a greater understanding of the molecular mechanisms underlying control of pluripotency, cell cycle and differentiation in the germ line and the initiation of germ cell derived testis tumours.