Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

MYB silencing in CD14-myeloblasts

ABSTRACT: The c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define the role of c-Myb during the terminal differentiation of hematopoietic precursors, we studied the effects of its silencing in human primary CD14-myeloblasts, which maintain a granulo-monocyte differentiation bipotentiality. c-Myb-silenced myeloblasts were blocked in the G1 phase of the cell cycle at 24 hours post-nucleofection and subsequently were forced towards macrophage differentiation, as demonstrated by immunophenotypic and morphological analysis. Indeed, c-Myb-silenced CD14- cells differentiate to macrophage even after the treatment with ATRA 10-6 M, demonstrating that the c-Myb knockdown strongly impairs the ability of myeloblasts to differentiate to granulocytes. Gene expression profiling of c-Myb-silenced CD14- cells identified some potential c-Myb targets that can account for these effects. To maximize siRNA transfection efficiency, we utilized the NucleofectorTM technology (Amaxa). CD14- cells were transfected with a mixture of 3 siRNAs targeting c-Myb mRNA, with a non-targeting siRNA as a negative control (NegCTR) and without siRNA (MOCK). c-Myb siRNAs were transfected at days 1 to 3 after the CD14- myeloblasts purification based on the observation that this timing represented a phase of phisiological increase in c-Myb expression in myeloblasts. Western Blot analysis at 24 hours post-nucleofection demonstrated the c-Myb protein knockdown in MYBsiRNA CD14- cells as compared with control samples (MOCK, NegCTR).

ORGANISM(S): Homo sapiens

SUBMITTER: Rossella Manfredini

PROVIDER: E-GEOD-21943 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

c-myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression.

Bianchi Elisa E Zini Roberta R Salati Simona S Tenedini Elena E Norfo Ruggiero R Tagliafico Enrico E Manfredini Rossella R Ferrari Sergio S

Blood 20100804 22

The c-myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define its role during the hematopoietic lineage commitment, we silenced c-myb in human CD34(+) hematopoietic stem/progenitor cells. Noteworthy, c-myb silencing increased the commitment capacity toward the macrophage and megakaryocyte lineages, whereas erythroid differentiation was impaired, as demonstrated by clonogenic assay, morphologic and immunophenotypic data. Ge ...[more]

PMID: 20686118

Similar Datasets

Project description:The transcription factor cMyb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that cMyb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which cmyb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. mRNA and miRNA expression for each sample were profiled by Affymetrix GeneAtlas U219 strip array and Exiqon Human miRNome PCR Panel, respectively. miRNA/mRNA data were integrated by Ingenuity Pathway Analysis. The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs downregulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p overexpression in CD34+ cells. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes downregulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as upregulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p upregulation and subsequent c-maf downregulation as a new molecular mechanism through which cMyb favours erythropoiesis while restraining megakaryopoiesis. RNA from CD34+ HPCs transfected with c-myb-targeting/non targeting control (NegCTR) synthetic siRNAs was collected 24 hours post-Nucleofection for a set of 5 independent experiments.

Project description:The transcription factor cMyb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that cMyb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which cmyb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. mRNA and miRNA expression for each sample were profiled by Affymetrix GeneAtlas U219 strip array and Exiqon Human miRNome PCR Panel, respectively. miRNA/mRNA data were integrated by Ingenuity Pathway Analysis. The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs downregulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p overexpression in CD34+ cells. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes downregulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as upregulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p upregulation and subsequent c-maf downregulation as a new molecular mechanism through which cMyb favours erythropoiesis while restraining megakaryopoiesis. RNA from CD34+ HPCs transfected once/twice/3 times with c-myb-targeting/non targeting control siRNAs was collected for a set of 5 independent experiments.

Project description:The transcription factor c-Myb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that c-Myb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which c-myb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs downregulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p overexpression in CD34+ cells. Moreover, in order to identify the mRNA target through which hsa-miR-486-3p affects lineage fate decision, we profiled the mRNA changes in mimic transfected CD34+ HPC by means of Affymetrix GeneAtlas U219 strip array. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes downregulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as upregulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p upregulation and subsequent c-maf downregulation as a new molecular mechanism through which cMyb favours erythropoiesis while restraining megakaryopoiesis. Gene expression profile (GEP) was performed on total RNA derived from three independent experiments at 24h after the last nucleofection.

Dataset Information

MYB silencing in CD14-myeloblasts

Publications

c-myb supports erythropoiesis through the transactivation of KLF1 and LMO2 expression.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets