Sox9 Activity Research Articles

Serinc5 Regulates Sequential Chondrocyte Differentiation by Inhibiting Sox9 Function in Pre-Hypertrophic Chondrocytes.

The growth plate is the primary site of longitudinal bone growth with chondrocytes playing a pivotal role in endochondral bone development. Chondrocytes undergo a series of differentiation steps, resulting in the formation of a unique hierarchical columnar structure comprising round, proliferating, pre-hypertrophic, and hypertrophic chondrocytes. Pre-hypertrophic chondrocytes, which exist in the transitional stage between proliferating and hypertrophic stages, are a critical cell population in the growth plate. However, the molecular basis of pre-hypertrophic chondrocytes remains largely undefined. Here, we employed scRNA-seq analysis on fluorescently labeled growth plate chondrocytes for their molecular characterization. Serine incorporator 5 (Serinc5) was identified as a marker gene for pre-hypertrophic chondrocytes. Histological analysis revealed that Serinc5 is specifically expressed in pre-hypertrophic chondrocytes, overlapping with Indian hedgehog (Ihh). Serinc5 represses cell proliferation and Col2a1 and Acan expression by inhibiting the transcriptional activity of Sox9 in primary chondrocytes. Chromatin profiling using ChIP-seq and ATAC-seq revealed an active enhancer of Serinc5 located in intron 1, with its chromatin status progressively activated during chondrocyte differentiation. Collectively, our findings suggest that Serinc5 regulates sequential chondrocyte differentiation from proliferation to hypertrophy by inhibiting Sox9 function in pre-hypertrophic chondrocytes, providing novel insights into the mechanisms underlying chondrocyte differentiation in growth plates.

Cell-integrated serum-induced signalling patterns can differentiate between hand and knee osteoarthritis patients.

OA is a very heterogeneous disease. Here, we aimed to differentiate OA patients based on their serum-induced cell-integrated signalling patterns. In order to monitor the activity of different cellular homeostasis-regulating pathways in response to patient serum, we analysed the response of human OA serum samples to sixteen cell-based transcription factor luciferase reporter assays. In this study we compared serum samples from 55 patients with knee OA, 56 patients with hand OA and 42 healthy controls. Differential serum-induced pathway activity was observed between samples from healthy controls, knee OA and hand OA patients: Serum of hand OA patients induced high MAPK-related AP1 activity whereas serum of knee OA patients induced more SRE, ISRE and SOX9 activity, which is related to ELK1-SRF, STAT1-STAT2 and SOX9 activity respectively. Principal component analysis revealed that these differences differentiate hand OA from knee OA. Both hand and knee OA clustered clearly in 2 different endotypes each, but no principle component could be identified of these subtypes within either the hand OA or the knee OA sample group. This study demonstrates that serum from hand OA and knee OA patients evokes diverse cellular signalling patterns that differentiates hand OA, knee OA and healthy controls. This underlines that the pathomolecular mechanisms of OA are likely significantly different between hand and knee OA, a finding that could lead to new insight into the pathobiology of OA endotypes and joint-specific therapies.

Dissecting SOX9 dynamics reveals its differential regulation in osteoarthritis.

The transcription factor SOX9 is integral to tissue homeostasis and is implicated in skeletal malformation, campomelic dysplasia, and osteoarthritis (OA). Despite extensive research, the complete regulatory landscape of SOX9 transcriptional activity, interconnected with signaling pathways (TGFβ, WNT, BMP, IHH, NFκB, and HIF), remains challenging to decipher. This study focuses on elucidating SOX9 signaling in OA pathology using Fluorescence Recovery After Photobleaching (FRAP) to assess SOX9 activity directly in live human primary chondrocytes (hPCs). Single cell FRAP data revealed two distinct subpopulations with differential SOX9 dynamics, showing varied distribution between healthy and OA hPCs. Moreover, inherently elevated SOX9-DNA binding was observed in healthy hPCs compared to preserved and OA counterparts. Anabolic factors (BMP7 and GREM1) and catabolic inhibitors (DKK1 and FRZb) were found to modulate SOX9 transcriptional activity in OA-hPCs. These findings provide valuable insights into the intricate regulation of SOX9 signaling in OA, suggesting potential therapeutic avenues for modulating SOX9 activity in diseased states.

SERPINA3 is a marker of cartilage differentiation and is essential for the expression of extracellular matrix genes during early chondrogenesis

Serine proteinase inhibitors (serpins) are a family of structurally similar proteins which regulate many diverse biological processes from blood coagulation to extracellular matrix (ECM) remodelling. Chondrogenesis involves the condensation and differentiation of mesenchymal stem cells (MSCs) into chondrocytes which occurs during early development. Here, and for the first time, we demonstrate that one serpin, SERPINA3 (gene name SERPINA3, protein also known as alpha-1 antichymotrypsin), plays a critical role in chondrogenic differentiation. We observed that SERPINA3 expression was markedly induced at early time points during in vitro chondrogenesis. We examined the expression of SERPINA3 in human cartilage development, identifying significant enrichment of SERPINA3 in developing cartilage compared to total limb, which correlated with well-described markers of cartilage differentiation. When SERPINA3 was silenced using siRNA, cartilage pellets were smaller and contained lower proteoglycan as determined by dimethyl methylene blue assay (DMMB) and safranin-O staining. Consistent with this, RNA sequencing revealed significant downregulation of genes associated with cartilage ECM formation perturbing chondrogenesis. Conversely, SERPINA3 silencing had a negligible effect on the gene expression profile during osteogenesis suggesting the role of SERPINA3 is specific to chondrocyte differentiation. The global effect on cartilage formation led us to investigate the effect of SERPINA3 silencing on the master transcriptional regulator of chondrogenesis, SOX9. Indeed, we observed that SOX9 protein levels were markedly reduced at early time points suggesting a role for SERPINA3 in regulating SOX9 expression and activity. In summary, our data support a non-redundant role for SERPINA3 in enabling chondrogenesis via regulation of SOX9 levels.

MDB-07. A SUICIDE GENE THERAPY TARGETING SOX9-POSITIVE THERAPY-RESISTANT MEDULLOBLASTOMA CELLS

Abstract The transcription factor SOX9 is a stem cell factor and a glial fate marker in the developing brain. SOX9 levels correlate with worse prognosis and relapse in Group 3 medulloblastoma, a malignant pediatric brain tumor with poor survival. SOX9 activation promotes epidermal to mesenchymal transition and leptomeningeal spread. It is also upregulated by and facilitates cisplatin resistance in medulloblastoma cells. Direct SOX9 inhibition has proven difficult and few targeted substances have emerged from drug screening efforts. SOX9 acts as a selective and potent activator of a minimal collagen type II alpha 1 enhancer element. We here show that cells with SOX9 activity can be efficiently and specifically labeled when this SOX9 response element is coupled to a minimal promoter (S9RE-minP) driving GFP or luciferase. We further show that S9RE-minP construct can be used to drive herpes simplex virus thymidine kinase (TK) in a suicide gene construct that can convert ganciclovir to a cytotoxic drug in mitotic SOX9-positive cells. The gene therapy worked efficiently together with cisplatin to eradicate SOX9-positive medulloblastoma cells in vitro and could be safely combined and synergize with fractionated radiation in vivo. We suggest that this could be an attractive approach to target therapy-resistant cells in malignant pediatric brain tumors.

Sox9-coordinated cellular neighborhoods generate fibrosis

Poorly regenerative organs deposit scar tissue to mend damage. Aggarwal etal. establish that transient Sox9 activity is necessary for early proximal tubule epithelial regeneration, while Trogisch etal. and Aggarwal etal. show that persistent Sox9 activity in epithelial and endothelial cells activates fibroblasts creating fibrotic microdomains in multiple organs.

SOX9 switch links regeneration to fibrosis at the single-cell level in mammalian kidneys.

The steps governing healing with or without fibrosis within the same microenvironment are unclear. After acute kidney injury (AKI), injured proximal tubular epithelial cells activate SOX9 for self-restoration. Using a multimodal approach for a head-to-head comparison of injury-induced SOX9 lineages, we identified a dynamic SOX9 switch in repairing epithelia. Lineages that regenerated epithelia silenced SOX9 and healed without fibrosis (SOX9on-off). By contrast, lineages with unrestored apicobasal polarity maintained SOX9 activity in sustained efforts to regenerate, which were identified as a SOX9on-on Cadherin6pos cell state. These reprogrammed cells generated substantial single-cell WNT activity to provoke a fibroproliferative response in adjacent fibroblasts, driving AKI to chronic kidney disease. Transplanted human kidneys displayed similar SOX9/CDH6/WNT2B responses. Thus, we have uncovered a sensor of epithelial repair status, the activity of which determines regeneration with or without fibrosis.

Investigation of the Sox-9 and Caspase-6 Immune Activity in Placentas of Pregnant Women with GDM

Aim: In this study, we investigated the immunohistochemical staining of Sox-9 and Caspase-6 expression in placentas of pregnant woman with gestational diabetes mellitus (GDM). Material and Methods: Placentas of 20 healthy and 20 women with GDM were processed for routine histological tissue processing. The biochemical and clinical parameters of patients were recorded. Placentas were stained with hematoxylin-eosin and Sox-9 and Caspase-6 immunostaining. Results: In control group, Sox-9 expression was negative in decidual and connective cells and endothelial cells. In GDM group, Sox-9 expression was increased especially in the decidual cells. For the Caspase-6 expression, Caspase-6 reaction was mainly in maternal region in control group. In GDM group, Caspase-6 reaction was increased in decidual cells, in endothelial cells and in the syncytial nodes. Conclusion: Expression of Sox-9 transformed the decidua cells and lead to apoptotic pathway via Caspase-6 expression.

Investigation of the Sox-9 and Cited-1 Immune Activity in Placentas of Women with Placenta Accreta

Aim: In this study, we investigated the immune activity of the Sox-9 and Cited-1 in women with placenta accreta. Material and Methods: 20 healthy and 20 placenta accreta were processed for routine histological tissue processing. Placentals samples were dissected and fixed in 10% formaldehyde solution. Samples were embedded in paraffin blocks. Clinical and biochemical parameters were recorded. Placental sections were cut from paraffin blocks and stained with Sox-9 and Cited-1 immunostaining. Results: In our study, control group showed negative Cited-1 expression in decidual cells, root villi and connective tissue areas in general. Placenta accreta group showed increased Cited-1 expression in degenerated decidual cells, fibroblastic cells and endothelium. In control group, Sox-9 expression was negative in the syncytial knots, in the vascular endothelial cells. In placenta accreta group, Sox-9 reaction was positive in the root villi, in the blood vessels, in the connective tissue. Conclusion: It was observed that the Sox-9 reaction was increased and inflammation was induced, depending on the differences in decidual cells, in the syncytial area and in the vascular endothelium in in placentas of women with placenta accreta. It is thought that Sox-9 signaling processes are being determined and Cited-1 may be stimulants that affect cell proliferation and angiogenesis regulation and affect placental development.

Differential activity of transcription factor Sox9 in early and adult oligodendroglial progenitor cells.

The high-mobility-group domain-containing transcription factor Sox9 confers glial competence to neuroepithelial precursors in the developing central nervous system and is an important determinant of astroglial and oligodendroglial specification. In oligodendroglial cells, it remains expressed in oligodendrocyte progenitor cells (OPCs) of the developing nervous system, but is shut off in differentiating oligodendrocytes as well as in OPCs that persist in the adult nervous system. To better understand the role of Sox9 in OPCs, we generated mouse models that allowed oligodendroglial expression of a Sox9 transgene during development or in the adult. With transgene expression beginning in the last trimester of pregnancy, the number of OPCs increased dramatically, followed by comparable gains in the number of pre-myelinating and myelinating oligodendrocytes as assessed by marker gene expression. This argues that Sox9 boosts oligodendrogenesis during ontogenetic development at all stages, including terminal oligodendrocyte differentiation. When Sox9 transgene expression started in the adult, many transgene-expressing OPCs failed to maintain their progenitor cell identity and instead converted into myelinating oligodendrocytes. As infrequent and inefficient differentiation of adult OPCs is one of the main obstacles to effective remyelination in demyelinating diseases such as Multiple Sclerosis, increased Sox9 levels in adult OPCs may substantially increase their remyelination capacity.

Sox9 is required in regeneration of pancreatic β cells following injury

The reduction of insulin secretion due to pancreatic β cell injury caused by autoimmune reaction is the pathological basis of Type 1 diabetes mellitus (T1DM). Therefore, seeking new molecular targets for alleviating pancreatic β cell injury will provide experimental basis for the prevention and treatment of T1DM. SRY-box 9 (Sox9) is not only an important molecule regulating the development of various organs, but also its high expression can aggravate the pathological process of various diseases. In addition, Sox9+ cells are also pancreatic progenitor cells, participating in pancreatic repair reaction induced by injury. In our study, elevated blood glucose and lack of pancreatic β cells almost returned to normal over time after streptozotocin (STZ)-induced pancreatic β cell damage, implying that pancreatic β cells were regenerated after STZ-induced injury. In particular, the expression of Sox9 was significantly elevated during pancreatic β cell regeneration. On this basis, we conducted in vitro experiments to verify whether overexpression of Sox9 could inhibit the damage of pancreatic β cells by inflammatory factors. Our results showed that overexpression of Sox9 alleviated the damage of pancreatic β cells by inflammatory factors and improved the inhibitory effect of inflammatory factors on insulin secretion of pancreatic β cells. Unsurprising, blood glucose levels, insulin content and pancreatic β cell number failed to return to near-normal levels timely after pancreatic β cells specific knockout Sox9 mice were treated with STZ, further confirming the importance of Sox9 in facilitating pancreatic β cell repair or regeneration. Our study indicate that enhanced Sox9 activity might protect pancreatic β cells from autoimmune induced damage and thus improve the pathological process of T1DM.

Abstract 2368: SOX9-binding proteins regulate SOX9 activity to control the growth of triple-negative breast cancer cells

Abstract Background: Triple-negative breast cancers (TNBCs) are the most aggressive types of breast cancer, which lack the expression of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR). TNBCs have a very poor prognosis and have few targeted therapy options. Therefore, the development of new TNBC treatment strategies is an urgent and unmet clinical need. Our previous studies have demonstrated that knockout of the SOX9 transcription factor decreased TNBC cell growth and tumor metastasis in vivo. Hypothesis: SOX9-binding proteins include upstream activators of this transcription factor which regulate SOX9 activity and control TNBC growth. Material and Methods: Immunoprecipitation (IP) in combination with mass spectrometry (IP-MS) analysis was used to identify SOX9 binding proteins, with support from MD Anderson’s proteomics core. The Mascot Score is a statistical score was used as the reference of targeted protein, and the data was summarized based on the Mascot Score. IP-Western Blotting analysis was used to confirm that the identified proteins bound SOX9 in TNBC cells. RNA-Seq analysis was used to identify SOX9 regulated genes in MDA MBA-231 and MDA MB-468 cells after SOX9 knockdown or in MCF-7 after SOX9 overexpression, with support from MD Anderson’s genomic biostatistics core. We then treated TNBC cells with or without siRNAs of these identified proteins to evaluate their effect on SOX9-regulated gene expression and TNBC growth. Cell growth was measured using an automated cell counting assay. Protein and mRNA levels were examined by western blotting and qRT-PCR assays. SOX9 transcriptional activity was measured using a reporter gene activity assay, and qRT-PCR assay. Data are presented as mean values ± SD. Statistical significance (p-values) was calculated using the Student’s t-test unless otherwise indicated. Results: Using immunoprecipitation in combination with mass spectrometry (IP-MS) analysis, we identified multiple SOX9 binding proteins. Among them, proteins that demonstrated high Mascot Scores include Valosin-Containing Protein (VCP), protein S100A10 (S100A10); Caveolin 1 (CAV1). Knockdown of each of these SOX9-binding proteins (VCP, S100A10, or CAV1) decreased TNBC cell growth in vitro. Using mRNA-Seq analysis, we next identified 38 genes down-regulated by SOX9 knockdown in TNBC cell lines, and up-regulated upon SOX9 overexpression in MCF-7 cells. Using these genes as markers of SOX9 transcriptional activity, all three of these SOX9-binding proteins regulate SOX9 activity. Thus, VCP, CAV1, and S100A10 all are potential upstream proteins of SOX9 that can be targeted to inhibit TNBC growth. Conclusion: Our results demonstrate that VCP, S100A10, and CAV1, are SOX9-binding proteins that regulate SOX9 activity and control TNBC cell growth. Implications: These proteins represent potential targets for the treatment of TNBCs. Citation Format: Yanxia Ma, David H. Hawke, Ganiraju Manyam, Wenyi Wang, Abhijit Mazumdar, Powel Brown. SOX9-binding proteins regulate SOX9 activity to control the growth of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2368.

Thrap3 promotes osteogenesis by inhibiting the degradation of Runx2.

The dysfunction of osteogenesis is a key character in the pathogenesis of osteoporosis, but the network of signaling mechanisms in controlling the differentiation of osteoblast remain unclear. Thrap3has been proved participating in various biological process, especially in the differentiation of stem cells. Here, we demonstrate that Thrap3 could promote osteogenesis through the inhibition of the degradation of Runx2, which is a key molecular structure in early osteoblast differentiation. Furthermore, we found that the osteogenesis enhancing capacity of Thrap3 was caused by physically binding with Sox9, inhibiting the transcriptional activity of Sox9, and then decreasing the decomposition-promoted effect of Sox9 on Runx2. Our data shows that Thrap3 promotes osteoblast differentiation through the Thrap3-Sox9-Runx2 axis. What we found may help for further clarifying the molecular mechanism of osteogenic differentiation and give a new potential therapeutic target for osteoporosis.

Truncating SOX9 Alterations Are Heterozygous Null Alleles in Genome-Stable Colorectal Cancer.

Truncating SOX9 Alterations Are Heterozygous Null Alleles in Genome-Stable Colorectal Cancer.

Repair impact of vibration exercise with different frequencies on articular cartilage of rats with early knee osteoarthritis and its JNK/NF-κB, SOX9 mechanisms

Objective: To investigate the repair effect and JNK/NF-κB,SOX9 mechanisms of vibration exercise with different frequencies on articular cartilage in rats with early knee osteoarthritis. Methods: Forty-eight adult male SD rats were randomly divided into six groups(n=8):model control group(MC),high frequency vibration group 1 (GP1,60 Hz),high frequency vibration 2 group (GP2,40 Hz),medium frequency vibration group (ZP,20 Hz),minor frequency group(DP,10 Hz)and normal control group(NC). Except for NC group,the rats in each group were made into early knee osteoarthritis model after six weeks of knee joint cavity injection of papain solution and 2% mixture l-cysteine on the 1st,4 th and 7th day. Each exercise group was subjected vibration to 40 minutes a day with amplitude of 2～5 mm and 5 days a week. Four weeks later, the articular cartilage of the lateral femoral condyle of the both back leg knee joints were detected by HE staining,serine O staining and Mankin scores for morphological observation. The expression levels of JNK,NF-κB p65 and Sox9 mRNA in articular cartilage of the medial femoral condyle were detected by RT-qPCR,and the protein expressions of JNK,NF-κB p65 and Sox9 were detected by Western blot. Results: Compared with the NC group,the Mankin score in other groups was significantly higher (P＜0.01). Compared with the MC group,the Mankin score of each vibration group was significantly lower(P＜0.05),the mRNA and protein expressions of JNK and NF-κB p65 in each vibration training group were significantly lower (P＜0.01),the expressions of Sox9 mRNA and protein in vibration training group were increased significantly (P＜0.01). Compared with the higher frequency group,the Mankin score,the mRNA and protein expressions of JNK and NF-κB p65 of lower frequency group were significantly lower (P＜0.05 or P＜0.01). But the expressions of Sox9 mRNA and protein were significantly higher (P＜ 0.05 or P＜0.01). Conclusion: Vibration exercise of different frequencies may present varying degrees of cartilage repair impact in rats with early knee osteoarthritis,and the cartilage repair by low-frequency vibration training is better than that by high-frequency vibration. This can be one of the mechanisms on controlling collagen synthesis by down-regulating JNK/NF-κB expression and increasing SOX9 activity of OA articular cartilage.

RIP140 Represses Intestinal Paneth Cell Differentiation and Interplays with SOX9 Signaling in Colorectal Cancer.

Simple SummaryIn the small intestine, the transcription factor SOX9 regulates the differentiation of Paneth cells, which are key actors in the intestinal antimicrobial defense and stem cell niche. SOX9 is also an important player in colon cancer development and progression. In this study, we demonstrate that the transcription coregulator RIP140 inhibits SOX9 expression and activity. Consequently, RIP140 and SOX9 exert opposite effects on Paneth cell differentiation and colon cancer cell proliferation. Their expression undergoes inverse regulation by the Wnt signaling pathway and inversely correlates with survival of patient with colon cancer. These results identify RIP140 as a major regulator of SOX9 signaling with functional relevance in intestinal physiopathology.RIP140 is a major transcriptional coregulator of gut homeostasis and tumorigenesis through the regulation of Wnt/APC signaling. Here, we investigated the effect of RIP140 on Paneth cell differentiation and its interplay with the transcription factor SOX9. Using loss of function mouse models, human colon cancer cells, and tumor microarray data sets we evaluated the role of RIP140 in SOX9 expression and activity using RT-qPCR, immunohistochemistry, luciferase reporter assays, and GST-pull down. We first evidence that RIP140 strongly represses the Paneth cell lineage in the intestinal epithelium cells by inhibiting Sox9 expression. We then demonstrate that RIP140 interacts with SOX9 and inhibits its transcriptional activity. Our results reveal that the Wnt signaling pathway exerts an opposite regulation on SOX9 and RIP140. Finally, the levels of expression of RIP140 and SOX9 exhibit a reverse response and prognosis value in human colorectal cancer biopsies. This work highlights an intimate transcriptional cross-talk between RIP140 and SOX9 in intestinal physiopathology.

The SOX9-MMS22L Axis Promotes Oxaliplatin Resistance in Colorectal Cancer.

BackgroundColorectal cancer (CRC) is estimated to be one of the most common cancers and the leading cause of cancer-related death worldwide. SOX9 is commonly overexpressed in CRC and participates in drug resistance. In addition, DNA damage repair confers resistance to anticancer drugs. However, the correlation between DNA damage repair and high SOX9 expression is still unclear. In this study, we aimed to investigate the function and the specific underlying mechanism of the SOX9-dependent DNA damage repair pathway in CRC.MethodsThe expression levels of SOX9 and MMS22L in CRC were examined by immunohistochemistry (IHC) and TCGA analysis. RNA sequencing was conducted in RKO SOX9-deficient cells and RKO shControl cells. Mechanistic studies were performed in CRC cells by modulating SOX9 and MMS22L expression, and we evaluated drug sensitivity and DNA damage repair signaling events. In addition, we investigated the effect of oxaliplatin in tumors with SOX9 overexpression and low expression of MMS22L in vivo.ResultsOur study showed that SOX9 has a higher expression level in CRC tissues than in normal tissues and predicts poor prognosis in CRC patients. Overexpression and knockdown of SOX9 were associated with the efficacy of oxaliplatin. In addition, SOX9 activity was enriched in the DNA damage repair pathway via regulation of MMS22L expression and participation in DNA double-strand break repair. SOX9 was upregulated and formed a complex with MMS22L, which promoted the nuclear translocation of MMS22L upon oxaliplatin treatment. Moreover, the xenograft assay results showed that oxaliplatin abrogated tumor growth from cells with MMS22L downregulation in mice.ConclusionsIn CRC, activation of the SOX9-MMS22L-dependent DNA damage pathway is a core pathway regulating oxaliplatin sensitivity. Targeting this pathway in oxaliplatin-resistant CRC cells is a promising therapeutic option.

SOX9 modulates cancer biomarker and cilia genes in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive form of cancer with high mortality. The cellular origins of PDAC are largely unknown; however, ductal cells, especially centroacinar cells (CACs), have several characteristics in common with PDAC, such as expression of SOX9 and components of the Notch-signaling pathway. Mutations in KRAS and alterations to Notch signaling are common in PDAC, and both these pathways regulate the transcription factor SOX9. To identify genes regulated by SOX9, we performed siRNA knockdown of SOX9 followed by RNA-seq in PANC-1s, a human PDAC cell line. We report 93 differentially expressed (DE) genes, with convergence on alterations to Notch-signaling pathways and ciliogenesis. These results point to SOX9 and Notch activity being in a positive feedback loop and SOX9 regulating cilia production in PDAC. We additionally performed ChIP-seq in PANC-1s to identify direct targets of SOX9 binding and integrated these results with our DE gene list. Nine of the top 10 downregulated genes have evidence of direct SOX9 binding at their promoter regions. One of these targets was the cancer stem cell marker EpCAM. Using whole-mount in situ hybridization to detect epcam transcript in zebrafish larvae, we demonstrated that epcam is a CAC marker and that Sox9 regulation of epcam expression is conserved in zebrafish. Additionally, we generated an epcam null mutant and observed pronounced defects in ciliogenesis during development. Our results provide a link between SOX9, EpCAM and ciliary repression that can be exploited in improving our understanding of the cellular origins and mechanisms of PDAC.

SUN-034 Combined CNV, Haplotyping and Whole Exome Sequencing Implicates Inherited Novel SRY Mutations Not Account for Familial 46,XY Sex Reversal

SRY is one of the important genes involved in the process of human sex determination. The disturbed sex determination caused by SRY mutation accounts for 10-15% cases with complete gonadal dysplasia (CGD), also known as 46, XY sex reversal. Recently, three distal enhancers are disclosed in the upstream of SOX9 gene. In an inherited 46, XY sex reversal pedigree with 5 patients, p.Arg76Leu mutation of SRY and p.G212S mutation of NR5A1were identified from the proband who present with primary amenorrhea and lack of puberty development. The missense mutation of NR5A1was found to be derived from the mother. Interestingly, the paternal inherited p.Arg76Leu mutation of SRY was revealed from other 2 CGD patients, as well as from apparent normal male family members with fertility. P.Arg76Leu variation was found have no effect to the transcriptional activity of target gene SOX9, neither alteration of the nuclear translocation of SRY. Whole exome sequencing also found SRY mutation, FGF10 mutation, GJB4 gene mutation, etc. with no segregation in the family, which suggested SNVs are not main cause of the disease in this pedigree. By copy number variation and SNP haplotype analysis, SOX9 gene far upstream deletion of 68kb was disclosed from the 3 patients in this family, containing one of the enhancers of SOX9. Real-time PCR confirmed that the heterozygous deletion of the region result in loss of SR-XY, but not eSR-B and eALDI. Therefore, single nucleotide variation (SNV) of SRY and NR5A1 are not main causes of severe phenotype of CGD, the enhancers of SOX9 should be investigated carefully in such patients.

Generation of a Quantitative Luciferase Reporter for Sox9 SUMOylation.

Sox9 is a master transcription factor for chondrogenesis, which is essential for chondrocyte proliferation, differentiation, and maintenance. Sox9 activity is regulated by multiple layers, including post-translational modifications, such as SUMOylation. A detection method for visualizing the SUMOylation in live cells is required to fully understand the role of Sox9 SUMOylation. In this study, we generated a quantitative reporter for Sox9 SUMOylation that is based on the NanoBiT system. The simultaneous expression of Sox9 and SUMO1 constructs that are conjugated with NanoBiT fragments in HEK293T cells induced luciferase activity in SUMOylation target residue of Sox9-dependent manner. Furthermore, the reporter signal could be detected from both cell lysates and live cells. The signal level of our reporter responded to the co-expression of SUMOylation or deSUMOylation enzymes by several fold, showing dynamic potency of the reporter. The reporter was active in multiple cell types, including ATDC5 cells, which have chondrogenic potential. Finally, using this reporter, we revealed a extracellular signal conditions that can increase the amount of SUMOylated Sox9. In summary, we generated a novel reporter that was capable of quantitatively visualizing the Sox9-SUMOylation level in live cells. This reporter will be useful for understanding the dynamism of Sox9 regulation during chondrogenesis.