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1
Han, Yu, and Véronique Lefebvre. "L-Sox5 and Sox6 Drive Expression of the Aggrecan Gene in Cartilage by Securing Binding of Sox9 to a Far-Upstream Enhancer." Molecular and Cellular Biology 28, no. 16 (2008): 4999–5013. http://dx.doi.org/10.1128/mcb.00695-08.
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ABSTRACT The Sry-related high-mobility-group box transcription factor Sox9 recruits the redundant L-Sox5 and Sox6 proteins to effect chondrogenesis, but the mode of action of the trio remains unclear. We identify here a highly conserved 359-bp sequence 10 kb upstream of the Agc1 gene for aggrecan, a most essential cartilage proteoglycan and key marker of chondrocyte differentiation. This sequence directs expression of a minimal promoter in both embryonic and adult cartilage in transgenic mice, in a manner that matches Agc1 expression. The chondrogenic trio is required and sufficient to mediate the activity of this enhancer. It acts directly, Sox9 binding to a critical cis-acting element and L-Sox5/Sox6 binding to three additional elements, which are cooperatively needed. Upon binding to their specific sites, L-Sox5/Sox6 increases the efficiency of Sox9 binding to its own recognition site and thereby robustly potentiates the ability of Sox9 to activate the enhancer. L-Sox5/Sox6 similarly secures Sox9 binding to Col2a1 (encoding collagen-2) and other cartilage-specific enhancers. This study thus uncovers critical cis-acting elements and transcription factors driving Agc1 expression in cartilage and increases understanding of the mode of action of the chondrogenic Sox trio.
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Smits, Patrick, Peter Dy, Srijeet Mitra, and Véronique Lefebvre. "Sox5 and Sox6 are needed to develop and maintain source, columnar, and hypertrophic chondrocytes in the cartilage growth plate." Journal of Cell Biology 164, no. 5 (2004): 747–58. http://dx.doi.org/10.1083/jcb.200312045.
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Sox5 and Sox6 encode Sry-related transcription factors that redundantly promote early chondroblast differentiation. Using mouse embryos with three or four null alleles of Sox5 and Sox6, we show that they are also essential and redundant in major steps of growth plate chondrocyte differentiation. Sox5 and Sox6 promote the development of a highly proliferating pool of chondroblasts between the epiphyses and metaphyses of future long bones. This pool is the likely cellular source of growth plates. Sox5 and Sox6 permit formation of growth plate columnar zones by keeping chondroblasts proliferating and by delaying chondrocyte prehypertrophy. They allow induction of chondrocyte hypertrophy and permit formation of prehypertrophic and hypertrophic zones by delaying chondrocyte terminal differentiation induced by ossification fronts. They act, at least in part, by down-regulating Ihh signaling, Fgfr3, and Runx2 and by up-regulating Bmp6. In conclusion, Sox5 and Sox6 are needed for the establishment of multilayered growth plates, and thereby for proper and timely development of endochondral bones.
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Cantù, Claudio, Rossella Ierardi, Ilaria Alborelli, et al. "Sox6 enhances erythroid differentiation in human erythroid progenitors." Blood 117, no. 13 (2011): 3669–79. http://dx.doi.org/10.1182/blood-2010-04-282350.
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Abstract Sox6 belongs to the Sry (sex-determining region Y)–related high-mobility-group–box family of transcription factors, which control cell-fate specification of many cell types. Here, we explored the role of Sox6 in human erythropoiesis by its overexpression both in the erythroleukemic K562 cell line and in primary erythroid cultures from human cord blood CD34+ cells. Sox6 induced significant erythroid differentiation in both models. K562 cells underwent hemoglobinization and, despite their leukemic origin, died within 9 days after transduction; primary erythroid cultures accelerated their kinetics of erythroid maturation and increased the number of cells that reached the final enucleation step. Searching for direct Sox6 targets, we found SOCS3 (suppressor of cytokine signaling-3), a known mediator of cytokine response. Sox6 was bound in vitro and in vivo to an evolutionarily conserved regulatory SOCS3 element, which induced transcriptional activation. SOCS3 overexpression in K562 cells and in primary erythroid cells recapitulated the growth inhibition induced by Sox6, which demonstrates that SOCS3 is a relevant Sox6 effector.
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Guimont, Philippe, Francine Grondin, and Claire M. Dubois. "Sox9-dependent transcriptional regulation of the proprotein convertase furin." American Journal of Physiology-Cell Physiology 293, no. 1 (2007): C172—C183. http://dx.doi.org/10.1152/ajpcell.00349.2006.
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The proprotein convertase furin participates in the maturation/bioactivation of a variety of proproteins involved in chondrogenesis events. These include parathyroid hormone-related peptide (PTHrP), an autocrine/paracrine factor that is crucial to both normal cartilage development and cartilage-related pathological processes. Despite the known importance of furin activity in the bioactivation of the polypeptides, the mechanisms that control furin regulation in chondrogenesis remain unknown. To gain insight into the molecular regulation of furin, we used the mouse prechondrogenic ATDC5 cell line, an established in vitro model of cartilage differentiation. Peak expression of both furin mRNA and furin PTHrP maturation was observed during chondrocyte nodule formation stage, an event that correlated with increased mRNA levels of Sox9, a potent high-mobility-group (HMG) box-containing transcription factor required for cartilage formation. Inhibition of furin activity led to a diminution in maturation of PTHrP, suggesting a relationship between Sox9-induced regulation of furin and chondrogenesis events. Transient transfection of Sox9 in nonchondrogenic cells resulted in a marked increase in furin mRNA and in the transactivation of the furin P1A promoter. Direct Sox9 action on the P1A promoter was narrowed down to a critical paired site with Sox9 binding capability in vitro and in vivo. Sox9 transactivation effect was inhibited by L-Sox5 and Sox-6, two Sox9 homologs also expressed in ATDC5 cells. Sox6 inhibitory effect was reduced when using Sox6-HMG-box mutants, indicating a repressive effect through direct HMG-box/DNA binding. Our work suggests a mechanism by which furin is regulated during chondrogenesis. It also adds to the complexity of Sox molecule interaction during gene regulation.
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Amano, Katsuhiko, Kenji Hata, Atsushi Sugita, et al. "Sox9 Family Members Negatively Regulate Maturation and Calcification of Chondrocytes through Up-Regulation of Parathyroid Hormone–related Protein." Molecular Biology of the Cell 20, no. 21 (2009): 4541–51. http://dx.doi.org/10.1091/mbc.e09-03-0227.
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Sox9 is a transcription factor that plays an essential role in chondrogenesis and has been proposed to inhibit the late stages of endochondral ossification. However, the molecular mechanisms underlying the regulation of chondrocyte maturation and calcification by Sox9 remain unknown. In this study, we attempted to clarify roles of Sox9 in the late stages of chondrocyte differentiation. We found that overexpression of Sox9 alone or Sox9 together with Sox5 and Sox6 (Sox5/6/9) inhibited the maturation and calcification of murine primary chondrocytes and up-regulated parathyroid hormone–related protein (PTHrP) expression in primary chondrocytes and the mesenchymal cell line C3H10T1/2. Sox5/6/9 stimulated the early stages of chondrocyte proliferation and development. In contrast, Sox5/6/9 inhibited maturation and calcification of chondrocytes in organ culture. The inhibitory effects of Sox5/6/9 were rescued by treating with anti-PTHrP antibody. Moreover, Sox5/6/9 bound to the promoter region of the PTHrP gene and up-regulated PTHrP gene promoter activity. Interestingly, we also found that the Sox9 family members functionally collaborated with Ihh/Gli2 signaling to regulate PTHrP expression and chondrocyte differentiation. Our results provide novel evidence that Sox9 family members mediate endochondral ossification by up-regulating PTHrP expression in association with Ihh/Gli2 signaling.
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Binlateh, Thunwa, Supita Tanasawet, Onnicha Rattanaporn, Wanida Sukketsiri, and Pilaiwanwadee Hutamekalin. "Metformin Promotes Neuronal Differentiation via Crosstalk between Cdk5 and Sox6 in Neuroblastoma Cells." Evidence-Based Complementary and Alternative Medicine 2019 (February 19, 2019): 1–13. http://dx.doi.org/10.1155/2019/1765182.
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Metformin has recently emerged as a key player in promotion of neuroblastoma differentiation and neurite outgrowth. However, molecular mechanisms of how metformin promotes cellular differentiation have not yet been fully elucidated. In this study, we investigated how metformin promotes cell differentiation, via an inhibition of cell proliferation, by culturing SH-SY5Y neuroblastoma cells with or without metformin. Pretreatment with reactive oxygen species (ROS) scavenger, NAC, revealed that ROS plays a crucial role in induction of cell differentiation. Cell differentiation was observed under various morphological criteria: extension of neuritic processes and neuronal differentiation markers. Treatment with metformin significantly increased neurite length, number of cells with neurite, and expression of neuronal differentiation markers, β-tubulin III and tyrosine hydroxylase (TH) compared with untreated control. Further investigation found that metformin significantly decreased Cdk5 but increased Sox6 during cell differentiation. Analysis of the mechanism underlying these changes using Cdk5 inhibitor, roscovitine, indicated that expressions of Cdk5 and Sox6 corresponded to metformin treatment. These results suggested that metformin produces neuronal differentiation via Cdk5 and Sox6. In addition, phosphorylated Erk1/2 was decreased while phosphorylated Akt was increased in metformin treatment. Taken together, these findings suggest that metformin promotes neuronal differentiation via ROS activation through Cdk5/Sox6 crosstalk, relating to Erk1/2 and Akt signaling.
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Haseeb, Abdul, and Véronique Lefebvre. "The SOXE transcription factors—SOX8, SOX9 and SOX10—share a bi-partite transactivation mechanism." Nucleic Acids Research 47, no. 13 (2019): 6917–31. http://dx.doi.org/10.1093/nar/gkz523.
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Abstract SOX8, SOX9 and SOX10 compose the SOXE transcription factor group. They govern cell fate and differentiation in many lineages, and mutations impairing their activity cause severe diseases, including campomelic dysplasia (SOX9), sex determination disorders (SOX8 and SOX9) and Waardenburg-Shah syndrome (SOX10). However, incomplete knowledge of their modes of action limits disease understanding. We here uncover that the proteins share a bipartite transactivation mechanism, whereby a transactivation domain in the middle of the proteins (TAM) synergizes with a C-terminal one (TAC). TAM comprises amphipathic α-helices predicted to form a protein-binding pocket and overlapping with minimal transactivation motifs (9-aa-TAD) described in many transcription factors. One 9-aa-TAD sequence includes an evolutionarily conserved and functionally required EΦ[D/E]QYΦ motif. SOXF proteins (SOX7, SOX17 and SOX18) contain an identical motif, suggesting evolution from a common ancestor already harboring this motif, whereas TAC and other transactivating SOX proteins feature only remotely related motifs. Missense variants in this SOXE/SOXF-specific motif are rare in control individuals, but have been detected in cancers, supporting its importance in development and physiology. By deepening understanding of mechanisms underlying the central transactivation function of SOXE proteins, these findings should help further decipher molecular networks essential for development and health and dysregulated in diseases.
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Dumitriu, Bogdan, Michael R. Patrick, Jane P. Petschek, et al. "Sox6 cell-autonomously stimulates erythroid cell survival, proliferation, and terminal maturation and is thereby an important enhancer of definitive erythropoiesis during mouse development." Blood 108, no. 4 (2006): 1198–207. http://dx.doi.org/10.1182/blood-2006-02-004184.
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Abstract Erythropoiesis, the essential process of hematopoietic stem cell development into erythrocytes, is controlled by lineage-specific transcription factors that determine cell fate and differentiation and by the hormone erythropoietin that stimulates cell survival and proliferation. Here we identify the Sry-related high-mobility-group (HMG) box transcription factor Sox6 as an important enhancer of definitive erythropoiesis. Sox6 is highly expressed in proerythroblasts and erythroblasts in the fetal liver, neonatal spleen, and bone marrow. Mouse fetuses and pups lacking Sox6 develop erythroid cells slowly and feature misshapen, short-lived erythrocytes. They compensate for anemia by elevating the serum level of erythropoietin and progressively enlarging their erythropoietic tissues. Erythroid-specific inactivation of Sox6 causes the same phenotype, demonstrating cell-autonomous roles for Sox6 in erythroid cells. Sox6 potentiates the ability of erythropoietin signaling to promote proerythroblast survival and has an effect additive to that of erythropoietin in stimulating proerythroblast and erythroblast proliferation. Sox6 also critically facilitates erythroblast and reticulocyte maturation, including hemoglobinization, cell condensation, and enucleation, and ensures erythrocyte cytoskeleton long-term stability. It does not control adult globin and erythrocyte cytoskeleton genes but acts by stabilizing filamentous actin (F-actin) levels. Sox6 thus enhances erythroid cell development at multiple levels and thereby ensures adequate production and quality of red blood cells.
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Li, Yi, Ming Xiao, and Fangchun Guo. "The role of Sox6 and Netrin-1 in ovarian cancer cell growth, invasiveness, and angiogenesis." Tumor Biology 39, no. 5 (2017): 101042831770550. http://dx.doi.org/10.1177/1010428317705508.
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SOX6 plays important roles in cell proliferation, differentiation, and cell fate determination. It has been confirmed that SOX6 is a tumor suppressor and downregulated in various cancers, including esophageal squamous cell carcinoma, hepatocellular carcinoma, and chronic myeloid leukemia. Netrin-1 is highly expressed in various human cancers and acts as an anti-apoptotic and proangiogenic factor to drive tumorigenesis. The role of SOX6 and netrin-1 in regulating the growth of ovarian tumor cells still remains unclear. Real-time polymerase chain reaction and western blot were used to determine the SOX6 messenger RNA and protein levels, respectively, in ovarian cancer cells and tumor tissues. Stable transfection of SOX6 was conducted to overexpress SOX6 in PA-1 and SW626 cells. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Invasion of ovarian cancer cells and migration of human umbilical vein endothelial cells were confirmed by Transwell assays. To overexpress netrin-1, ovarian cancer cells with SOX6 restoration was transduced with netrin-1 lentiviral particles. PA-1 xenografts in a nude mice model were used to conduct in vivo evaluation of the role of SOX6 and its relationship with netrin-1 in tumor growth and angiogenesis. In this study, we found significantly reduced SOX6 levels in PA-1, SW626, SK-OV-3, and CaoV-3 ovarian cancer cell lines and human tumor tissues in comparison with normal human ovarian epithelial cells or matched non-tumor tissues. SOX6 overexpression by stable transfection dramatically inhibited proliferation and invasion of PA-1 and SW626 cells. Also, conditioned medium from PA-1 and SW626 cells with SOX6 restoration exhibited reduced ability to induce human umbilical vein endothelial cells migration and tube formation compared with conditioned medium from the cells with transfection control. Furthermore, an inverse relationship between SOX6 and netrin-1 expression was observed in PA-1 and SW626 cells. Overexpression of netrin-1 in ovarian cancer cells with forced SOX6 expression remarkably abrogated the inhibitory effect of SOX6 on proliferation, invasion of the cells, and tumor xenograft growth and vascularity in vivo. Human umbilical vein endothelial cell migration and tube formation were enhanced in the conditioned medium from the ovarian cancer cells transduced with netrin-1 lentivirus particles. Our observations revealed that SOX6 is a tumor suppressor in ovarian cancer cells, and SOX6 exerts an inhibitory effect on the proliferation, invasion, and tumor cell-induced angiogenesis of ovarian cancer cells, whereas nerin-1 plays an opposite role and its expression is inversely correlated with SOX6. Moreover, our findings suggest a new role of SOX6 and netrin-1 for understanding the progression of ovarian cancer and have the potential for the development of new diagnosis and treatment strategies for ovarian cancer.
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Suzuki, Hidetsugu, Yoshiaki Ito, Masahiro Shinohara, et al. "Gene targeting of the transcription factor Mohawk in rats causes heterotopic ossification of Achilles tendon via failed tenogenesis." Proceedings of the National Academy of Sciences 113, no. 28 (2016): 7840–45. http://dx.doi.org/10.1073/pnas.1522054113.
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Cell-based or pharmacological approaches for promoting tendon repair are currently not available because the molecular mechanisms of tendon development and healing are not well understood. Although analysis of knockout mice provides many critical insights, small animals such as mice have some limitations. In particular, precise physiological examination for mechanical load and the ability to obtain a sufficient number of primary tendon cells for molecular biology studies are challenging using mice. Here, we generated Mohawk (Mkx)−/− rats by using CRISPR/Cas9, which showed not only systemic hypoplasia of tendons similar to Mkx−/− mice, but also earlier heterotopic ossification of the Achilles tendon compared with Mkx−/− mice. Analysis of tendon-derived cells (TDCs) revealed that Mkx deficiency accelerated chondrogenic and osteogenic differentiation, whereas Mkx overexpression suppressed chondrogenic, osteogenic, and adipogenic differentiation. Furthermore, mechanical stretch stimulation of Mkx−/− TDCs led to chondrogenic differentiation, whereas the same stimulation in Mkx+/+ TDCs led to formation of tenocytes. ChIP-seq of Mkx overexpressing TDCs revealed significant peaks in tenogenic-related genes, such as collagen type (Col)1a1 and Col3a1, and chondrogenic differentiation-related genes, such as SRY-box (Sox)5, Sox6, and Sox9. Our results demonstrate that Mkx has a dual role, including accelerating tendon differentiation and preventing chondrogenic/osteogenic differentiation. This molecular network of Mkx provides a basis for tendon physiology and tissue engineering.
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Zhang, Zihao, Shudai Lin, Wen Luo, et al. "Sox6 Differentially Regulates Inherited Myogenic Abilities and Muscle Fiber Types of Satellite Cells Derived from Fast- and Slow-Type Muscles." International Journal of Molecular Sciences 23, no. 19 (2022): 11327. http://dx.doi.org/10.3390/ijms231911327.
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Adult skeletal muscle is primarily divided into fast and slow-type muscles, which have distinct capacities for regeneration, metabolism and contractibility. Satellite cells plays an important role in adult skeletal muscle. However, the underlying mechanisms of satellite cell myogenesis are poorly understood. We previously found that Sox6 was highly expressed in adult fast-type muscle. Therefore, we aimed to validate the satellite cell myogenesis from different muscle fiber types and investigate the regulation of Sox6 on satellite cell myogenesis. First, we isolated satellite cells from fast- and slow-type muscles individually. We found that satellite cells derived from different muscle fiber types generated myotubes similar to their origin types. Further, we observed that cells derived from fast muscles had a higher efficiency to proliferate but lower potential to self-renew compared to the cells derived from slow muscles. Then we demonstrated that Sox6 facilitated the development of satellite cells-derived myotubes toward their inherent muscle fiber types. We revealed that higher expression of Nfix during the differentiation of fast-type muscle-derived myogenic cells inhibited the transcription of slow-type isoforms (MyH7B, Tnnc1) by binding to Sox6. On the other hand, Sox6 activated Mef2C to promote the slow fiber formation in slow-type muscle-derived myogenic cells with Nfix low expression, showing a different effect of Sox6 on the regulation of satellite cell development. Our findings demonstrated that satellite cells, the myogenic progenitor cells, tend to develop towards the fiber type similar to where they originated. The expression of Sox6 and Nfix partially explain the developmental differences of myogenic cells derived from fast- and slow-type muscles.
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Anderson, Douglas M., Rajani George, Marcus B. Noyes, et al. "Characterization of the DNA-binding Properties of the Mohawk Homeobox Transcription Factor." Journal of Biological Chemistry 287, no. 42 (2012): 35351–59. http://dx.doi.org/10.1074/jbc.m112.399386.
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The homeobox transcription factor Mohawk (Mkx) is a potent transcriptional repressor expressed in the embryonic precursors of skeletal muscle, cartilage, and bone. MKX has recently been shown to be a critical regulator of musculoskeletal tissue differentiation and gene expression; however, the genetic pathways through which MKX functions and its DNA-binding properties are currently unknown. Using a modified bacterial one-hybrid site selection assay, we determined the core DNA-recognition motif of the mouse monomeric Mkx homeodomain to be A-C-A. Using cell-based assays, we have identified a minimal Mkx-responsive element (MRE) located within the Mkx promoter, which is composed of a highly conserved inverted repeat of the core Mkx recognition motif. Using the minimal MRE sequence, we have further identified conserved MREs within the locus of Sox6, a transcription factor that represses slow fiber gene expression during skeletal muscle differentiation. Real-time PCR and immunostaining of in vitro differentiated muscle satellite cells isolated from Mkx-null mice revealed an increase in the expression of Sox6 and down-regulation of slow fiber structural genes. Together, these data identify the unique DNA-recognition properties of MKX and reveal a novel role for Mkx in promoting slow fiber type specification during skeletal muscle differentiation.
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Ji, Jing, Ya-Qin Sun, Zheng Zha, et al. "Bu Shen Yi Sui Capsules Promote Remyelination by Regulating MicroRNA-219 and MicroRNA-338 in Exosomes to Promote Oligodendrocyte Precursor Cell Differentiation." Evidence-Based Complementary and Alternative Medicine 2022 (April 13, 2022): 1–19. http://dx.doi.org/10.1155/2022/3341481.
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Remyelination is a refractory feature of demyelinating diseases such as multiple sclerosis (MS). Studies have shown that promoting oligodendrocyte precursor cell (OPC) differentiation, which cannot be achieved by currently available therapeutic agents, is the key to enhancing remyelination. Bu Shen Yi Sui capsule (BSYSC) is a traditional Chinese herbal medicine over many years of clinical practice. We have found that BSYSC can effectively treat MS. In this study, the effects of BSYSC in promoting OPCs differentiation and remyelination were assessed using an experimental autoimmune encephalomyelitis (EAE) model in vivo and cultured OPCs in vitro. The results showed that BSYSC reduced clinical function scores and increased neuroprotection. The expression of platelet-derived growth factor receptor α (PDGFR-α) was decreased and the level of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) was increased in the brains and spinal cords of mice as well as in OPCs after treatment with BSYSC. We further found that BSYSC elevated the expression of miR-219 or miR-338 in the serum exosomes of mice with EAE, thereby suppressing the expression of Sox6, Lingo1, and Hes5, which negatively regulate OPCs differentiation. Therefore, serum exosomes of BSYSC-treated mice (exos-BSYSC) were extracted and administered to OPCs in which miR-219 or miR-338 expression was knocked down by adenovirus, and the results showed that Sox6, Lingo1, and Hes5 expression was downregulated, MBP expression was upregulated, OPCs differentiation was increased, and the ability of OPCs to wrap around neuronal axons was improved. In conclusion, BSYSC may exert clinically relevant effects by regulating microRNA (miR) levels in exosomes and thus promoting the differentiation and maturation of OPCs.
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Dinh, Minhan L., Yao Dong, and Nobuko Hagiwara. "Evolutionary conservation of the role of Sox6 in terminal differentiation of skeletal muscle." Developmental Biology 344, no. 1 (2010): 533. http://dx.doi.org/10.1016/j.ydbio.2010.05.402.
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Chang, Yao-Jen, Zhifu Kang, Jiayuan Bei, et al. "Generation of TRIM28 Knockout K562 Cells by CRISPR/Cas9 Genome Editing and Characterization of TRIM28-Regulated Gene Expression in Cell Proliferation and Hemoglobin Beta Subunits." International Journal of Molecular Sciences 23, no. 12 (2022): 6839. http://dx.doi.org/10.3390/ijms23126839.
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TRIM28 is a scaffold protein that interacts with DNA-binding proteins and recruits corepressor complexes to cause gene silencing. TRIM28 contributes to physiological functions such as cell growth and differentiation. In the chronic myeloid leukemia cell line K562, we edited TRIM28 using CRISPR/Cas9 technology, and the complete and partial knockout (KO) cell clones were obtained and confirmed using quantitative droplet digital PCR (ddPCR) technology. The amplicon sequencing demonstrated no off-target effects in our gene editing experiments. The TRIM28 KO cells grew slowly and appeared red, seeming to have a tendency towards erythroid differentiation. To understand how TRIM28 controls K562 cell proliferation and differentiation, transcriptome profiling analysis was performed in wild-type and KO cells to identify TRIM28-regulated genes. Some of the RNAs that encode the proteins regulating the cell cycle were increased (such as p21) or decreased (such as cyclin D2) in TRIM28 KO cell clones; a tumor marker, the MAGE (melanoma antigen) family, which is involved in cell proliferation was reduced. Moreover, we found that knockout of TRIM28 can induce miR-874 expression to downregulate MAGEC2 mRNA via post-transcriptional regulation. The embryonic epsilon-globin gene was significantly increased in TRIM28 KO cell clones through the downregulation of transcription repressor SOX6. Taken together, we provide evidence to demonstrate the regulatory network of TRIM28-mediated cell growth and erythroid differentiation in K562 leukemia cells.
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Snyder, Marylynn, Xin-Yun Huang, and J. Jillian Zhang. "Stat3 is essential for neuronal differentiation through direct transcriptional regulation of the Sox6 gene." FEBS Letters 585, no. 1 (2010): 148–52. http://dx.doi.org/10.1016/j.febslet.2010.11.030.
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Li, Xianhui, Jiaji Wang, Zhuqing Jia, et al. "MiR-499 Regulates Cell Proliferation and Apoptosis during Late-Stage Cardiac Differentiation via Sox6 and Cyclin D1." PLoS ONE 8, no. 9 (2013): e74504. http://dx.doi.org/10.1371/journal.pone.0074504.
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Li, Wang, Jie Fang, Jingxuan Shen, et al. "MicroRNA-135a-5p promotes neuronal differentiation of pluripotent embryonal carcinoma cells by repressing Sox6/CD44 pathway." Biochemical and Biophysical Research Communications 509, no. 2 (2019): 603–10. http://dx.doi.org/10.1016/j.bbrc.2018.12.162.
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Shearstone, Jeffrey R., Olga Golonzhka, Apurva Chonkar, and Matthew Jarpe. "Pharmacological Inhibition of Histone Deacetylases 1 and 2 (HDAC1/2) Induces Fetal Hemoglobin (HbF) through Activation of Gata2." Blood 124, no. 21 (2014): 335. http://dx.doi.org/10.1182/blood.v124.21.335.335.
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Abstract Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease, and could also be effective in treating beta-thalassemia. Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of the fetal beta-like globin gene (HbG) transcript (Bradner JE, Proc Natl Acad Sci, 2010). We have previously shown that selective chemical inhibitors of HDAC1/2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). In this work, we have utilized our proof of concept molecule ACY-957, a selective inhibitor of HDAC1/2, to discover a novel role for Gata2 in the activation of HbG. To identify genes affected by HDAC1/2 inhibition, CD34+ bone marrow cells undergoing erythroid differentiation were treated with ACY-957 or vehicle, followed by mRNA expression profiling. Among the genes differentially regulated by both pharmacological inhibition and genetic ablation of HDAC1/2 were Bcl11a and Sox6, known HbG repressors, and Gata2, a potential HbG activator. Quantitative real time PCR (QRT-PCR) time course experiments confirmed that ACY-957 treatment leads to a 2-fold and 10-fold decrease in Bcl11A and Sox6, respectively, and an 8-fold increase in Gata2 mRNA. Unlike Bcl11a and Sox6, Gata2 induction by ACY-957 was highly correlated with HbG induction, suggesting a possible role for this transcription factor in the direct activation of HbG. To investigate this possibility, lentiviral infection was utilized to overexpress full length Gata2 transcript in differentiating primary erythroblasts. After 5 days of differentiation, Gata2 overexpression resulted in a 2.5-fold increase in HbG mRNA, while the level of the major adult beta-like globin chain (HbB) mRNA was unaffected. HbG mRNA remained elevated by Gata2 overexpression at day 7 of differentiation, while HbB was reduced by 1.6-fold. Gata2 overexpression appeared to have minimal effect on cell differentiation, as determined by the cell surface markers CD71 and GlycophorinA, a finding consistent with observations in ACY-957 treated cells with elevated Gata2. Furthermore, lentiviral delivery of short hairpin RNA (shRNA) targeting Gata2, attenuated HbG induction by ACY-957. These data suggest that elevated levels of Gata2 resulting from HDAC1/2 inhibition is sufficient to induce HbG at early stages of erythroid cell differentiation. To understand how HDAC1/2 inhibition drives Gata2 activation, chromatin immunoprecipitation coupled with either next generation sequencing (ChIP-seq) or QRT-PCR was performed in ACY-957 and vehicle treated cells. HDAC1 and HDAC2 were present throughout the Gata2 gene body and promoter regions, and HDAC1/2 binding levels were highly correlated, suggesting co-occupancy of these enzymes at this locus. ACY-957 treatment led to elevated histone acetylation at previously described Gata2 gene regulatory regions (Bresnick et. al. 2010, J Biol Chem). Specifically, the -1.8 kb and -2.8 kb regulatory regions showed a 6-fold increase in histone H3K9 and H2BK5 acetylation, while the +9.5 kb and -3.9 kb regions showed a 3-fold increase. The Gata2 protein showed increased binding at these regulatory regions in response to ACY-957 treatment, with a maximum increase of 3-fold at the -1.8 kb region. This finding is consistent with the known positive autoregulation of the Gata2 gene. Taken together, these data suggest that selective inhibition of HDAC1/2 leads to elevated Gata2 through acetylation-induced activation of a positive autoregulatory loop. The tight temporal correlation between Gata2 and HbG activation following HDAC1/2 inhibition argues that Gata2 may affect the beta-globin locus directly. ChIP-seq data across the 70-kb beta-globin locus demonstrated that ACY-957 treatment altered Gata2 binding only at a single region, lying within the promoter for delta globin. This region is suspected in playing a role in switching from fetal to adult globin during development, as naturally occurring deletions of this region are associated with elevated fetal hemoglobin in adults (Sankaran et. al. 2011, NEJM). Whether the change in GATA2 binding to this region is responsible for the increased expression of HbG in cells treated with HDAC1/2-selective inhibitors is under investigation. Disclosures Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Golonzhka:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Chonkar:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.
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Zhang, Zhilong, Min Chu, Qi Bao, et al. "Two Different Copy Number Variations of the SOX5 and SOX8 Genes in Yak and Their Association with Growth Traits." Animals 12, no. 12 (2022): 1587. http://dx.doi.org/10.3390/ani12121587.
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Copy number variation (CNV) is a structural variant with significant impact on genetic diversity. CNV has been widely used in breeding for growth traits, meat production or quality, and coat color. SRY-like box genes (SOXs) are a class of transcription factors that play a regulatory role in cell fate specification and differentiation. SOX5 and SOX8 belong to subgroups D and E of the SOXs, respectively. Previous studies have shown that SOX5 and SOX8 are essential in the development of bones. In this study, we explored the association between the growth traits and CNVs of SOX5 and SOX8 in 326 Ashidan yaks and detected mRNA expression levels in different tissues. Our results illustrated that CNVs of SOX5 and SOX8 were significantly associated with withers height at 18 months of age and chest girth at 30 months of age (p < 0.05). The CNV combination of SOX5 and SOX8 was significantly associated with withers height at 18 months of age (p < 0.01). SOX5 expression in the lung was significantly higher than in the heart, spleen, kidney, and muscle (p < 0.05). SOX8 expression in the lung was significantly higher than in the liver and muscle (p < 0.05). Our results provide evidence that the CNVs of SOX5 and SOX8 genes could be used as new markers for the selection of yak growth traits.
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Dulmovits, Brian M., Abena O. Appiah-Kubi, Julien Papoin, et al. "Pomalidomide Transcriptionally Reprograms Adult Erythroid Progenitors Independently of Ikaros Proteasomal Degradation." Blood 126, no. 23 (2015): 160. http://dx.doi.org/10.1182/blood.v126.23.160.160.
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Abstract Pomalidomide, a second-generation immunomodulatory drug, is a fetal hemoglobin (HbF) inducing agent with potential implications for the treatment of β-hemoglobinopathies such as sickle cell disease (SCD). However, its mechanism of action remains unknown. Through an in-depth characterization of human erythropoiesis and globin gene regulatory networks, we now provide evidence that pomalidomide alters transcription networks involved in erythropoiesis and globin switching, thereby leading to a partial reprogramming of adult hematopoietic progenitors toward fetal-like erythropoiesis. Adult peripheral blood CD34+ cells from normal individuals were differentiated toward the red cell lineage using an adapted 3-phase culture system. At day 14 of culture, we observed a reciprocal globin gene switch at the mRNA and protein levels. These results were confirmed by high performance liquid chromatography of hemolysates (HbF/(HbF+HbA): 31.7 ± 1.4% vs. 6.5 ± 0.7% pomalidomide and vehicle, respectively). Next, we studied erythroid differentiation using flow cytometric analyses of the cell surface markers interleukin-3R (IL-3R), glycophorin A (GPA), CD34 and CD36 for early erythroid precursors (BFU-E and CFU-E) as well as GPA, α4-integrin and band3 for terminal erythroid differentiation. While there were no changes in terminal erythroblast maturation, an accumulation of BFU-E in pomalidomide-treated cultures at days 2 and 4 of differentiation was seen, indicating a delay at the BFU-E to CFU-E transition, and also, that pomalidomide exerts its effect in the early-stages of erythropoiesis. Indeed, treatment with pomalidomide during the phase of the culture system that generates erythroid progenitors led to significantly more γ-globin expression than treatment during the phase which proerythroblasts undergo terminal erythroid differentiation. At the molecular level, pomalidomide was found to rapidly and robustly decrease Ikaros (IKZF1) expression exclusively by post-translational targeting to the proteasome. Moreover, pomalidomide selectively reduced the expression of components of key globin regulatory pathways including BCL11A, SOX6, KLF1, GATA1 and LSD1 while not affecting others (e.g. CoREST, GATA2, GFI1B, and HDAC1). Pomalidomide had a transient effect on GATA1 and KLF1 expression. While shRNA knockdown of Ikaros using two different lentiviral constructs delayed erythroid differentiation, it failed to appreciably stimulate HbF production or alter BCL11A expression. These results suggest that the loss of Ikaros alone is insufficient to recapitulate the phenotype observed in pomalidomide-treated conditions. We next compared the expression levels of proteins involved in globin gene regulation among untreated peripheral blood, pomalidomide-treated peripheral blood and untreated cord blood-derived erythroid cells. We found striking similarities between cord blood and pomalidomide-treated adult cells at day 4 of differentiation. Indeed, BCL11A, KLF1, SOX6, LSD1 and GATA1 showed decreased expression levels both in cord blood and pomalidomide-treated adult peripheral blood, while the levels of CoREST, HDAC1 and GATA2 remained unchanged indicating that pomalidomide partially reprograms adult erythroid cells to a fetal-like state. Taken together, our results show that the mechanism underlying reactivation of HbF by pomalidomide involves Ikaros-independent reprogramming of adult erythroid progenitors. Finally, we found that this mechanism is conserved in SCD-derived CD34+ cells. Our work has broad implications for globin switching, as we provide direct evidence that Ikaros does not play a major role in the repression of γ-globin during adult erythropoiesis, and further supports the previously held notion that globin chain production is determined prior to or at the level of CFU-E. Disclosures Allen: Celgene: Research Funding; Bristol Myers Squibb: Equity Ownership; Onconova: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.
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Dulmovits, Brian M., Abena O. Appiah-Kubi, Julien Papoin, et al. "Pomalidomide Modulates Transcription Networks Regulating Human Erythropoiesis and Globin Switching: Implications for Treatment of Hemoglobinopathies." Blood 124, no. 21 (2014): 1375. http://dx.doi.org/10.1182/blood.v124.21.1375.1375.
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Abstract Sickle cell disease (SCD) represents a major challenge in hematology, with approximately 100,000 Americans afflicted and the annual number of newborns with SCD set to rise over the next 40 years worldwide. Current treatment approaches rely on increasing levels of fetal hemoglobin (HbF) to prevent painful vaso-occlusive crises and hemolysis secondary to red cell sickling. Hydroxyurea remains the only pharmacologic intervention approved for SCD; however, it has limited efficacy and carries significant side effects such as myelosuppression. Thus, there is a critical need to develop drugs that enhance HbF production without similar dose limiting side effects. Second generation immunomodulatory drugs, such as pomalidomide, are a class of emerging HbF inducers both in vitro and in vivo. Recent work from our laboratory revealed that hydroxyurea and pomalidomide differentially regulate HbF production in CD34+ cells undergoing erythroid differentiation using a 3-phase culture system. Pomalidomide, but not hydroxyurea, was found to decrease BCL11A expression through a yet to be defined mechanism. In the present study, we sought to characterize erythropoiesis and the expression of key transcription factor networks in this 3-phase culture system to determine the mechanisms underlying pomalidomide’s effect. Following a four day expansion period, isolated CD34+ cells from the peripheral blood of SCD or normal individuals were differentiated along erythroid lineage in the presence of pomalidomide (1μM) or DMSO (vehicle) for 14 days. As an additional control, CD34+ cells were also treated with hydroxyurea (10μM). Proliferation and erythroid differentiation were assessed at 7, 11 and 14 days of culture. Although a 50% decrease in cell growth was noted in cells treated with hydroxyurea, no such decrement was found in control, DMSO and pomalidomide-treated cells. Moreover, pomalidomide produced a transient delay in erythroid differentiation between days 6 and 11 of culture phenotypically documented by flow cytometric analysis using glycophorin-A, α-4 integrin and band 3 as surface markers monitoring erythroid differentiation as well as morphologically by May-Grunwald Giemsa staining. In contrast, cells treated with hydroxyurea demonstrated accelerated differentiation, compared to the control cultures. However, by day 14 of culture, no significant difference was observed under any condition, suggesting that the delayed cells eventually finished terminal differentiation. In terms of HbF induction, we confirmed elevated production in the cultures with pomalidomide by measuring the number of F-cells by flow cytometry. We also evaluated the production of γ-globin chains by qRT-PCR and western blot at D4 and D11 and found a dramatic increase in the production of γ-globin, in both SCD and normal samples treated with pomalidomide. We posited that pomalidomide might foster changes in transcription factors known to play a role in both erythropoiesis and globin switching. To this end, we evaluated the expression kinetics of BCL11A, SOX6, KLF1, MI2β, GATA1 and FOG1 via qRT-PCR and western blot analyses. In DMSO-treated cultures the above transcription factors were maximally expressed between days 6-8, and their levels diminished during the remainder of the culture. Conversely, pomalidomide markedly decreased BCL11A, SOX6, KLF1 and MI2β between days 4 and 6 in cultures of both SCD and normal samples. In line with our results, MI2β acts as a positive regulator of BCL11A and KLF1, and previous studies have shown that its knock down in CD34 cells leads to decreased levels of BCL11A and KLF1. Western blot analyses confirmed the qRT-PCR data. Further, the divergent expression patterns correlated temporally with the differentiation delay suggesting that pomalidomide modulates expression of members of the BCL11Atranscription factor complex, thereby augmenting γ-globin production. Taken together, these data provide evidence that pomalidomide influences erythropoiesis by modulating transcription factor expression in CD34+ cells differentiated in the 3-phase culture system, leading to a decrease in BCL11A and activation of γ-globin production. Importantly, further exploration of these pathways that function to regulate erythropoiesis and promote HbF silencing, may help elucidate the mechanism of action of pomalidomide as well as identify additional druggable molecules. Disclosures No relevant conflicts of interest to declare.
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Shearstone, Jeffrey R., John H. van Duzer, Simon S. Jones, and Matthew Jarpe. "Mechanistic Insights Into Fetal Hemoglobin (HbF) Induction Through Chemical Inhibition Of Histone Deacetylase 1 and 2 (HDAC1/2)." Blood 122, no. 21 (2013): 2253. http://dx.doi.org/10.1182/blood.v122.21.2253.2253.
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Abstract Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). Fetal beta-like globin gene (HbG) expression is silenced in adults partly through the nucleosome remodeling and histone deacetylase (NuRD) complex, which contains HDAC1/2 (Sankaran VG, Science, 2008). Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of HbG expression (Bradner JE, Proc Natl Acad Sci, 2010). Furthermore, we have previously shown that selective chemical inhibitors of HDAC1 and 2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). However, the mechanism through which HDAC1/2 inhibition leads to activation of HbG remains largely unknown. In this work, we have utilized our proof of concept molecule, ACY-957, to investigate changes in gene expression and chromatin organization that result from inhibition of HDAC1/2. Gene expression profiling was performed on cells treated with ACY-957 (n=3) or vehicle (n=3) using Affymetrix PrimeView GeneChips. Treatment of early erythroblasts (CD71+, GlyA-) resulted in the up and down regulation of 1294 and 681 transcript probe sets, respectively. In comparison, treatment of late erythroblasts (CD71+, GlyA+) resulted in a total of 255 transcript probe set changes. This finding is consistent with follow-up experiments demonstrating that ACY-957 is unable to induce HbG in cells positive for both CD71 and GlyA. Taken together, these results suggest that erythroblasts become less responsive to HDAC inhibition as they mature. Gene set enrichment analysis using public domain data revealed that genes up- or down-regulated by HDAC1/2 shRNA knockdown are significantly overrepresented in the list of genes induced or repressed by ACY-957, respectively; suggesting pharmacologic inhibition of HDAC1/2 recapitulates genetic ablation. We also identified significant enrichment in other gene sets involving targets linked to HbG regulation, including lysine-specific demethylase 1 (LSD1) (Shi L, Nature Medicine, 2012). GeneChip and quantitative real-time PCR time course experiments show ACY-957 treatment leads to a decrease in Bcl11A (2-fold) and Sox6 (10-fold) mRNA, known repressors of fetal globin synthesis, and an increase in Klf2 (2-fold) and Gata2 (8-fold) mRNA, proposed fetal globin activators. This result is consistent with work by others that show Gata2 is suppressed, in part, by the NuRD complex (Hong W, EMBO Journal, 2005) and that Gata2 binding at the HbG promoter leads to increased levels of HbG expression (Zhu X, PLoS One, 2012). Interestingly, Gata2 induction preceded Sox6 suppression in ACY-957 treated cells and the Sox6 promoter contains 8 canonical WGATAR binding sites and one Gata2-specific binding motif, raising the possibility suppression of Sox6 by ACY-957 is mediated by Gata2 induction. To investigate these possibilities, we have performed chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) for HDAC1, HDAC2, Gata2, and the HDAC2-specific histone modification H3K56 in ACY-957 and vehicle treated cells. These experiments will be discussed. ChIP-seq data, both by itself and in combination with gene expression data, will provide further insight into the mechanism through which HDAC1/2 regulates HbF synthesis. Disclosures: Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.
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Kishi, M., K. Mizuseki, N. Sasai, et al. "Requirement of Sox2-mediated signaling for differentiation of early Xenopus neuroectoderm." Development 127, no. 4 (2000): 791–800. http://dx.doi.org/10.1242/dev.127.4.791.
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From early stages of development, Sox2-class transcription factors (Sox1, Sox2 and Sox3) are expressed in neural tissues and sensory epithelia. In this report, we show that Sox2 function is required for neural differentiation of early Xenopus ectoderm. Microinjection of dominant-negative forms of Sox2 (dnSox2) mRNA inhibits neural differentiation of animal caps caused by attenuation of BMP signals. Expression of dnSox2 in developing embryos suppresses expression of N-CAM and regional neural markers. We have analyzed temporal requirement of Sox2-mediated signaling by using an inducible dnSox2 construct fused to the ligand-binding domain of the glucocorticoid receptor. Attenuation of Sox2 function both from the late blastula stage and from the late gastrula stage onwards causes an inhibition of neural differentiation in animal caps and in whole embryos. Additionally, dnSox2-injected cells that fail to differentiate into neural tissues are not able to adopt epidermal cell fate. These data suggest that Sox2-class genes are essential for early neuroectoderm cells to consolidate their neural identity during secondary steps of neural differentiation.
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Hamada-Kanazawa, Michiko, Kyoko Ishikawa, Kaori Nomoto, et al. "Sox6 overexpression causes cellular aggregation and the neuronal differentiation of P19 embryonic carcinoma cells in the absence of retinoic acid." FEBS Letters 560, no. 1-3 (2004): 192–98. http://dx.doi.org/10.1016/s0014-5793(04)00086-9.
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Hamada-Kanazawa, Michiko, Kyoko Ishikawa, Daisuke Ogawa, et al. "Suppression of Sox6 in P19 cells leads to failure of neuronal differentiation by retinoic acid and induces retinoic acid-dependent apoptosis." FEBS Letters 577, no. 1-2 (2004): 60–66. http://dx.doi.org/10.1016/j.febslet.2004.09.063.
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Zhao, Youshan, Feng Xu, Juan Guo, Sida Zhao, Chunkang Chang, and Xiao Li. "Dysregulation of ANKRD11 Influenced Hematopoisis By Histone Acetylation-Mediated Gene Expression in Myelodysplastic Syndrome." Blood 128, no. 22 (2016): 4292. http://dx.doi.org/10.1182/blood.v128.22.4292.4292.
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Abstract Background and Object In addition to histone deacetylation, the importance of histone over-acetylation induced oncogene transcription in initiation and progression of myelodysplastic syndrome (MDS) has been proposed recently. Our previous whole-exome sequencing identified a new somatic mutation, ANKRD11, an important factor in histone acetylation regulation. Its roles in MDS pathophysiology need to be clarified. Methods The next generation target sequencing (Including ANKRD11) was carried out in 320 patients with MDS using the MiSeq Benchtop Sequencer. ANKRD11 mRNA expression in bone marrow of MDS was measured by real-time PCR. Loss and gain of function assay were carried out in myeloid cell lines K562, MEG-01£¬or SKM-1 to observe the influence on cell proliferation and differentiation . The levels of histone acetylation at H3 and H4 were detected by Western blot. Results Target sequencing in a cohort of 320 MDS patients identified 14 of ANKRD11 mutations (4.38%, Fig.1), which were confirmed by Sanger sequencing. Meanwhile, no ANKRD11 mutations in 100 normal controls were defined. ANKRD11 mutations occurred frequently in exons 10 and 9. The mRNA expression levels of ANKRD11 were significantly decreased in MDS patients, especially in ANKRD11mutant patients (Fig.2). ANKRD11 knockdown in K562 and MEG-1 resulted in growth inhibition, cell cycle arrest and erythroid/megakaryocytic differentiation retardant. In MDS cell line SKM-1, the arrested differentiation was rescued by over-expression of ANKRD11. Consistent with a role for ANKRD11 in histone acetylation, ANKRD11 KD increased acetylation of histones H3 and H4 at H3K14 and H4K5 and resulted in the upregulation of genes involved in differentiation inhibilation (SOX6, P21, et al). Finally, the ANKRD11 KD-mediated influence on cell proliferation and differentiation were reversed by inhibiting histone acetyltransferase activity. Conclusion Our assay defined that ANKRD11 was a crucial chromatin regulator that suppress histone acetylation and then decrease gene expression during myeloid differentiation, providing a likely explanation for its role in MDS pathogenesis. This study further support histone acetylase inhibitor as a potential treatment in MDS. Figure ANKRD11mutation distribution (a) and coexist with other mutations (b). Figure. ANKRD11mutation distribution (a) and coexist with other mutations (b). Figure The mRNA expression levels of ANKRD11in our MDS (A, C) subset and GEO data (B). Figure. The mRNA expression levels of ANKRD11in our MDS (A, C) subset and GEO data (B). Changes of histone acetylation in ANKRD11-KD cell line (MEG-01). ANKRD11 KD significantly increased acetylation of histones H3 and H4 at H3K14 and H4K5. Changes of histone acetylation in ANKRD11-KD cell line (MEG-01). ANKRD11 KD significantly increased acetylation of histones H3 and H4 at H3K14 and H4K5. Disclosures No relevant conflicts of interest to declare.
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Pongpaksupasin, Phitchapa, Tiwaporn Nualkaew, Suradej Hongeng, Suthat Fucharoen, Natee Jearawiriyapaisarn та Orapan Sripichai. "Lysine-Specific Histone Demethylase 1 Inhibition Enhances Robust Fetal Hemoglobin Induction in Human β0-Thalassemia/Hemoglobin E Rrythroid Cells". Hematology Reports 13, № 4 (2021): 9215. http://dx.doi.org/10.4081/hr.2021.9215.
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Induction of fetal hemoglobin (HbF) ameliorates the clinical severity of β-thalassemias. Histone methyltransferase LSD1 enzyme removes methyl groups from the activating chromatin mark histone 3 lysine 4 at silenced genes, including the γ-globin genes. LSD1 inhibitor RN-1 induces HbF levels in cultured human erythroid cells. Here, the HbF-inducing activity of RN-1 was investigated in erythroid progenitor cells derived from β0-thalassemia/HbE patients. The significant and reproducible increases in γ-globin transcript and HbF expression upon RN-1 treatment was demonstrated in erythroid cells with divergent HbF baseline levels, the average of HbF induction was 17.7 + 0.8%. RN-1 at low concentration did not affect viability and proliferation of erythroid cells, but decreases in cell number was observed in cells treated with RN-1 at high concentration. Delayed terminal erythroid differentiation was revealed in β0-thalassemia/HbE erythroid cells treated with RN-1 as similar to other compounds that target LSD1 activity. Downregulation of repressors of γ-globin expression; NCOR1 and SOX6, was observed in RN-1 treatment. These findings provide a proof of concept that a LSD1 epigenetic enzymes is a potential therapeutic target for β0-thalassemia/HbE patients.
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Danopoulos, Soula, Irving Alonso, Matthew E. Thornton, et al. "Human lung branching morphogenesis is orchestrated by the spatiotemporal distribution of ACTA2, SOX2, and SOX9." American Journal of Physiology-Lung Cellular and Molecular Physiology 314, no. 1 (2018): L144—L149. http://dx.doi.org/10.1152/ajplung.00379.2017.
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Lung morphogenesis relies on a number of important processes, including proximal-distal patterning, cell proliferation, migration and differentiation, as well as epithelial-mesenchymal interactions. In mouse lung development, SOX2+ cells are localized in the proximal epithelium, whereas SOX9+ cells are present in the distal epithelium. We show that, in human lung, expression of these transcription factors differs, in that during the pseudoglandular stage distal epithelial progenitors at the tips coexpress SOX2 and SOX9. This double-positive population was no longer present by the canalicular stages of development. As in mouse, the human proximal epithelial progenitors express solely SOX2 and are surrounded by smooth muscle cells (SMCs) both in the proximal airways and at the epithelial clefts. Upon Ras-related C3 botulinum toxin substrate 1 inhibition, we noted decreased branching, as well as increased SMC differentiation, attenuated peristalsis, and a reduction in the distal double-positive SOX2/SOX9 progenitor cell population. Thus, the presence of SOX2/SOX9 double-positive progenitor cells in the distal epithelium during the pseudoglandular stage of human lung development appears to be critical to proximal-distal patterning and lung branching. Moreover, SMCs promote a SOX2 proximal phenotype and seem to suppress the SOX9+ population.
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Li, Biaoru, Lianghao Ding, Chinrang Yang, Michael Story, and Betty S. Pace. "Transcription Factor Networks Involved in Fetal Stem Cell Erythropoiesis." Blood 120, no. 21 (2012): 3444. http://dx.doi.org/10.1182/blood.v120.21.3444.3444.
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Abstract Abstract 3444 CD34+ fetal stem cells (FSC) are primitive cells which can be isolated from umbilical cord blood. They have hematopoietic potential and can reconstitute the different cell lineages of the bone marrow similar to adult CD34+ stem cells. There are gaps in knowledge related to mechanisms of FSC differentiation which can be used to develop therapy for genetic diseases such as the hemoglobinopathies. Insights into fetal erythropoiesis can be gained by understanding mechanisms of globin gene regulation in the human β-globin locus where five functional genes (ε, Aγ, Gγ, δ, β-globin) are expressed in a developmentally regulated fashion. Globin gene expression during development is controlled in part by the locus control region however genomic controls during fetal erythropoiesis have not been clearly elucidated. Therefore, we utilized FSC isolated from umbilical cord blood induced to undergo differentiation as a model to characterize the transcriptome associated with the γ/β globin switch during fetal erythropoiesis. FSC (0.5 million) were grown in the one-phase liquid culture system containing stem cell factor (50ng/ml), interleukin-3 (10ng/ml) and erythropoietin (4U/ml). We observed a 30-fold higher proliferative capacity of FSC compared to adult progenitors reaching 10 billion cells by day-56 in culture. Erythroid differentiation was confirmed by Giemsa staining and increased expression of the differentiation biomarkers CD71 and CD235a and decline of CD34 was observed. Moreover, the γ/β globin switch occurred around day 45 in FSC compared to day 21 for adult stem cells (Li et al. BMC Genomics13:153, 2012). These data suggest that the γ-globin gene is activated for a longer period in the FSC environment in contrast to adult progenitors supporting different mechanisms of gene regulation in the two systems. To gain further insights, microarray analysis was performed on the HumanHT-12 v4 Expression BeadChip containing 47,000 probes, using triplicate RNA samples harvested on day 21, 42, 49 and 56. After data normalization by model-based background correction method, profile-1 genes with decreased expression from day-21 to day-56 (similar to γ-globin silencing) or profile-2 similar to β-globin activation from day-21 to day-56 were defined using Principal Component Analysis. Sixty-five profile-1 genes were subsequently identified by Gene Set Enrichment Analysis (GSEA) for erythroid-specific gene subsets. DAVID (Database for Annotation Visualization and Integrated Discovery) Ontology analysis confirmed 23 out of 65 erythroid genes including known regulators of γ-globin such as KLF4, KLF11, BCL11A and SIRT3. A similar analysis of profile-2 genes demonstrated 155 erythroid genes by GSEA and 28 factors were confirmed by DAVID platform related with β-globin regulation such as KLF1, GATA1, MXl1 and SOX6. To define transcription factor networks related to globin gene expression in FSC, the confirmed gene subsets were analyzed using the Michigan Molecular Interaction plugin for Cytoscape. A transcription factor network centered on KLF4 and GATA2 was defined; KLF4 positively regulates CREB, KLF6, GATA2, POU1F1, JUN and STAT3 while repressing Sp1. We previously demonstrated the ability of KLF4 to trans-activate γ-globin in adult stem cells. How these factors regulate γ-globin expression in FSC will be explored further. Similar studies for profile-2 genes revealed KLF1 and GATA1 as a major network hub. The special characteristics of fetal stem cell including high proliferative capacity and prolonged growth and γ-globin expression combined with a novel transcription factor network will be used to define genomic mechanisms of fetal erythropoiesis. Disclosures: No relevant conflicts of interest to declare.
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Akinyemi, Mabel O., Jessica Finucan, Anastasia Grytsay, et al. "Molecular Evolution and Inheritance Pattern of Sox Gene Family among Bovidae." Genes 13, no. 10 (2022): 1783. http://dx.doi.org/10.3390/genes13101783.
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Sox genes are an evolutionarily conserved family of transcription factors that play important roles in cellular differentiation and numerous complex developmental processes. In vertebrates, Sox proteins are required for cell fate decisions, morphogenesis, and the control of self-renewal in embryonic and adult stem cells. The Sox gene family has been well-studied in multiple species including humans but there has been scanty or no research into Bovidae. In this study, we conducted a detailed evolutionary analysis of this gene family in Bovidae, including their physicochemical properties, biological functions, and patterns of inheritance. We performed a genome-wide cataloguing procedure to explore the Sox gene family using multiple bioinformatics tools. Our analysis revealed a significant inheritance pattern including conserved motifs that are critical to the ability of Sox proteins to interact with the regulatory regions of target genes and orchestrate multiple developmental and physiological processes. Importantly, we report an important conserved motif, EFDQYL/ELDQYL, found in the SoxE and SoxF groups but not in other Sox groups. Further analysis revealed that this motif sequence accounts for the binding and transactivation potential of Sox proteins. The degree of protein–protein interaction showed significant interactions among Sox genes and related genes implicated in embryonic development and the regulation of cell differentiation. We conclude that the Sox gene family uniquely evolved in Bovidae, with a few exhibiting important motifs that drive several developmental and physiological processes.
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Formeister, Eric J., Ayn L. Sionas, David K. Lorance, Carey L. Barkley, Ginny H. Lee, and Scott T. Magness. "Distinct SOX9 levels differentially mark stem/progenitor populations and enteroendocrine cells of the small intestine epithelium." American Journal of Physiology-Gastrointestinal and Liver Physiology 296, no. 5 (2009): G1108—G1118. http://dx.doi.org/10.1152/ajpgi.00004.2009.
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SOX transcription factors have the capacity to modulate stem/progenitor cell proliferation and differentiation in a dose-dependent manner. SOX9 is expressed in the small intestine epithelial stem cell zone. Therefore, we hypothesized that differential levels of SOX9 may exist, influencing proliferation and/or differentiation of the small intestine epithelium. Sox9 expression levels in the small intestine were investigated using a Sox9 enhanced green fluorescent protein ( Sox9 EGFP) transgenic mouse. Sox9 EGFP levels correlate with endogenous SOX9 levels, which are expressed at two steady-state levels, termed Sox9 EGFPLO and Sox9 EGFPHI. Crypt-based columnar cells are Sox9 EGFPLO and demonstrate enriched expression of the stem cell marker, Lgr5. Sox9 EGFPHI cells express chromogranin A and substance P but do not express Ki67 and neurogenin3, indicating that Sox9 EGFPHI cells are postmitotic enteroendocrine cells. Overexpression of SOX9 in a crypt cell line stopped proliferation and induced morphological changes. These data support a bimodal role for SOX9 in the intestinal epithelium, where low SOX9 expression supports proliferative capacity, and high SOX9 expression suppresses proliferation.
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Steinberg Shemer, Orna, Marta Byrska-Bishop, Jacob C. Ulirsch, et al. "Temporally Distinct Developmental Waves of Erythropoiesis from Human Pluripotent Stem Cells." Blood 126, no. 23 (2015): 1170. http://dx.doi.org/10.1182/blood.v126.23.1170.1170.
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Abstract Mammalian erythropoiesis during embryogenesis occurs in several distinct stages or "waves" that vary according to timing, site of production, gene expression and physiology. The ontogeny of mammalian erythropoiesis is most thoroughly studied in mice where the earliest circulating erythroblasts released from the yolk sac are termed primitive. Later, the first definitive erythroid lineage is established by erythro-myeloid progenitors (EMPs) that originate in the yolk sac and migrate to the fetal liver for terminal differentiation. A second wave of definitive erythropoiesis is established from hematopoietic stem/progenitor cells that originate in the dorsal aorta and migrate to later stage fetal liver for terminal differentiation. Finally around birth, definitive erythropoiesis shifts to the bone marrow. The ontogeny of erythropoiesis overlaps in mice and humans, although less is known about the latter, as hematopoietic tissues from precisely staged early human embryos are difficult to obtain. We hypothesized that the initial steps of human erythroid ontogeny could be recapitulated by induced pluripotent stem cells (iPSCs) induced to undergo hematopoietic differentiation. We used a serum- and feeder-free protocol to differentiate iPSCs into embryoid bodies (EBs) that produced two sequential waves of distinctly different erythroid precursors. At day 8 of differentiation, EBs began to release hematopoietic precursors. Thereafter, erythroid precursors were released from the EBs in the presence of stem cell factor (SCF), erythropoietin (EPO) and insulin-like growth factor 1 (IGF-1). Erythroid precursors produced during wave 1 (days 12-23 of differentiation) were relatively large and expressed embryonic-type globins (zeta and epsilon), resembling those produced during primitive erythropoiesis. In contrast, wave 2 erythroblasts (day 27 and later) were smaller and expressed mainly gamma and alpha globins with some beta globin, suggestive of fetal-type definitive erythropoiesis. To investigate further the similarity of wave 1 and wave 2 erythroblasts to cells at the primitive and definitive stages of ontogeny, respectively, we used Affymetrix Genechips to analyze the global transcriptomes of stage-matched (CD235+ CD71high) cells. As primary human primitive erythroblasts were not available for comparison, we compared the transcriptomes from the iPSC-derived erythroblasts with those of primary murine definitive and primitive erythroblasts that were flow cytometry-purified from embryonic day 15.5 (E15.5) fetal liver and E10.5 bloodstream, respectively. The comparisons showed that wave 1 erythroblasts from human pluripotent cells resembled more closely the erythroid primitive lineage from mice, while wave 2 erythroblasts from the human cells resembled the erythroid definitive lineage of mice (P-value < 0.05 by a modified Kolmogorov-Smirnov test). For example, SOX6 and BCL11A, preferentially expressed during definitive erythropoiesis, were expressed at relatively high levels in wave 2 erythroblasts. In addition, gene set enrichment analysis (GSEA) demonstrated that wave 2 human iPSC-derived erythroblasts and primary murine definitive erythroblasts expressed numerous genes related to immune/inflammatory pathways that were shown previously to be important for the formation of definitive hematopoietic stem and progenitor cells in zebrafish and mouse embryos. Our findings demonstrate that human iPSC-derived embryoid bodies recapitulate early stages of erythroid ontogeny with respect to the timing of emerging lineages and their gene expression. Additionally, gene expression studies of human iPSC-derived primitive and definitive erythroblasts indicate inflammatory signaling as a potential regulator of the later stage of erythroid development. Disclosures No relevant conflicts of interest to declare.
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Graham, JD, SM Hunt, N. Tran, and CL Clarke. "Regulation of the expression and activity by progestins of a member of the SOX gene family of transcriptional modulators." Journal of Molecular Endocrinology 22, no. 3 (1999): 295–304. http://dx.doi.org/10.1677/jme.0.0220295.
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The mammalian testis-determining gene Sry and the related Sox genes define a family of transcriptional regulators widely expressed during embryogenesis. Tightly controlled temporal profiles of expression are a feature of the Sox gene family and may be required for initiation of a cascade of gene expression, yet the molecular mechanisms that control Sox gene expression are unknown. We now show that human SOX4 is expressed in the normal breast and in breast cancer cells. In these cells SOX4 is a progesterone-regulated gene, the expression of which is increased by progestins, leading to a marked increase in SOX-mediated transcriptional activity. Treatment of T-47D breast cancer cells with the synthetic progestin ORG 2058 directly increased SOX4 transcription, resulting in a 4-fold increase in SOX4 mRNA levels within 4 h of treatment. No effect of ORG 2058 was noted on other SOX genes measured, nor were other hormone-regulated HMG box proteins detected in this system, suggesting that the observed ability of progestin to increase SOX mRNA expression was confined to SOX4. The increase in SOX4 transcription was reflected in increased SOX4 protein expression, as progestin treatment of T-47D cells transfected with a SOX-responsive reporter resulted in a marked increase in reporter gene expression. Progesterone is essential for normal development and differentiation of the female reproductive system, plays an essential role in regulating growth and differentiation of the mammary gland and is required for opposing the proliferative effects of estrogen in specific cell types. The detection of SOX4 expression in the normal and malignant breast and the demonstration that SOX4 expression is under progesterone control suggests that changes in SOX4 gene expression may play a role in commitment to the differentiated phenotype in the normal and malignant mammary gland.
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Kervarrec, Thibault, Mahtab Samimi, Sonja Hesbacher, et al. "Merkel Cell Polyomavirus T Antigens Induce Merkel Cell-Like Differentiation in GLI1-Expressing Epithelial Cells." Cancers 12, no. 7 (2020): 1989. http://dx.doi.org/10.3390/cancers12071989.
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Merkel cell carcinoma (MCC) is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV). It is still under discussion, in which cells viral integration and MCC development occurs. Recently, we demonstrated that a virus-positive MCC derived from a trichoblastoma, an epithelial neoplasia bearing Merkel cell (MC) differentiation potential. Accordingly, we hypothesized that MC progenitors may represent an origin of MCPyV-positive MCC. To sustain this hypothesis, phenotypic comparison of trichoblastomas and physiologic human MC progenitors was conducted revealing GLI family zinc finger 1 (GLI1), Keratin 17 (KRT 17), and SRY-box transcription factor 9 (SOX9) expressions in both subsets. Furthermore, GLI1 expression in keratinocytes induced transcription of the MC marker SOX2 supporting a role of GLI1 in human MC differentiation. To assess a possible contribution of the MCPyV T antigens (TA) to the development of an MC-like phenotype, human keratinocytes were transduced with TA. While this led only to induction of KRT8, an early MC marker, combined GLI1 and TA expression gave rise to a more advanced MC phenotype with SOX2, KRT8, and KRT20 expression. Finally, we demonstrated MCPyV-large T antigens’ capacity to inhibit the degradation of the MC master regulator Atonal bHLH transcription factor 1 (ATOH1). In conclusion, our report suggests that MCPyV TA contribute to the acquisition of an MC-like phenotype in epithelial cells.
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Chew, Joon-Lin, Yuin-Han Loh, Wensheng Zhang, et al. "Reciprocal Transcriptional Regulation of Pou5f1 and Sox2 via the Oct4/Sox2 Complex in Embryonic Stem Cells." Molecular and Cellular Biology 25, no. 14 (2005): 6031–46. http://dx.doi.org/10.1128/mcb.25.14.6031-6046.2005.
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ABSTRACT Embryonic stem cells (ESCs) are pluripotent cells that can either self-renew or differentiate into many cell types. Oct4 and Sox2 are transcription factors essential to the pluripotent and self-renewing phenotypes of ESCs. Both factors are upstream in the hierarchy of the transcription regulatory network and are partners in regulating several ESC-specific genes. In ESCs, Sox2 is transcriptionally regulated by an enhancer containing a composite sox-oct element that Oct4 and Sox2 bind in a combinatorial interaction. It has previously been shown that Pou5f1, the Oct4 gene, contains a distal enhancer imparting specific expression in both ESCs and preimplantation embryos. Here, we identify a composite sox-oct element within this enhancer and show that it is involved in Pou5f1 transcriptional activity in ESCs. In vitro experiments with ESC nuclear extracts demonstrate that Oct4 and Sox2 interact specifically with this regulatory element. More importantly, by chromatin immunoprecipitation assay, we establish that both Oct4 and Sox2 bind directly to the composite sox-oct elements in both Pou5f1 and Sox2 in living mouse and human ESCs. Specific knockdown of either Oct4 or Sox2 by RNA interference leads to the reduction of both genes' enhancer activities and endogenous expression levels in addition to ESC differentiation. Our data uncover a positive and potentially self-reinforcing regulatory loop that maintains Pou5f1 and Sox2 expression via the Oct4/Sox2 complex in pluripotent cells.
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de Vasconcellos, Jaira F., Colleen Byrnes, Y. Terry Lee, et al. "Targeted Reduction of Let-7a miRNA Increases Fetal Hemoglobin in Human Adult Erythroblasts." Blood 124, no. 21 (2014): 451. http://dx.doi.org/10.1182/blood.v124.21.451.451.
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Abstract MicroRNAs (miRNAs) are a class of small, noncoding RNAs that bind and regulate target messenger RNAs (mRNAs). The let-7 family consists of twelve genes encoding nine highly conserved miRNAs that are involved in developmental timing events in multicellular organisms. Previous studies showed regulation during the fetal-to-adult transition in the erythroid lineage with significant increases in let-7 miRNAs from adult compared to umbilical cord blood reticulocytes (1). Further studies indicated that reduced expression of let-7 in adult CD34+ cells by “sponge” targeting the miRNA family seed region caused increased fetal hemoglobin (HbF), but the mean level of HbF remained less than 20% of the total hemoglobin (2). Increased expression of LIN28A (a major regulator of all let-7 miRNAs) caused greater increases in HbF (greater than 30% of the total) in cultured erythrocytes from pediatric patients with HbSS genotype (3). However, these studies did not address the potential for targeting an individual let-7 miRNA family member to regulate HbF expression. For this purpose, we initially determined the expression levels of mature let-7 family members in purified cell populations sorted from peripheral blood. The total levels of let-7 miRNAs in peripheral blood cells were as follows: reticulocytes: 1.7E+08 ± 1.0E+08 copies/ng; neutrophils: 2.0E+07 ± 1.1E+07 copies/ng; lymphocytes: 1.1E+07 ± 6.2E+06 copies/ng and monocytes: 3.5E+06 ± 2.7E+06 copies/ng. Among the individual species, let-7a was identified as a predominantly expressed let-7 family member in reticulocytes. As such, we hypothesized that specifically targeting let-7a may be sufficient to regulate HbF levels. To study the effects of let-7a miRNAs upon erythropoiesis and globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a was compared with empty vector controls. Transductions were performed in CD34+ cells from five adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Down-regulation of let-7a was confirmed by Q-RT-PCR at day 14 (control: 1.4E+07 ± 2.4E+06 copies/ng; let-7a-TuD: 1.6E+06 ± 4.6E+05 copies/ng; p=0.0003). Cell proliferation and differentiation were comparable in let-7a-TuD versus control transductions. Expression levels of globin genes were evaluated upon let-7a-TuD by Q-RT-PCR. Let-7a-TuD transductions caused significantly increased gamma-globin mRNA expression levels compared to control transductions (control: 1.2E+06 ± 6.8E+05 copies/ng; let-7a-TuD: 1.1E+07 ± 4.5E+06 copies/ng; p=0.004). HPLC analyses at the end of the culture period demonstrated robust increases in HbF levels after let-7a-TuD transduction (HbF control: 4.7 ± 0.6%; let-7a-TuD: 38.2 ± 3.8%; p=0.00003). In addition, the expression patterns of the erythroid transcription factors BCL11A, KLF1 and SOX6 were investigated. Let-7a-TuD decreased BCL11A mRNA expression levels (control: 1.7E+03 ± 4.5E+02 copies/ng; let-7a-TuD: 4.3E+02 ± 1.8E+02 copies/ng; p=0.003), but major changes in KLF1 or SOX6 were not detected. In summary, we report here that the let-7 miRNA family is differentially expressed in purified cell populations from adult human blood, and that let-7a is a predominantly expressed species in reticulocytes. Further, targeted reduction of let-7a in erythroblasts is sufficient to cause robust increases in gamma-globin mRNA expression and HbF to mean levels around 35-40% of the total hemoglobin produced. Targeting of individual let-7 genes or RNA transcripts may be useful for therapeutic induction of HbF expression in patients with sickle cell disease or other beta-hemoglobinopathies. 1) Noh SJ et al. J Transl Med. 7:98 (2009). 2) Lee YT et al. Blood. 122:1034-41 (2013). 3) Vasconcellos JF et al. Blood. 122: Abstract 313 (2013). Disclosures No relevant conflicts of interest to declare.
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Lindeman, Robin E., Mark W. Murphy, Kellie S. Agrimson, et al. "The conserved sex regulator DMRT1 recruits SOX9 in sexual cell fate reprogramming." Nucleic Acids Research 49, no. 11 (2021): 6144–64. http://dx.doi.org/10.1093/nar/gkab448.
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Abstract Mammalian sexual development commences when fetal bipotential progenitor cells adopt male Sertoli (in XY) or female granulosa (in XX) gonadal cell fates. Differentiation of these cells involves extensive divergence in chromatin state and gene expression, reflecting distinct roles in sexual differentiation and gametogenesis. Surprisingly, differentiated gonadal cell fates require active maintenance through postnatal life to prevent sexual transdifferentiation and female cell fate can be reprogrammed by ectopic expression of the sex regulator DMRT1. Here we examine how DMRT1 reprograms granulosa cells to Sertoli-like cells in vivo and in culture. We define postnatal sex-biased gene expression programs and identify three-dimensional chromatin contacts and differentially accessible chromatin regions (DARs) associated with differentially expressed genes. Using a conditional transgene we find DMRT1 only partially reprograms the ovarian transcriptome in the absence of SOX9 and its paralog SOX8, indicating that these factors functionally cooperate with DMRT1. ATAC-seq and ChIP-seq show that DMRT1 induces formation of many DARs that it binds with SOX9, and DMRT1 is required for binding of SOX9 at most of these. We suggest that DMRT1 can act as a pioneer factor to open chromatin and allow binding of SOX9, which then cooperates with DMRT1 to reprogram sexual cell fate.
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Diks, Sander H., Robert J. Bink, Sandra van de Water, et al. "The novel gene asb11: a regulator of the size of the neural progenitor compartment." Journal of Cell Biology 174, no. 4 (2006): 581–92. http://dx.doi.org/10.1083/jcb.200601081.
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From a differential display designed to isolate genes that are down-regulated upon differentiation of the central nervous system in Danio rerio embryos, we isolated d-asb11 (ankyrin repeat and suppressor of cytokine signaling box–containing protein 11). Knockdown of the d-Asb11 protein altered the expression of neural precursor genes sox2 and sox3 and resulted in an initial relative increase in proneural cell numbers. This was reflected by neurogenin1 expansion followed by premature neuronal differentiation, as demonstrated by HuC labeling and resulting in reduced size of the definitive neuronal compartment. Forced misexpression of d-asb11 was capable of ectopically inducing sox2 while it diminished or entirely abolished neurogenesis. Overexpression of d-Asb11 in both a pluripotent and a neural-committed progenitor cell line resulted in the stimulus-induced inhibition of terminal neuronal differentiation and enhanced proliferation. We conclude that d-Asb11 is a novel regulator of the neuronal progenitor compartment size by maintaining the neural precursors in the proliferating undifferentiated state possibly through the control of SoxB1 transcription factors.
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Huang, Kevin Y., and Enrico Petretto. "Cross-species integration of single-cell RNA-seq resolved alveolar-epithelial transitional states in idiopathic pulmonary fibrosis." American Journal of Physiology-Lung Cellular and Molecular Physiology 321, no. 3 (2021): L491—L506. http://dx.doi.org/10.1152/ajplung.00594.2020.
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Single-cell transcriptomics analyses of the fibrotic lung uncovered two cell states critical to lung injury recovery in the alveolar epithelium—a reparative transitional cell state in the mouse and a disease-specific cell state ( KRT5−/ KRT17+) in human idiopathic pulmonary fibrosis (IPF). The murine transitional cell state lies between the differentiation from type 2 (AT2) to type 1 pneumocyte (AT1), and the human KRT5−/ KRT17+ cell state may arise from the dysregulation of this differentiation process. We review major findings of single-cell transcriptomics analyses of the fibrotic lung and reanalyzed data from seven single-cell RNA sequencing studies of human and murine models of IPF, focusing on the alveolar epithelium. Our comparative and cross-species single-cell transcriptomics analyses allowed us to further delineate the differentiation trajectories from AT2 to AT1 and AT2 to the KRT5−/ KRT17+ cell state. We observed AT1 cells in human IPF retain the transcriptional signature of the murine transitional cell state. Using pseudotime analysis, we recapitulated the differentiation trajectories from AT2 to AT1 and from AT2 to KRT5−/ KRT17+ cell state in multiple human IPF studies. We further delineated transcriptional programs underlying cell-state transitions and determined the molecular phenotypes at terminal differentiation. We hypothesize that in addition to the reactivation of developmental programs ( SOX4, SOX9), senescence ( TP63, SOX4) and the Notch pathway ( HES1) are predicted to steer intermediate progenitors to the KRT5−/ KRT17+ cell state. Our analyses suggest that activation of SMAD3 later in the differentiation process may explain the fibrotic molecular phenotype typical of KRT5−/ KRT17+ cells.
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Western, P., J. Van Den Bergen, D. Miles, R. Ralli, and A. Sinclair. "002. REGULATION OF PLURIPOTENCY AND CELL CYCLE IN FETAL GERM CELLS." Reproduction, Fertility and Development 21, no. 9 (2009): 2. http://dx.doi.org/10.1071/srb09abs002.
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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.
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Wan, HaiXia, Howard C. H. Chow, Tsz-Kan Fung, et al. "Lineage-Specific Sox7 Expression In Hematopoietic Progenitor Cells Derived From Human Umbilical Cord Blood." Blood 116, no. 21 (2010): 4781. http://dx.doi.org/10.1182/blood.v116.21.4781.4781.
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Abstract Abstract 4781 Introduction: The SOX (Sry-related HMG box) genes belong to a family of transcription factors containing a High-Mobility-Group box domain. In an initial screen of SOX genes in human leukemias, SOX7 is uniquely down-regulated in acute myeloid leukemia, myelodysplastic syndrome and chronic myelogenous leukemia but up-regulated in most cases of acute lymphoblastic leukemia. The observation led to the proposition that SOX7 may play a role in lineage differentiation in hematopoiesis. In this study, we examined SOX7 expression in human umbilical cord blood (UCB) with a view to understand its role in hematopoietic lineage specification. Methods: Mononuclear cells (MNC) were isolated from UCB and fractionated into CD34+, CD34-, CD34+CD38+ and CD34+CD38- populations by immunomagnetic selection and fluorescence activated cell sorting (FACS). 0.1–0.4 × 106 CD34+ UCB cells were transplanted intravenously into sub-lethally irradiated NOD/SCID mice with or without prior anti-CD122 antibody injection. Donor engraftment was assessed after 7–8 weeks and engrafting human cells were sorted for CD34+, CD33+, CD19+ expression. CD34+ UCB cells were also plated in colony-forming unit (CFU) assay. SOX7 expression was evaluated by quantitative real-time reverse-transcriptase polymerase chain reaction (Q-RT-PCR). Results: The relative SOX7 expression in CD34+ and CD34+CD38- UCB cells was 9.15±2.18 and 4.53±1.09 fold higher than in CD34- (P=0.0001) and CD34+CD38+ (P=0.014) respectively. SOX7 expression in CD34+ cells (arbitrarily set as 1.0) was down-regulated upon differentiation in CFU assay (CFU-GM/G: 0.09±0.06 fold, P=0.007; BFU-E: 0.05±0.03 fold, P=0.003). CD34+ cells engrafted into NOD/SCID mice as enumerated by human CD45+ cells in mouse BM (with anti-CD122: 51±6.3%; without anti-CD122: 28±9%; p=0.017) and gave rise to predominantly B-lymphoid (CD19+) (53.1±8.18%) and to a less extent myeloid (CD33+) (2.9±0.54%) differentiated cells. Prior injection with anti-CD122 antibody had no effect on lineage differentiation and the results were pooled. When SOX7 expression in engrafting myeloid and B-lymphoid differentiated cells (arbitrarily set as 1.0) were compared, it was significantly down-regulated in the myeloid series (0.28±0.08 fold, p = 0.036). Conclusion SOX7 is selectively expressed in human HSC from UCB. It is preferentially down-regulated during myeloid differentiation both in-vivo and in-vitro. The function of SOX7 during lineage specification and its link to myeloid malignancies is currently investigated in our laboratory. Acknowledgments The project was supported by a grant from the strategic Research Theme of cancer stem cells in the HKU. Disclosures: No relevant conflicts of interest to declare.
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Hoffmann, S. A., D. Hos, M. Kuspert, et al. "Stem cell factor Sox2 and its close relative Sox3 have differentiation functions in oligodendrocytes." Development 141, no. 1 (2013): 39–50. http://dx.doi.org/10.1242/dev.098418.
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Manshaei, Saba, Thea L. Willis, Virinder Reen, et al. "RF13 | PMON143 BRF1-Mediated Paracrine Signalling by a Subset of SOX2-Expressing Stem Cells is Required for Normal Development of the Stem Cell Compartment and Terminal Differentiation of Pituitary Committed Progenitors." Journal of the Endocrine Society 6, Supplement_1 (2022): A580—A581. http://dx.doi.org/10.1210/jendso/bvac150.1203.
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Abstract Introduction Hormone-producing pituitary cell lineages are derived from a population of embryonic precursors expressing SOX2. These cells maintain multipotency into early postnatal life, acting as the resident population of pituitary stem cells (PSCs) and contributing extensively to all the endocrine cell lineages. In addition to this direct contribution to pituitary turnover, paracrine signalling from PSCs has been shown to be important for cell proliferation of neighbouring progenitors (PMC7803373). It is not known if SOX2+ PSCs are involved in the regulation of additional cell attributes during normal physiology and if there is functional heterogeneity among the SOX2+ PSC population. Experimental Methods We have carried out single-cell RNA-Sequencing of SOX2+ PSCs from Sox2Egfp/+ mouse pituitaries at three postnatal stages from P3 to P56 and used computational approaches to analyse their molecular signatures. A novel conditional mouse model expressing a constitutively active mutant form of the RNA binding factor BRF1 (R26stop-mBRF1) has been used to attenuate the expression of several cytokines and chemokines in SOX2+ cells embryonically and postnatally (PMC4589897). Results We show that the SOX2+ PSC population consists of three subgroups (SC1, SC2 and SC3). We reveal that SC1-SC2 express abundant cytokines and secreted factors, suggesting paracrine function. In contrast, SC3 is characterised by robust expression of Lef1, is identified as a committing PSC cluster, and its presence diminishes with age. Key markers of PSC clusters SC1-SC2 include the RNA binding factor BRF1. We show that BRF1 is highly expressed in PSCs and validate its expression by immunohistochemistry in both mouse and human pituitaries. Secondly, we show that the dysregulation of BRF1 in embryonic SOX2+ cells using the Hesx1-Cre driver (PMC3461924) results in pituitary hypoplasia and severe hypopituitarism due to a failure of the PIT1 and SF1 cell-lineage committed progenitors to terminally differentiate into hormone-producing cells. Additionally, there is a significant reduction of the stem cell compartment, manifested by lower numbers of SOX2/SOX9+ stem cells. This phenotype is recapitulated when using a Sox2-CreERT2 driver (PMID24094324). The differentiation failure can be rescued in vitro through co-culture of mutant cells with wild-type stem cells, as well as in vivo, in mutant pituitaries where activation of constitutively active BRF1 is restricted to few SOX2+ PSCs in a mosaic manner. Finally, we identify key ligands underlying this differentiation phenotype and demonstrate a partial restoration of terminal differentiation in the mutant, when cultured in the presence of these ligands. Conclusion We provide evidence indicating the presence of functionally distinct groups of SOX2+ pituitary stem cells and reveal a critical role for a PSC subset in the development of the stem cell compartment and in driving terminal differentiation of committed progenitors. Presentation: Sunday, June 12, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.
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Ahmed, Emad A., and Abdullah M. Alzahrani. "SOXC Transcription Factors as Diagnostic Biomarkers and Therapeutic Targets for Arthritis." International Journal of Molecular Sciences 24, no. 4 (2023): 4215. http://dx.doi.org/10.3390/ijms24044215.
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Osteoarthritis (OA) and rheumatoid arthritis (RA) are two common disorders that disrupt the quality of life of millions of people. These two chronic diseases cause damage to the joint cartilage and surrounding tissues of more than 220 million people worldwide. Sex-determining region Y-related (SRY) high-mobility group (HMG) box C, SOXC, is a superfamily of transcription factors that have been recently shown to be involved in various physiological and pathological processes. These include embryonic development, cell differentiation, fate determination, and autoimmune diseases, as well as carcinogenesis and tumor progression. The SOXC superfamily includes SOX4, SOX11, and SOX12, all have a similar DNA-binding domain, i.e., HMG. Herein, we summarize the current knowledge about the role of SOXC transcription factors during arthritis progression and their potential utilization as diagnostic biomarkers and therapeutic targets. The involved mechanistic processes and signaling molecules are discussed. SOX12 appears to have no role in arthritis, however SOX11 is dysregulated and promotes arthritic progression according to some studies but supports joint maintenance and protects cartilage and bone cells according to others. On the other hand, SOX4 upregulation during OA and RA was documented in almost all studies including preclinical and clinical models. Molecular details have indicated that SOX4 can autoregulate its own expression besides regulating the expression of SOX11, a characteristic associated with the transcription factors that protects their abundance and activity. From analyzing the currently available data, SOX4 seems to be a potential diagnostic biomarker and therapeutic target of arthritis.
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Suto, Akira, Shigeru Tanaka, and Hiroshi Nakajima. "Sox5 and Th17 cell differentiation." Oncotarget 6, no. 24 (2015): 19952–53. http://dx.doi.org/10.18632/oncotarget.4784.
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Lizárraga-Verdugo, Erik, Erika Ruiz-García, César López-Camarillo, et al. "Cell Survival Is Regulated via SOX9/BCL2L1 Axis in HCT-116 Colorectal Cancer Cell Line." Journal of Oncology 2020 (April 29, 2020): 1–10. http://dx.doi.org/10.1155/2020/5701527.
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Colorectal cancer (CRC) is one of the most frequent types of malignancies and one of the major causes of cancer-related death worldwide. Sex-determining region Y (SRY)-box 9 protein (SOX9) is a member of the SOX family of transcription factors which are involved in the regulation of differentiation and development. Recently, several reports suggest an important role of SOX9 in tumorigenesis since its overexpression correlates with tumor progression and poor outcome in several types of cancer; however, its role in CRC is not clear until now. Therefore, in this work, we searched for novel SOX9-regulated genes involved in cell survival of CRC. We silenced SOX9 in the poorly differentiated HCT-116 cell line, using a specific siRNA, to identify differential expressed genes by DNA microarrays and analyzed the role or candidate genes in apoptosis and autophagy. Transcriptome analysis showed that diverse cellular pathways, associated with CRC carcinogenesis such as Wnt/β-catenin, MAPK, TGF-β, and mTOR, were modulated after SOX9 silencing. Interestingly, we found that SOX9 silencing promotes downregulation of BCL2L1 and overexpression of CASP3, proteins related to apoptosis, which was further confirmed in SW-480, a moderated-differentiated cell line, but not in HT-29, well-differentiated cell line. Moreover, inhibition of BCL2L1 by ABT-737 (BH3 mimetic) in SOX9-silenced HCT-116 cells resulted in an increased apoptosis percentage. However, downregulation of BCL2L1 was not enough to induce autophagy. This is the first report, suggesting that cell survival in poorly and moderated-differentiated CRC cells lines is regulated by SOX9/BCL2L1 axis, but not in well-differentiated cell lines.
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Soleas, John P., Linwen Huang, Elisa D’Arcangelo, et al. "Guided Self-Assembly of ES-Derived Lung Progenitors into Biomimetic Tube Structures That Impact Cell Differentiation." Bioengineering 8, no. 12 (2021): 209. http://dx.doi.org/10.3390/bioengineering8120209.
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Chemically directed differentiation of pluripotent stem cells (PSCs) into defined cell types is a potent strategy for creating regenerative tissue models and cell therapies. In vitro observations suggest that physical cues can augment directed differentiation. We recently demonstrated that confining human PSC-derived lung progenitor cells in a tube with a diameter that mimics those observed during lung development results in the alteration of cell differentiation towards SOX2−SOX9+ lung cells. Here we set out to assess the robustness of this geometric confinement effect with respect to different culture parameters in order to explore the corresponding changes in cell morphometry and determine the feasibility of using such an approach to enhance directed differentiation protocols. Culture of progenitor cells in polydimethylsiloxane (PDMS) tubes reliably induced self-organization into tube structures and was insensitive to a variety of extracellular matrix coatings. Cellular morphology and differentiation status were found to be sensitive to the diameter of tube cells that were cultured within but not to seeding density. These data suggest that geometric cues impose constraints on cells, homogenize cellular morphology, and influence fate status.
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Bayram, Keziban Korkmaz, Juliette Fitremann, Arslan Bayram, et al. "Gene Expression of Mouse Hippocampal Stem Cells Grown in a Galactose-Derived Molecular Gel Compared to In Vivo and Neurospheres." Processes 9, no. 4 (2021): 716. http://dx.doi.org/10.3390/pr9040716.
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Background: N-heptyl-D-galactonamide (GalC7) is a small synthetic carbohydrate derivative that forms a biocompatible supramolecular hydrogel. In this study, the objective was to analyze more in-depth how neural cells differentiate in contact with GalC7. Method: Direct (ex vivo) cells of the fresh hippocampus and culture (In vitro) of the primary cells were investigated. In vitro, investigation performed under three conditions: on culture in neurospheres for 19 days, on culture in GalC7 gel for 7 days, and on culture in both neurospheres and GalC7 gel. Total RNA was isolated with TRIzol from each group, Sox8, Sox9, Sox10, Dcx, and Neurod1 expression levels were measured by qPCR. Result: Sox8 and Sox10, oligodendrocyte markers, and Sox9, an astrocyte marker, were expressed at a much higher level after 7 days of culture in GalC7 hydrogel compared to all other conditions. Dcx, a marker of neurogenesis, and Neurod1, a marker of neuronal differentiation, were expressed at better levels in the GalC7 gel culture compared to the neurosphere. Conclusions: These results show that the GalC7 hydrogel brings different and interesting conditions for inducing the differentiation and maturation of neural progenitor cells compared with polymer-based scaffolds or cell-only conditions. The differences observed open new perspectives in tissue engineering, induction, and transcript analysis.
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Jiang, Yanwen, Katerina Hatzi, Olivier Elemento, and Ari Melnick. "Enhancer Profiling Reveals SOX9 As a Novel Transcription Regulator of B Cell Activation and DLBCL Transformation." Blood 120, no. 21 (2012): 527. http://dx.doi.org/10.1182/blood.v120.21.527.527.
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Abstract Abstract 527 Antigen stimulation of naïve B cells (NBC) induces differentiation with a phenotype characterized by robust proliferation and genomic instability tolerance to enable activated germinal center B cells (GCB) to undergo immunoglobulin affinity maturation. Aberrant genetic events resulting from this process lead to malignant transformation and diffuse large B cell lymphoma (DLBCL). Phenotypic progression from quiescent NBC to activated GCB and malignant DLBCL involves major shifts in gene expression. Recent studies suggest that enhancers play a key role in mediating cell type-specific gene regulation. We therefore postulated that enhancers are involved in dictating the gene expression programs that govern normal and malignant B cell phenotypes; and systematic discovery of enhancers coupled with bioinformatic analysis would uncover key enhancer-binding transcription factors (TFs) that regulate these cell states. To test this hypothesis, we performed ChIP-seq on enhancer histone marks, i.e. H3K4me2, H3K27Ac, and H3K4me3, in primary NBC and GCB, and in DLBCL cell lines in biological replicates. We defined enhancers by the criterion of H3K4me2hiH3K4me3low. We observed a striking pattern of enhancer re-organization between cell types. First, we found a larger number of enhancers in primary B-cells (∼20,000) than in DLBCL (∼12,000). Second, we confirmed that enhancers are cell type-specific. For example, 11,492 out of 20,173 NBC enhancers were lost during transition to GCB (loss of H3K4me2 enrichment), while 13,088 new enhancers were gained in GCB. A similar phenomenon was also observed in DLBCL when compared to either NBC or GCB. This re-organization of enhancers suggests that cells may have dynamic gene regulatory programs during differentiation or malignant transformation. To discover TFs that act through enhancers, we used bioinformatic analyses, including FIRE and MEME, to search for TF consensus binding sequences within enhancers. Over-represented DNA motifs included motifs of SPI1, RUNX1, STAT3, RELA and SOX9, etc. SOX9 motif was significantly enriched in GCB specific enhancers (p=3.07e-15). SOX9 belongs to the SOX family TFs and plays an important role in cartilage development, sex determination, and intestinal differentiation but has not been implicated in B cell development. To investigate the role of SOX9 in B cell activation and malignant transformation, we first examined the expression of SOX9 in these cells. RNA-seq performed on human tonsilar NBC and GCB showed more than 20-fold increase of SOX9 mRNA in GCB as compared to NBC (6.75±0.80 vs 0.29±0.14, RPKM, p=0.0002). In addition, SOX9 expression was maintained in plasma B cells (2.88±0.49, RPKM). To understand how SOX9 regulates transcriptional programming in GCB, we performed SOX9 ChIP-seq in GCB to look for its targets. We found that SOX9 binds to 1,668 upstream distal enhancer regions (-5 to -100 kb of TSS) associated with 963 genes. These target genes were significantly enriched in many important pathways including cell cycle regulation (CCND2, CDC25B, CDK1), transcription regulation (BCOR, NCOR2), epigenetic regulation (BMI1, DNMT3A, MLL2, SUZ12, TET3), and MAPK signaling (MAP2K3, MAP3K7) (p<0.001). One of the SOX9 targets is PRMD1, a TF that controls the transition from GCB to plasma cells, suggesting that SOX9 may be involved in B cell terminal differentiation. To our surprise, we did not detect SOX9 mRNA in 10 out of 12 DLBCL cell lines by RNA-seq. Moreover, SOX9 was not expressed in the majority of primary malignant non-Hodgkin's lymphoma cases studied by IHC in the Human Protein Atlas project. To examine whether reduced SOX9 expression could induce malignant transformation, we used shRNA to knockdown Sox9 in mouse BCL1 lymphoma cells and subjected them to colony forming assay in semi-solid methylcellulose. Knockdown of Sox9 increased BCL1 colony forming ability by 50% as compared to scramble, suggesting that loss of SOX9 expression maybe important for lymphomagenesis. In summary, we identified a novel germinal center TF, SOX9, by examining enrichment of TF motifs within enhancer regions uncovered by ChIP-seq. Our current data suggest that SOX9 may play an important role in germinal center reaction and subsequent terminal differentiation by regulating key factors, such as PRDM1, and that loss of SOX9 may contribute to DLBCL malignant transformation by potentially blocking the terminal differentiation of mature GCB. Disclosures: No relevant conflicts of interest to declare.