SRA

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. Overall design: Chromatin immunoprecipitation was carried out in K562 cells for GFI1B, Beta-Catenin, LSD1, H3, H3K4me1, H3K9me2. ChIP was carried in both the presence and absence of Wnt3a treatment