“…5b). Previous studies also demonstrated this sequence characteristic of Sox9 binding in adult hair follicle stem cells [11] and in prostate cancer [10]. Together, these observations suggest a substantial divergence of the Sox9 consensus motif.Fig.…”
BackgroundDistinctive structures called crypts harbor intestinal epithelial stem cells (IESCs) which generate progenitor and terminally differentiated cells in the intestinal epithelium. Mammalian IESCs and their daughter cells require the participation of DNA methylation and the transcription factor Sox9 for proliferation and differentiation. However, the association between Sox9 and DNA methylation in this process remains elusive.MethodsThe DNA methylation of small intestinal epithelial crypts in db/db mice was detected via combining methylated DNA immunoprecipitation with microarray hybridization. DNA methylation of Sox9 promoter in crypts and IESCs was validated using bisulfite sequence analysis. The target sequence of the transcription factor Sox9 in IESCs was investigated via chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq).ResultsIncreased Sox9 expression is accompanied by the loss of methylation in its promoter in IESCs. Sox9 targets the enhancers of the Wnt signaling pathway-related genes. Sox9 predominantly acts as a transcriptional activator at proximal enhancers of Wnt4, Tab2, Sox4, and Fzd8, but also functions as a potential transcriptional inhibitor at a distant enhancer of Cdk1. Lack of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway leads to the loss of intrinsic inhibitory action and ultimately produces overactivation of this pathway in db/db mice.ConclusionsOur study sheds light on the connections among DNA methylation, transcription factor modulation, and Wnt signaling in IESCs in the diabetic state. Hypomethylation in the Sox9 promoter is correlated to increased Sox9 expression in db/db IESCs. Although there is increased expression of Sox9 in db/db IESCs, the loss of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway might result in abnormalities in this pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0507-4) contains supplementary material, which is available to authorized users.
“…5b). Previous studies also demonstrated this sequence characteristic of Sox9 binding in adult hair follicle stem cells [11] and in prostate cancer [10]. Together, these observations suggest a substantial divergence of the Sox9 consensus motif.Fig.…”
BackgroundDistinctive structures called crypts harbor intestinal epithelial stem cells (IESCs) which generate progenitor and terminally differentiated cells in the intestinal epithelium. Mammalian IESCs and their daughter cells require the participation of DNA methylation and the transcription factor Sox9 for proliferation and differentiation. However, the association between Sox9 and DNA methylation in this process remains elusive.MethodsThe DNA methylation of small intestinal epithelial crypts in db/db mice was detected via combining methylated DNA immunoprecipitation with microarray hybridization. DNA methylation of Sox9 promoter in crypts and IESCs was validated using bisulfite sequence analysis. The target sequence of the transcription factor Sox9 in IESCs was investigated via chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq).ResultsIncreased Sox9 expression is accompanied by the loss of methylation in its promoter in IESCs. Sox9 targets the enhancers of the Wnt signaling pathway-related genes. Sox9 predominantly acts as a transcriptional activator at proximal enhancers of Wnt4, Tab2, Sox4, and Fzd8, but also functions as a potential transcriptional inhibitor at a distant enhancer of Cdk1. Lack of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway leads to the loss of intrinsic inhibitory action and ultimately produces overactivation of this pathway in db/db mice.ConclusionsOur study sheds light on the connections among DNA methylation, transcription factor modulation, and Wnt signaling in IESCs in the diabetic state. Hypomethylation in the Sox9 promoter is correlated to increased Sox9 expression in db/db IESCs. Although there is increased expression of Sox9 in db/db IESCs, the loss of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway might result in abnormalities in this pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0507-4) contains supplementary material, which is available to authorized users.
“…The MAPK/ERK pathway is a positive regulator of Sox9 in nontumoral and tumoral cells (Ling et al, ; Murakami, Kan, McKeehan, & de Crombrugghe, ; Song et al, ) and this could also be the case in astrocytoma tumors which show a substantial number of p‐Erk+ cells (Figure A). Astrocytomas have a worse prognosis compared to oligodendrogliomas (Cancer Genome Atlas Research Network et al, ; Sabha et al, ; Suzuki et al, ) which could be linked to Sox9 expression as this transcription factor is an important driver in several cancers (Larsimont et al, ) through activating the Wnt signaling (Liu et al, ; Ma et al, ; Wang et al, ). The expression and critical role of Sox9 has been reported in GBM (Liu et al, ,b; ) and this transcription factor could play a similar role in diffuse low grade IDH1‐mutated astrocytomas.…”
Diffuse low grade gliomas (DLGG, grade II gliomas) are slowly-growing brain tumors that often progress into high grade gliomas. Most tumors have a missense mutation for IDH1 combined with 1p19q codeletion in oligodendrogliomas or ATRX/TP53 mutations in astrocytomas. The phenotype of tumoral cells, their environment and the pathways activated in these tumors are still ill-defined and are mainly based on genomics and transcriptomics analysis. Here we used freshly-resected tumors to accurately characterize the tumoral cell population and their environment. In oligodendrogliomas, cells express the transcription factors MYT1, Nkx2.2, Olig1, Olig2, Sox8, four receptors (EGFR, PDGFRα, LIFR, PTPRZ1) but not the co-receptor NG2 known to be expressed by oligodendrocyte progenitor cells. A variable fraction of cells also express the more mature oligodendrocytic markers NOGO-A and MAG. DLGG cells are also stained for the young-neuron marker doublecortin (Dcx) which is also observed in oligodendrocytic cells in nontumoral human brain. In astrocytomas, MYT1, PDGFRα, PTPRZ1 were less expressed whereas Sox9 was prominent over Sox8. The phenotype of DLGG cells is overall maintained in culture. Phospho-array screening showed the absence of EGFR and PDGFRα phosphorylation in DLGG but revealed the strong activation of p44/42 MAPK/ERK which was present in a fraction of tumoral cells but also in nontumoral cells. These results provide evidence for the existence of close relationships between the cellular phenotype and the mutations found in DLGG. The slow proliferation of these tumors may be associated with the absence of activation of PDGFRα/EGFR receptors.
“…Lineage − BM cells were isolated by depleting lineage‐positive cells from total BM cells using biotinylated antibodies and Dynabeads M280 Streptavidin (Thermo Fisher Scientific). The Lin − BM cells were cultured in Iscove's Modified Dulbecco's Medium with 15% fetal bovine serum (Thermo Fisher Scientific), 50 µM 2‐ME (Stemcell Technologies, Vancouver, BC, Canada), 100 µg/ml streptomycin and penicillin (Thermo Fisher Scientific), 50 ng/ml stem cell factor (Peprotech, Rocky Hill, NJ, USA), and 20 ng/ml thrombopoietin (Peprotech) in a 96‐well flat‐bottom plate at the density of 0.5 × 10 6 /ml with 40 ng/ml rmWnt3a (R&D Systems, Minneapolis, MN, USA) (27).…”
Hematopoietic stem cells (HSCs) have the capacity for self‐renewal to maintain the HSCs' pool and the ability for multilineage differentiation, which are responsible for sustained production of multiple blood lineages. The regulation of HSC development is controlled precisely by complex signal networks and hematopoietic microenvironment, which has been termed the HSCs' niche. The Wnt signaling pathway is one of a variety of signaling pathways that have been involved in HSC self‐renewal and maintenance. Previous studies are indeterminant on the regulation of adult HSCs upon canonical Wnt signaling pathways because of the different experimental systems and models used. In this study, we generated the conditional knockout Wnt coreceptor low‐density lipoprotein receptor‐related protein 5 (Lrp5) and low‐density lipoprotein receptor‐related protein 6 (Lrp6) mice in adult hematopoiesis via Vav‐Cre Loxp system. Inactivation of Lrp5 and ‐6 in a hematopoietic system diminished the pool of HSCs, but there were no obvious defects in mature immune cells. Lrp5 and ‐6 double deficiency HSCs showed intrinsic defects in self‐renewal and differentiation due to reduced proliferation and increased quiescence of the cell cycle. Analysis of HSC gene expression suggested that the quiescence regulators were significantly up‐regulated, such as Egr1, Cdkn1a, Nr4a1, Gata2, Junb and Btg2, and the positive cell cycle regulators were correspondingly down‐regulated, such as Ccna2 and Ranbp1. Taken together, we investigated the roles of Lrp5 and ‐6 in HSCs by functional and bioinformatic assays, and we demonstrated that Lrp5 and ‐6 are required for the self‐renewal and differentiation of adult HSCs. The canonical Wnt pathway may contribute to maintaining the HSC pool and regulate the differentiation of adult HSCs by controlling cell cycle gene regulatory module.—Liu, J., Cui, Z., Wang, F., Yao, Y., Yu, G., Liu, J., Cao, D., Niu, S., You, M., Sun, Z., Lian, D., Zhao, T., Kang, Y., Zhao, Y., Xue, H.‐H., Yu, S. Lrp5 and Lrp6 are required for maintaining self‐renewal and differentiation of hematopoietic stem cells. FASEB J. 33, 5615–5625 (2019). http://www.fasebj.org
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