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ChIP-Seq analysis for GFI1B, Beta-Catenin, LSD1 and histone marks in response to Wnt treatment

ABSTRACT: Gfi1b is a DNA binding transcriptional repressor highly expressed in hematopoietic stem cells (HSCs) and megakaryocytes (MKs). Gfi1b deficiency leads to the expansion of both cell types and abrogates the ability of MKs to respond to integrin substrates with spreading and movement. Here we show that Gfi1b is present in complexes with ²-catenin and its co-factors Pontin52, CHD8, TLE3 and CtBP1 and can regulate Wnt/²-catenin dependent gene expression. In reporter assays, Gfi1b can activate TCF-dependent transcription and this activation is enhanced upon treatment with Wnt3a. This requires the interaction between Gfi1b and lysine demethylase 1 (LSD1), but is independent of the ability of Gfi1b to bind DNA. This suggests that a tripartite ²-catenin/Gfi1b/LSD1 complex exists that regulates Wnt/²-catenin target genes. Consistently, expression of many canonical Wnt/²-catenin target genes is deregulated in Gfi1b-deficient cells and many of these target genes are co-occupied by Gfi1b, ²-catenin and LSD1 at promoter sites. When Gfi1b deficient cells were treated with Wnt3a, their normal cellularity was restored and Gfi1b-deficient MKs regained their ability to spread on integrin substrates. This suggests that Gfi1b controls both the cellularity and functional integrity of HSCs and MKs by regulating Wnt/²-catenin signaling pathway.

ORGANISM(S): Homo sapiens

PROVIDER: GSE117944 | GEO | 2019/02/21

REPOSITORIES: GEO

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Project description:Wnt/M-NM-2-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and M-NM-2-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However, both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of M-NM-2-catenin signaling during myogenic differentiation remain unknown. Using C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of M-NM-2-catenin/Tcf complex formation, reduced basal M-NM-2-catenin in cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased membrane-bound M-NM-2-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated M-NM-2-catenin (Tyr654) during myogenic differentiation. These results suggest that various Wnt ligands control subcellular M-NM-2-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via M-NM-2-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation. Control cells (day 0) prior to differentiation induction with n=4; differentiated for two days with n=3; differentiated for four days with n=3.

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ChIP-Seq analysis for GFI1B, Beta-Catenin, LSD1 and histone marks in response to Wnt treatment

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