Regulates Sox2 Expression Research Articles

Wu Mei Wan suppresses colorectal cancer stemness by regulating Sox9 expression via JAK2/STAT3 pathway

Ethnopharmacological relevanceWu Mei Wan (WMW) is a traditional Chinese herbal formula with a long-standing history in Chinese medicine, valued for its therapeutic properties. However, its potential anti-cancer effects, especially against colorectal cancer (CRC), have not been fully elucidated. Aim of the studyThis study aims to investigate the effects of WMW on colorectal cancer stemness and to elucidate the underlying molecular mechanisms, focusing on the modulation of Sox9 expression via the JAK2/STAT3 signaling pathway. Materials and methodsWMW was prepared and analyzed using UPLC-MS to identify their main components. To study the therapeutic effects of WMW, AOM/DSS-induced CRC mouse models were established. A comprehensive suite of experimental techniques, including in vivo imaging, cell culture, transfection, CCK-8 assays, colony formation assays, wound healing assays, cell migration assays, Western blotting, dot blot analysis, RT-qPCR, immunohistochemistry, cell transcriptome sequencing, and gene set enrichment analysis, were utilized to explore the pharmacological effects and mechanisms of WMW. ResultsWMW significantly inhibited CRC cell viability, proliferation, invasion, and migration in vitro. Mechanistically, WMW suppressed CRC stemness by downregulating Sox9 expression through the JAK2/STAT3 signaling pathway. Additionally, the regulation of methylation and demethylation mediated by TET1 and DNMT3a expression was directly associated with the JAK2/STAT3 pathway's modulation of Sox9 expression. In vivo, WMW treatment attenuated CRC progression and metastasis with minimal toxicity. ConclusionThese findings suggest that WMW exerts potent anti-CRC stemness effects by regulating Sox9 via the JAK2/STAT3 signaling pathway, underscoring its potential as a promising therapeutic agent for CRC treatment.

SRC Phosphorylation of EPS8 Regulates SOX2 Expression and HNSCC Stemness

SRC Phosphorylation of EPS8 Regulates SOX2 Expression and HNSCC Stemness

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.

Chromatin profiling identifies chondrocyte-specific Sox9 enhancers important for skeletal development.

The transcription factor SRY-related HMG box 9 (Sox9) is essential for chondrogenesis. Mutations in and around SOX9 cause campomelic dysplasia (CD) characterized by skeletal malformations. Although the function of Sox9 in this context is well studied, the mechanisms that regulate Sox9 expression in chondrocytes remain to be elucidated. Here, we have used genome-wide profiling to identify 2 Sox9 enhancers located in a proximal breakpoint cluster responsible for CD. Enhancer activity of E308 (located 308 kb 5' upstream) and E160 (located 160 kb 5' upstream) correlated with Sox9 expression levels, and both enhancers showed a synergistic effect in vitro. While single deletions in mice had no apparent effect, simultaneous deletion of both E308 and E160 caused a dwarf phenotype, concomitant with a reduction of Sox9 expression in chondrocytes. Moreover, bone morphogenetic protein 2-dependent chondrocyte differentiation of limb bud mesenchymal cells was severely attenuated in E308/E160 deletion mice. Finally, we found that an open chromatin region upstream of the Sox9 gene was reorganized in the E308/E160 deletion mice to partially compensate for the loss of E308 and E160. In conclusion, our findings reveal a mechanism of Sox9 gene regulation in chondrocytes that might aid in our understanding of the pathophysiology of skeletal disorders.

CHD7 and SOX2 act in a common gene regulatory network during mammalian semicircular canal and cochlear development

Inner ear morphogenesis requires tightly regulated epigenetic and transcriptional control of gene expression. CHD7, an ATP-dependent chromodomain helicase DNA-binding protein, and SOX2, an SRY-related HMG box pioneer transcription factor, are known to contribute to vestibular and auditory system development, but their genetic interactions in the ear have not been explored. Here, we analyzed inner ear development and the transcriptional regulatory landscapes in mice with variable dosages of Chd7 and/or Sox2. We show that combined haploinsufficiency for Chd7 and Sox2 results in reduced otic cell proliferation, severe malformations of semicircular canals, and shortened cochleae with ectopic hair cells. Examination of mice with conditional, inducible Chd7 loss by Sox2CreER reveals a critical period (~E9.5) of susceptibility in the inner ear to combined Chd7 and Sox2 loss. Data from genome-wide RNA-sequencing and CUT&Tag studies in the otocyst show that CHD7 regulates Sox2 expression and acts early in a gene regulatory network to control expression of key otic patterning genes, including Pax2 and Otx2. CHD7 and SOX2 directly bind independently and cooperatively at transcription start sites and enhancers to regulate otic progenitor cell gene expression. Together, our findings reveal essential roles for Chd7 and Sox2 in early inner ear development and may be applicable for syndromic and other forms of hearing or balance disorders.

EFFECT OF PPARG INHIBITION ON HUMAN BMSC CELL FATE

The effects of dexamethasone (dex), during in vitro human osteogenesis, are contrasting. Indeed, dex downregulates SOX9 during osteogenic differentiation of human bone marrow mesenchymal stromal cells (HBMSCs). However, dex also promotes PPARG expression, resulting in the formation of adipocyte-like cells within the osteogenic monolayers. The regulation of both SOX9 and PPARG seems to be downstream the transactivation activity of the glucocorticoid receptor (GR), thus the effect of dex on SOX9 downregulation is indirect. This study aims at determining whether PPAR-γ regulates SOX9 expression levels, as suggested by several studies.HBMSCs were isolated from bone marrow of patients with written informed consent. HBMSCs were cultured in different osteogenic induction media containing 10 or 100 nM dex. Undifferentiated cells were used as controls. Cells were treated either with a pharmacological PPAR-γ inhibitor T0070907 (donors n=4) or with a PPARG-targeting siRNA (donors n=2). Differentiation markers or PPAR-γ target genes were analysed by RT-qPCR. Mineral deposition was assessed by ARS staining. Two-way ANOVA followed by a Tukey's multiple comparison test compared the effects of treatments.At day 7, T0070907 downregulated ADIPOQ and upregulated CXCL8, respectively targets of PPAR-γ-mediated transactivation and transrepression. RUNX2 and SOX9 were also significantly downregulated in absence of dex. PPARG was successfully downregulated by siRNA. ADIPOQ expression was also inhibited, while CXCL8 did not show any significant difference between siRNA treatment groups. RUNX2 was downregulated by the PPARG-siRNA treatment in presence of 100 nM dexamethasone, while SOX9 levels were not affected. ARS showed no change in the mineralization levels when PPARG expression or activity was inhibited.Understanding how dex regulates HBMSC differentiation is of pivotal importance to refine current in vitro models. These results suggest that PPARG does not mediate SOX9 downregulation. Unexpectedly, RUNX2 expression was also unaltered or even downregulated after PPAR-γ inhibition.Acknowledgements: AO Foundation, AO Research Institute (CH) and PRIN 2017 MUR (IT) for financial support.

Mechanical force regulates Sox9 expression at the developing enthesis.

Entheses transmit force from tendons and ligaments to the skeleton. Regional organization of enthesis extracellular matrix (ECM) generates differences in stiffness required for force transmission. Two key transcription factors co-expressed in entheseal tenocytes, scleraxis (Scx) and Sox9, directly control production of enthesis ECM components. Formation of embryonic craniofacial entheses in zebrafish coincides with onset of jaw movements, possibly in response to the force of muscle contraction. We show dynamic changes in scxa and sox9a mRNA levels in subsets of entheseal tenocytes that correlate with their roles in force transmission. We also show that transcription of a direct target of Scxa, Col1a, in enthesis ECM is regulated by the ratio of scxa to sox9a expression. Eliminating muscle contraction by paralyzing embryos during early stages of musculoskeletal differentiation alters relative levels of scxa and sox9a in entheses, primarily owing to increased sox9a expression. Force-dependent TGF-β (TGFβ) signaling is required to maintain this balance of scxa and sox9a expression. Thus, force from muscle contraction helps establish a balance of transcription factor expression that controls specialized ECM organization at the tendon enthesis and its ability to transmit force.

LncRNA XIST Inhibits Gastric Cancer Progression Through the Wnt Pathway by Regulating the miR-30a-5p/SOX9 Axis

We aimed to investigate the impact of XIST on the pathogenesis of gastric cancer (GC). We used quantitative real-time polymerase chain reaction (RT-qPCR) to determine the expression levels of XIST, SOX9, and miR-30a-5p in GC cells. Western blot assay was used to measure the protein levels of SOX9, β-catenin, and c-Myc. We also performed MTT and transwell assays to assess proliferation, apoptosis, and invasion. Dual-luciferase reporter assays and RNA immunoprecipitation (RIP) assays were used to confirm the interaction between miR-30a-5p and XIST or SOX9. We found that GC tumor cells express high levels of XIST and SOX9, which promote cell proliferation, invasion, and resistance to apoptosis. Specifically, we observed that XIST acts as a sponge for miR-30a-5p in GC cell line SGC-7901, while miR-30a-5p targets SOX9. SOX9 expression is regulated by XIST via miR-30a-5p acting as a competitive endogenous RNA. Furthermore, we identified a miR-30a-5p/SOX9 axis that modulates the Wnt/β-catenin signaling pathway. In conclusion, our study demonstrates that LncXIST interacts with miR-30a-5p as a competitive endogenous RNA to promote GC cell proliferation, migration, invasion, and inhibition of apoptosis through the regulation of SOX9 expression.

Structural elements promote architectural stripe formation and facilitate ultra-long-range gene regulation at a human disease locus

Enhancer clusters overlapping disease-associated mutations in Pierre Robin sequence (PRS) patients regulate SOX9 expression at genomic distances over 1.25 Mb. We applied optical reconstruction of chromatin architecture (ORCA) imaging to trace 3D locus topology during PRS-enhancer activation. We observed pronounced changes in locus topology between cell types. Subsequent analysis of single-chromatin fiber traces revealed that these ensemble-average differences arise through changes in the frequency of commonly sampled topologies. We further identified two CTCF-bound elements, internal to the SOX9 topologically associating domain, which promote stripe formation, are positioned near the domain's 3D geometric center, and bridge enhancer-promoter contacts in a series of chromatin loops. Ablation of these elements results in diminished SOX9 expression and altered domain-wide contacts. Polymer models with uniform loading across the domain and frequent cohesin collisions recapitulate this multi-loop, centrally clustered geometry. Together, we provide mechanistic insights into architectural stripe formation and gene regulation over ultra-long genomic ranges.

P02.10.A CRISPR/Cas9 deletion of SOX2 Regulatory Region 2 (SRR2) decreases SOX2 malignant activity in glioblastoma

Abstract Background SOX2 is a transcription factor associated with stem cell activity in several tissues. SOX2 expression is elevated in a large proportion of cancers, such as glioblastoma (GB), the most common malignant primary brain tumor in adults. SOX2 acts as a relevant driver of GB progression and high levels are associated with the population of tumor initiating cells and poor patient outcome. Transcriptional regulation of SOX2 is complex and not fully understood. The SOX2 regulatory region 2 (SRR2) is located downstream of the SOX2 coding region and mediates SOX2 expression by the association of p21CIP1 and p27KIP1 to SRR2. In this study we dissected the role of SRR2 on SOX2 activity in GB. Material and Methods We deleted SRR2 (SRR2del) by CRISPR/Cas9 technology in U373 GB cells. We analyzed the expression of SOX2 and performed in vitro functional experiments. We accomplished rescue experiments overexpressing SOX2 in SRR2del cells and we studied the link between SOX2, p21CIP1 and p27KIP1. Finally, we carried out an in vivo carcinogenesis assay by the injection of SRR2del cells in immunodeficient mice. Results We report that the deletion of SRR2 in GB cells leads to a reduction of SOX2 expression, which was accompanied with an impairment of cell growth and proliferation as well as with a reduction of self-renewal capacity in vitro. These data reveal that SRR2 is required for SOX2 expression and that its deletion results in impaired tumorigenic capacity of GB cells. These effects correlated with an increased expression of p21CIP1 and p27KIP1 together with high levels of p53 in SRR2del cells. On the contrary, SRR2del cells presented decreased levels of stem cell markers, supporting the idea that SRR2 is necessary for the SOX2-mediated regulation of stemness pathways. Furthermore, ectopic overexpression of SOX2 in SRR2del cells restored SOX2 expression as well as proliferation and stem cell properties, indicating that the expression and the oncogenic activity of SOX2 is regulated by SRR2. Additionally, our xenograft models revealed that the lack of SRR2 impairs tumor initiation and growth in vivo. Tumors derived from SRR2del cells were significantly smaller compared to controls and exhibit low levels of SOX2, Ki67 and BMI1 but high levels of p21CIP1 and p27KIP1. Conclusion Our data confirm that the SRR2 regulates SOX2 expression and reveal that SRR2 deletion reduces SOX2 levels and halts malignant activity driven by SOX2 in GB.

SOX9 and SRY binding sites on mouse mXYSRa/Enh13 enhancer redundantly regulate Sox9 expression to varying degrees.

Sox9 plays an essential role in mammalian testis formation. It has been reported that gene expression in the testes is regulated by enhancers. Among them, mXYSRa/Enh13-which is located at far upstream of the transcription start site-plays a critical role, wherein its deletion causes complete male-to-female sex reversal in mice. It has been proposed that the binding sites (BSs) of SOX9 and SRY, the latter of which is the sex determining gene on the Y chromosome, are associated with mXYSRa/Enh13. They function as an enhancer, whereby the sequences are evolutionarily conserved and in vivo binding of SOX9 and SRY to mXYSRa/Enh13 has been demonstrated previously. However, their precise in vivo functions have not been examined to date. To this end, this study generated mice with substitutions on the SOX9 and SRY BSs to reveal their in vivo functions. Homozygous mutants of SOX9 and SRY BS were indistinguishable from XY males, whereas double mutants had small testes, suggesting that these functions are redundant and that there is another functional sequence on mXYSRa/Enh13, since mXYSRa/Enh13 deletion mice are XY females. In addition, the majority of hemizygous mice with substitutions in SOX9 BS and SRY BS were female and male, respectively, suggesting that SOX9 BS contributes more to SRY BS for mXYSRa/Enh13 to function. The additive effect of SOX9 and SRY via these BSs was verified using an in vitro assay. In conclusion, SOX9 BS and SRY BS function redundantly in vivo, and at least one more functional sequence should exist in mXYSRa/Enh13.

Resveratrol maintain Human Iliac Bone Marrow Mesenchymal Stem Cells Stemness through Sirtuin 1 Mediated Regulation of SRY-Box Transcription Factor 2: an in vitro and in silico study

Sirtuin 1 (Sirt-1) - SRY-Box Transcription Factor 2 (sox2) axis maintains the stemness of human MSCs. Resveratrol may maintain stemness of human iliac bone marrow (BM)-MSCs. The aim of this study to investigate resveratrol effect on sox2 to maintain BM-MSCs stemness through an in silico and in vitro study. BM-MSCs was aspirated from orthopedic patients then, cultured in vitro. The study groups were into a control group, resveratrol group at doses of 0.1 μM and 1 μM. The characterization human iliac BM-MSCs was examined by immunocytochemistry analysis cluster of differentiation (CD)73, CD90, CD105 and CD45. The proliferation of human iliac BM-MSCs in each group was analyzed by MTT assay with various dose of resveratrol 0.01 μM; 0.05 μM; 0.1 μM; 0.5 μM; 1 μM respectively. A molecular docking was done to evaluate the interactions between resveratrol, sirt1 and Sox2 in silico. Resveratrol act as Sirt1 activator with high binding affinity between Sirt1 and Sox2 was -883.9 kcal/mol in silico. BM-MSCs at third, fourth, fifth and sixth sub-cultured with administrated resveratrol at dose 1 μM showed more confluent, less apoptosis and less senescence cells than control group. The characterization of human iliac BM-MSCs at third sub-culture showed that positive expression of CD73, CD90 and CD105 but lack of CD45 expression. There was no significant different of BM-MSCs viability percentage after administration of resveratrol with various doses (p>0.05). Resveratrol has an effect to regulate Sox2 expression that can maintain human illiac BM-MSCs proliferation, self-renewal and stemness in silico and in vitro.

Matrix stiffness-induced upregulation of histone acetyltransferase KAT6A promotes hepatocellular carcinoma progression through regulating SOX2 expression.

Lysine acetyltransferase 6 A (KAT6A) is a MYST-type histone acetyltransferase (HAT) enzyme, which contributes to histone modification and cancer development. However, its biological functions and molecular mechanisms, which respect to hepatocellular carcinoma (HCC), are still largely unknown. Immunohistochemical, western blot and qRT-PCR analysis of KAT6A were performed. A series of in vitro and in vivo experiments were conducted to reveal the role of KAT6A in the progression of HCC. We demonstrated that KAT6A expression was upregulated in HCC tissues and cell lines. Clinical analysis showed that increased KAT6A was significantly associated with malignant prognostic features and shorter survival. Gain- and loss-of-function experiments indicated that KAT6A promoted cell viability, proliferation and colony formation of HCC cells in vitro and in vivo. We confirmed that KAT6A acetylates lysine 23 of histone H3 (H3K23), and then enhances the association of the nuclear receptor binding protein TRIM24 and H3K23ac. Consequently, TRIM24 functions as a transcriptional activator to activate SOX2 transcription and expression, leading to HCC tumorigenesis. Restoration of SOX2 at least partially abolished the biological effects of KAT6A on HCC cells. Overexpression of KAT6A acetyltransferase activity-deficient mutants or TRIM24 mutants lacking H3K23ac binding sites did not affect SOX2 expression and HCC biological function. Moreover, matrix stiffness can upregulate the expression of KAT6A in HCC cells. Our data support the first evidence that KAT6A plays an oncogenic role in HCC through H3K23ac/TRIM24-SOX2 pathway, and represents a promising therapeutic strategy for HCC patients.

Circ_0075825 promotes gastric cancer progression via adsorbing miR-432-5p to modulate SOX9

MethodsCirc_0075825 expression in adjacent tissues and GC tissues was evaluated by bioinformatics method and quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). How circ_0075825 regulated GC cell growth, migration, invasion, and apoptosis were investigated by cell counting kit-8 assay, transwell assay and flow cytometry. The targeted interplays among circ_0075825, and miR-432-5p and Sex-Determining Region Y-related high-mobility group box 9 (SOX9) were explored by bioinformatics analysis and luciferase reporter gene assay. The regulatory effects of circ_0075825 and miR-432-5p on SOX9 protein expression were probed by western blot. ResultsCirc_0075825 expression was raised in GC tissues and cell lines. Circ_0075825 overexpression promoted the proliferative, migrative and invasive abilities of GC cells, while inhibiting apoptosis, while depletion of circ_0075825 suppressed the malignant biological behaviors of GC cells. SOX9 was identified as one of the direct target genes of miR-432-5p, and circ_0075825 repressed the expression of miR-432-5p, to induce the expression of SOX9. Furthermore, miR-432-5p overexpression counteracted the promoting effect of circ_0075825 on the malignancy of GC cells. ConclusionCirc_0075825 promotes GC progression via sponging miR-432-5p to regulate SOX9 expression level, and it may be a novel therapeutic target for treating GC.

H3K36 methyltransferase NSD1 regulates chondrocyte differentiation for skeletal development and fracture repair

Chondrocyte differentiation is a critical process for endochondral ossification, which is responsible for long bone development and fracture repair. Considerable progress has been made in understanding the transcriptional control of chondrocyte differentiation; however, epigenetic regulation of chondrocyte differentiation remains to be further studied. NSD1 is a H3K36 (histone H3 at lysine 36) methyltransferase. Here, we showed that mice with Nsd1 deficiency in Prx1+ mesenchymal progenitors but not in Col2+ chondrocytes showed impaired skeletal growth and fracture healing accompanied by decreased chondrogenic differentiation. Via combined RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we identified sex determining region Y box 9 (Sox9), the key transcription factor of chondrogenic differentiation, as a functional target gene of NSD1. Mechanistically, NSD1 regulates Sox9 expression by modulating H3K36me1 and H3K36me2 levels in the Sox9 promoter region, constituting a novel epigenetic regulatory mechanism of chondrogenesis. Moreover, we found that NSD1 can directly activate the expression of hypoxia-inducible factor 1α (HIF1α), which plays a vital role in chondrogenic differentiation through its regulation of Sox9 expression. Collectively, the results of our study reveal crucial roles of NSD1 in regulating chondrogenic differentiation, skeletal growth, and fracture repair and expand our understanding of the function of epigenetic regulation in chondrogenesis and skeletal biology.

SOX9 in prostate cancer is upregulated by cancer-associated fibroblasts to promote tumor progression through HGF/c-Met-FRA1 signaling.

Transcription factor SOX9 was a biomarker for prostate cancer (Pca) with poor prognosis. Nevertheless, the regulatory mechanism underlying SOX9 upregulation still remains unclear. Several cytokines have been reported to be involved in the regulation of SOX9, suggesting that cancer-associated fibroblasts (CAFs), one of the main sources of secreted factors in the tumor microenvironment (TME), may play a role in regulating SOX9 expression. Herein, an invitro model of paracrine interaction between primary CAFs and Pca cells was applied to investigate the molecular mechanism of SOX9 upregulation during Pca progression. The regulatory axis was validated by invivo experiments and The Cancer Genome Atlas data. Conditional medium of CAFs (CAF-CM) upregulated the expression of SOX9, which was mutually proved to be essential for CAF-induced tumor progression. Further analysis showed that hepatocyte growth factor (HGF) secreted by CAFs was responsible for SOX9 elevation in Pca cells, via the activation of c-Met signaling. Mechanistically, HGF/c-Met signaling specifically activated MEK1/2-ERK1/2 pathway, which induced phosphorylation and upregulation of FRA1, which then transcriptionally upregulated SOX9 by binding to the promoter of SOX9 gene. Moreover, we identified that HGF/c-Met-ERK1/2-FRA1-SOX9 axis was relatively conserved between human and mouse species by validating in mouse Pca cells. Our results reveal a novel insight into the molecular mechanism that SOX9 in Pca cells is promoted by CAFs through HGF/c-Met-ERK1/2-FRA1 axis. Furthermore, SOX9 may serve as an alternative marker for the activated HGF/c-Met signaling to enroll the optimal Pca patients for HGF/c-Met inhibition treatment, since it is much more stable and easier to detect.

Light-induced local gene expression in primary chick cell culture system.

The ability to manipulate gene expression at a specific region in a tissue or cell culture system is critical for analysis of target gene function. For chick embryos/cells, several gene introduction/induction methods have been established such as those involving retrovirus, electroporation, sonoporation, and lipofection. However, these methods have limitations in the accurate induction of localized gene expression. Here we demonstrate the effective application of a recently developed light‐dependent gene expression induction system (LightOn system) using the Neurospora crassa photoreceptor Vivid fused with a Gal4 DNA binding domain and p65 activation domain (GAVPO) that alters its activity in response to light stimulus in a primary chicken cell culture system. We show that the gene expression level and induction specificity in this system are strongly dependent on the light irradiation conditions. Especially, the irradiation interval is an important parameter for modulating gene expression; for shorter time intervals, higher induction specificity can be achieved. Further, by adjusting light irradiation conditions, the expression level in primary chicken cells can be regulated in a multiple step manner, in contrast to the binary expression seen for gene disruption or introduction (i.e., null or overexpression). This result indicates that the light‐dependent expression control method can be a useful technique in chick models to examine how gene function is affected by gradual changes in gene expression levels. We applied this light induction system to regulate Sox9 expression in cultures of chick limb mesenchyme cells and showed that induced SOX9 protein could modulate expression of downstream genes.

CRISPR/Cas9 Deletion of SOX2 Regulatory Region 2 (SRR2) Decreases SOX2 Malignant Activity in Glioblastoma.

Simple SummaryUnderstanding how SOX2, a major driver of cancer stem cells, is regulated in cancer cells is relevant to tackle tumorigenesis. In this study, we deleted the SRR2 regulatory region in glioblastoma cells. Our data confirm that the SRR2 enhancer regulates SOX2 expression in cancer and reveal that SRR2 deletion halts malignant activity of SOX2.SOX2 is a transcription factor associated with stem cell activity in several tissues. In cancer, SOX2 expression is increased in samples from several malignancies, including glioblastoma, and high SOX2 levels are associated with the population of tumor-initiating cells and with poor patient outcome. Therefore, understanding how SOX2 is regulated in cancer cells is relevant to tackle tumorigenesis. The SOX2 regulatory region 2 (SRR2) is located downstream of the SOX2 coding region and mediates SOX2 expression in embryonic and adult stem cells. In this study, we deleted SRR2 using CRISPR/Cas9 in glioblastoma cells. Importantly, SRR2-deleted glioblastoma cells presented reduced SOX2 expression and decreased proliferative activity and self-renewal capacity in vitro. In line with these results, SRR2-deleted glioblastoma cells displayed decreased tumor initiation and growth in vivo. These effects correlated with an elevation of p21CIP1 cell cycle and p27KIP1 quiescence regulators. In conclusion, our data reveal that SRR2 deletion halts malignant activity of SOX2 and confirms that the SRR2 enhancer regulates SOX2 expression in cancer.

Med23 Regulates Sox9 Expression during Craniofacial Development

The etiology and pathogenesis of craniofacial birth defects are multifactorial and include both genetic and environmental factors. Despite the identification of numerous genes associated with congenital craniofacial anomalies, our understanding of their etiology remains incomplete, and many affected individuals have an unknown genetic diagnosis. Here, we show that conditional loss of a Mediator complex subunit protein, Med23 in mouse neural crest cells (Med23 fx/fx ;Wnt1-Cre), results in micrognathia, glossoptosis, and cleft palate, mimicking the phenotype of Pierre Robin sequence. Sox9 messenger RNA and protein levels are both upregulated in neural crest cell–derived mesenchyme surrounding Meckel’s cartilage and in the palatal shelves in Med23 fx/fx ;Wnt1-Cre mutant embryos compared to controls. Consistent with these observations, we demonstrate that Med23 binds to the promoter region of Sox9 and represses Sox9 expression in vitro. Interestingly, Sox9 binding to β-catenin is enhanced in Med23 fx/fx ;Wnt1-Cre mutant embryos, which, together with downregulation of Col2a1 and Wnt signaling target genes, results in decreased proliferation and altered jaw skeletal differentiation and cleft palate. Altogether, our data support a cell-autonomous requirement for Med23 in neural crest cells, potentially linking the global transcription machinery through Med23 to the etiology and pathogenesis of craniofacial anomalies such as micrognathia and cleft palate.

Circular RNA circ_0032821 contributes to oxaliplatin (OXA) resistance of gastric cancer cells by regulating SOX9 via miR-515-5p.

Chemoresistance is one of the major obstacles for gastric cancer (GC) treatment. Exosome-mediated transfer of circular RNAs (circRNAs) is associated with the drug-resistance in GC. Circ_0032821 has been reported as an oncogene in GC. This study is designed to explore the function and mechanism of Exosomal circ_0032821 in oxaliplatin (OXA) resistance of GC. Circ_0032821 was highly expressed in OXA-resistant GC cells, and exosomes secreted by OXA-resistant GC cells. Moreover, circ_0032821-containing exosomes secreted by OXA-resistant GC cells could boost OXA resistance, proliferation, migration, and invasion in OXA-sensitive GC cells. The mechanical analysis discovered that circ_0032821 acted as a sponge of miR-515-5p to regulate SOX9 expression. Circ_0032821 silencing and OXA treatment repressed tumor growth in the GC mice model. Exosomal circ_0032821 boosted OXA resistance of GC cells partly by the miR-515-5p/SOX9 axis, hinting a promising therapeutic target for GC treatment.