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Growth factor independence 1b (Gfi1b) is required for erythroid cell maturation and regulates embryonic globin expression [Mouse430_2]

ABSTRACT: Growth factor independence 1b (Gfi1b) is a DNA binding repressor of transcription with vital functions in hematopoiesis. Gfi1b-null embryos die at midgestation very likely due to defects in erythro- and megakaryopoiesis. To analyze the full functionality of Gfi1b in erythropoiesis, we used conditionally deficient mice that harbor floxed Gfi1b alleles and the Mx-Cre transgene inducible by pIpC treatment. We used bone marow cells from 12 weeks old wild type C57BL/6 mice or conditional Gfi1b knock-out mice, both carrying the Mx-cre transgene induced by five treatments with pIpC (500µg each) every other day and sacrificed 42 days after the first injection. Ter119 positive erythroid cells were isolated by flow cytometry and RNA from these cells was isolated using the Trizol procedure according to the manufacturers instructions for gene expression analysis on Affymetrix MOE_430 2.0 expression arrays (GPL 1261)

ORGANISM(S): Mus musculus

PROVIDER: GSE54205 | GEO | 2014/12/31

SECONDARY ACCESSION(S): PRJNA235885

REPOSITORIES: GEO

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Growth factor independence 1b (Gfi1b) is required for erythroid cell maturation and regulates embryonic globin expression [MoGene-1_0]

Project description:Growth factor independence 1b (Gfi1b) is a DNA binding repressor of transcription with vital functions in hematopoiesis. Gfi1b-null embryos die at midgestation very likely due to defects in erythro- and megakaryopoiesis. To analyze the full functionality of Gfi1b in embryonic erythropoiesis, we used conditionally deficient mice that harbor floxed Gfi1b alleles and one EpoR-Cre knock-in allele. We used fetal liver cells from wild type C57BL/6 embryos or conditional Gfi1b knock-out embryos at day 14.5 p.c., both carrying one EpoR-cre knock-in allele. Ter119 positive erythroid cells were isolated by flow cytometry and RNA from these cells was isolated using the Trizol procedure according to the manufacturers instructions for gene expression analysis on Affymetrix Gene 1.0 ST arrays (GPL 6246)

2014-12-31 | GSE54204 | GEO

Effect of loss of Gfi1b on hematopietic stem cells

Project description:Using Gfi1b conditional mice, deletion of gfi1b in the hematopietic system was induced by injecting MxCre tg Gfi1bfl/fl mice with pIpC. 30 days after injection, Cd150 pos, Cd 48 neg, Lin neg Sca and c-kit pos stem cells were sortrted from Gfi1bfl/fl and Mxcre tg Gfi1bfl/fl mice and analysed. We used the mouse Affymetrix Gene ST Array. The study should determine whether loss of Gfi1 alters the gene expression pattern in the hematopietic stem cells.

2010-09-03 | E-GEOD-20655 | biostudies-arrayexpress

Effect of loss of Gfi1b on hematopietic stem cells

2010-09-03 | GSE20655 | GEO

RNA-Seq Analysis of Gfi1b KO Megakaryocytes

Project description:Megakaryocytes isolated from Gfi1b flox/flox mice carrying PF4-Cre or not, and from Gfi1b flox/flox mice carrying ROSA-Cre-ERT with or without tamoxifen injection were analyzed for differential expression by RNA-Seq

2017-01-21 | GSE71310 | GEO

RNA-Seq Analysis of Gfi1b KO Hematopoietic Stem Cells

Project description:The zinc finger protein and SNAG domain transcription factor Gfi1b is highly expressed in hematopoietic stem cells (HSC) and in megakaryocytes (MKs). Deletion of Gfi1b in mice leads to a drastic expansion of both cell types, suggesting that Gfi1b controls their proliferation. Here we present evidence that Gfi1b exerts this control by modulating the Wnt/beta-catenin signaling pathway. We can show that Gfi1b binds to beta-catenin and is part of a larger complex that contains β-catenin co-factors. Gfi1b activates beta-catenin/TCF mediated transcription and is required for the activity of beta-catenin target gene promoter driven reporter genes in vivo. This is dependent of the ability of Gfi1b to recruit the histone modifying enzyme lysine demethylase 1 (LSD1) to β-catenin containing complexes. Moreover, LSD1 enhances the Gfi1b-mediated activation of beta-catenin/TCF dependent transcription. Both Gfi1b deficient HSCs and MKs show de-regulation of expression of many sets on Wnt/β-catenin target genes and Gfi1b and β-catenin co-occupy the promoters of many common target genes. Finally, activating the Wnt/β-catenin signaling pathway in Gfi1b deficient HSCs and MKs significantly reduces their expansion, which confirms that Gfi1b is a critical factor controlling the cellularity of HSCs and MKs and exerts this function by regulating the Wnt/β-catenin pathway in these cells.

2017-09-01 | GSE85737 | GEO

Effect of Dot1l deletion on LSKs and GMPs

Project description:We wanted to investigate the effects of Dot1l deletion on gene expression in LSKs and GMPs of C57/BL6 mice Aberrant Hox gene activation is a recurrent feature in several different types of human leukemia, including leukemias with rearrangements of the mixed lineage leukemia (MLL) gene. In this study, we demonstrate that Hox gene expression is controlled by higher degree H3K79 methylation in acute myeloid leukemia (AML). We show that the deposition of progressive H3K79 methylation states at the genomic loci of critical Hox genes is dependent on the interaction of the H3K79 methyltransferase Dot1l with Af10, a protein that is found in the Dot1l complex isolated from diverse cell types. Furthermore, abrogation of the Dot1l-Af10 interaction reverses aberrant epigenetic profiles found in the leukemia epigenome and impairs the transforming ability of mechanistically distinct AML oncogenes. Lineage negative Sca-1 positive Kit positive (LSK) cells and granulocyte macrophage progenitors (GMPs) were sorted from Dot1 wt/wt x Mx-Cre mice or Dot1l fl/fl x Mx-Cre mice were injected with PIPC. PIPC injection induced biallelic deletion of the Dot1l allele in the Dot1l fl/fl mice but not the Dot1l wt/wt mice. The Dot1l wt/wt LSKs and GMPs were compared to the Dot1l -/- counterparts by RNA extraction and Microarrays.

2014-09-08 | E-GEOD-54498 | biostudies-arrayexpress

GFI1B and LSD1 repress myeloid traits during megakaryocyte differentiation

Project description:The transcription factor Growth Factor Independence 1B (GFI1B) recruits Lysine Specific Demethylase 1A (LSD1/KDM1A) to stimulate gene programs relevant for megakaryocyte and platelet biology. Inherited pathogenic GFI1B variants result in thrombocytopenia and bleeding propensities with varying intensity. Whether these affect similar gene programs is unknow. Here we studied transcriptomic effects of four patient-derived GFI1B variants (GFI1BT174N,H181Y,R184P,Q287*) in MEG01 megakaryoblasts. Compared to normal GFI1B, each variant affected different gene programs with GFI1BQ287* uniquely failing to repress myeloid traits. In line with this, single cell RNA-sequencing of induced pluripotent stem cell (iPSC)-derived megakaryocytes revealed a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in GFI1BQ287* versus normal conditions. Inhibiting the GFI1B-LSD1 interaction with small molecule GSK-LSD1 resulted in activation of myeloid genes in normal iPSC-derived megakaryocytes similar as observed for GFI1BQ287* iPSC-derived megakaryocytes. Thus, GFI1B and LSD1 facilitate gene programs relevant for megakaryopoiesis while simultaneously repressing programs that induce myeloid differentiation.

2024-03-28 | PXD050401 | Pride

Overexpression of Gfi1b in a pro-B Abelson murine leukemia virus transformed cell line

Project description:We have conducted a screen for factors that downregulate expression of the genes encoding the V(D)J recombinase (RAG1 and RAG2) during B cell development. We have identified the transcription factor Gfi1B as being one of the proteins capable of decreasing RAG transcription when overexpressed in Ableson transormed ProB cell lines. We have yet to determine whether the overexpression of Gfi1B downregulates the RAGs directly, or whether it initiates a signalling programme that results in RAG downregulation. We hypothesize that by comparing global gene expression patterns in cells that overexpress Gfi1B and those that do not, we can distinguish between these possibilities and additionally gain insight into the broader genetic program that may be influenced by Gfi1B during hematopoiesis. Abelson pro-B cells were infected with a retrovirus encoding Gfi1b fused to the estrogen receptor domain. Gfi1b expression was induced by adding tamoxifen to the culture medium for 12h. Three biological replicates of untreated and treated cells were analyzed.

2011-12-13 | E-GEOD-33709 | biostudies-arrayexpress

Pathogenesis of dominant-negative mutant GFI1B and associated proteomic abnormalities in megakaryocytes and platelets

Project description:Dominant-negative mutations in transcription factor Growth Factor Independence-1B (GFI1B) cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that normal and mutant GFI1B interacted most strongly with the LSD1-RCOR-HDAC corepressor complex in megakaryoblasts. Sequestration of this complex by mutant GFI1B and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1B-mutant induced pluripotent stem cells (iPSC) also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant iPSC-derived megakaryocytes identified a multitude of deregulated pathways downstream of mutant GFI1B. Proteome studies on primary normal and GFI1B-mutant platelets showed reduced expression of proteins implicated in platelet function, and sustained expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B regulates a broad developmental program during megakaryopoiesis. Mutant GFI1B deregulates this program through LSD1-RCOR-HDAC sequestering.

2019-01-25 | PXD009020 | Pride

Transcriptomic changes induced by GFI1B variants

Project description:Mutations in transcription factor Growth Factor Independence 1B (GFI1B) cause inherited bleedings with varying intensity possibly caused by different effects on the transcriptional function of GFI1B. We studied transcriptomic changes of normal and GFI1BT174N,H181Y,R184P,Q287* mutants in megakaryoblast MEG01 cells using RNA-sequencing. Compared to normal GFI1B each variant affected different gene modules with limited overlap between variants. Remarkably, GFI1BQ287* specifically activated a myeloid-associated gene module. Based on this finding we studied megakaryocyte differentiation using normal and GFI1BQ287* patient-derived induced pluripotent stem cells (IPSC) followed by single cell RNA-sequencing. This revealed a 45-fold decrease in the megakaryocyte/myeloid cell ratio in the GFI1BQ287* versus control condition. Moreover, myeloid specific genes were expressed within GFI1BQ287* but not normal megakaryocytes. Finally, we studied how megakaryocyte development was affected by inhibiting binding of GFI1B to Lysine Specific Demethylase 1 (LSD1), an interaction required for megakaryocyte development. Treatment of both MEG01 cells and normal IPSC-derived developing megakaryocytes with the small-molecule inhibitor GSK-LSD1 resulted in profound activation of myeloid gene programs within megakaryocytes, while the IPSC-derived megakaryocyte/myeloid ratio dropped two-fold. We conclude that GFI1B and LSD1 suppress myeloid differentiation allowing for proper megakaryocyte development. LSD1 inhibitor and GFI1BQ287*-mediated perturbation of this function causes myeloid skewing during megakaryocyte development.

2024-03-28 | GSE244756 | GEO

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