Gene expression signatures in Tif1b, Hp1a or Hp1g knockdown hematopoietic progenitors
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Satoru Miyagi
PROVIDER: E-GEOD-48897 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Gene expression analyses of Tif1b, Hp1a or Hp1g knockdown hematopoietic progenitors. Growth of hematopoietic stem cells (HSCs) are significantly impaired upon knockdown of Hp1a and Hp1g. Results provide insight into the role of these factors in hematopoiesis.
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Project description:The transcription factor Meis1 is preferentially expressed in hematopoietic stem cells (HSCs) and over-expressed in certain leukemias. However, the functions of Meis1 in hematopoiesis remain largely unknown. Using inducible knock-out mice, we found that Meis1 is required for the maintenance of hematopoiesis under stress and over long term while steady-state hematopoiesis was sustained in the absence of Meis1. Bone marrow cells of Meis1 deficient mice showed reduced colony formation, contained significantly fewer numbers of long- term HSCs and these Meis1-deficient HSCs exhibited loss of quiescence. Further, we found that Meis1 deletion led to the accumulation of reactive oxygen species (ROS) in HSCs and decreased expression of genes implicated in hypoxia response. Finally, ROS scavenging by N-acetyl cysteine or stabilization of hypoxia-signaling by knockdown of the VHL protein led to reversal of the effects of Meis1-deletion. Taken together, these results demonstrate that Meis1 protects and preserves HSCs by restricting oxidative metabolism. Lineage negative cells were harvested from Meis1-flox/CreER or control mice and incubated with 4-OHT for 48 hours. Total RNA was extracted and used for the profiling experiment.
2012-09-26 | E-GEOD-41133 | biostudies-arrayexpress
Project description:We have used a retroviral integration screen to search for novel genes that regulate hematopoietic stem cell (HSC) function. One of the genes that conferred HSC dominance when overexpressed due to an adjacent retroviral insertion was Musashi 2 (Msi2), an RNA binding protein that can act as a translational inhibitor. A gene trap mouse model that inactivates the gene shows that Msi2 is more highly expressed in long term (LT) and short term (ST) HSCs as well as in lymphoid myeloid primed progenitors (LMPPs), but much less in intermediate progenitors and mature cells. Mice lacking Msi2 are fully viable up to more than a year but exhibit severe defects in primitive precursors, most significantly a reduction in the number of ST-HSCs and LMPPs and a decrease of leukocyte numbers, effects that are exacerbated with age. Cell cycle and gene expression analyses suggest that the main hematopoietic defect in Msi2 defective mice consists in a decreased proliferation capacity of ST-HSCs and LMPPs. In addition, HSCs lacking Msi2 are severely impaired in competitive repopulation experiments, being overgrown by wild type cells even when mutant cells were provided in excess. Our data indicate that Msi2 maintains the stem cell compartment mainly by regulating the proliferation of primitive progenitors downstream of LT-HSCs. RNA from 2 different cell populations (LSK cells and KIT+ cells) were analyzed for both wildtype and Msi2-/- null samples. Two technical replicates were analyzed for the KIT+ samples and three technical replicates were analyzed for the LSK samples.
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Project description:Hematopoietic stem cells (HSCs) are located in the bone marrow in a specific microenvironment referred as the hematopoietic stem cell niche, where HSCs interact with a variety of stromal cells. Though several components of the stem cell niche have been identified, the regulatory mechanisms through which such components regulate the stem cell fate are still unknown. In order to address this issue, we investigated how osteoblasts (OBs) can affect the molecular and functional phenotype of HSCs and vice versa. Our data showed that CD34+ cells cultured with OBs give rise to higher total cell numbers, produce more CFU and maintain a higher percentage of CD34+CD38- cells compared to control culture. Moreover, clonogenic assay and long-term culture results showed that OBs enhance HSC differentiation towards the mono/macrophage lineage at the expense of the granulocytic and erythroid ones. Finally, GEP analysis allowed us to identify several cytokine-receptor networks, such as WNT pathway, and transcription factors, as TWIST1 and FOXC1, that could be activated by co-culture with OBs and could be responsible for the biological effects reported above. Altogether our results indicate that OBs are able to affect both HPC maintenance and differentiation capacity by modulating mono/macrophage and erythroid commitment. We set up a co-culture system composed of human CD34+ cells in culture with human OBs. After coculture, CD34+ cells and the hematopoietic cell fraction were separated from OBs and analyzed by gene expression profiling (GEP), clonogenic assay and long-term culture.
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Project description:CB CD34+ cells were isolated by Miltenyi miniMACS column. Cells were prestimulated in HPGM with 100 ng/ml KITL, FLT#L and TPO for 48 hrs. Cells were transduced with control MiNR1 or STAT5A-ER in three rounds over 48 hrs. Hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and megakaryocyte/erythroid progenitors (MEPs) were sorted (for details see Blood 2011, Fatrai et al). cells were stimulated with 100 ng/ml 4OHT for 24 hrs after which RNA was isolated for Illumina beadhchiop arrays HT12 v3 CB CD34+ cells were isolated by Miltenyi miniMACS column. Cells were prestimulated in HPGM with 100 ng/ml KITL, FLT#L and TPO for 48 hrs. Cells were transduced with control MiNR1 or STAT5A-ER in three rounds over 48 hrs. Hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and megakaryocyte/erythroid progenitors (MEPs) were sorted (for details see Blood 2011, Fatrai et al). cells were stimulated with 100 ng/ml 4OHT for 24 hrs after which RNA was isolated for Illumina beadhchiop arrays HT12 v3
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Project description:Gene expression analyses of hematopoietic stem cells (HSCs), progenitor cells (HPCs), and differentiated cell. Gene expressions of long-term HSCs (CD34-ckit+Sca1+Lineage-), short term HSCs (CD34+ckit+Sca1+Lineage-), Progenitor cells (ckit+Sca1- Lineage-), and differentiated cels (Lineage+) were examined by microarray. Results provide insight into the mechanism of hematopoietic cell differentiation. Long-term HSCs (CD34-ckit+Sca1+Lineage-), Short term-HSCs (CD34+ckit+Sca1+Lineage-), Progenitor cells (ckit+Sca1- Lineage-, and Lineage+), and differentiated cell (Lineage+) were sorted from mouse bone marraw and were examined by microarray. Results provide insight into the mechanism of hematopoietic cell differentiation.
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Project description:Gene expression signatures in Tif1b, Hp1a or Hp1g knockdown hematopoietic progenitors
| PRJNA212232 | ENA
Project description:Hematopoietic differentiation is strictly regulated by complex network of transcription factors that are controlled by ligands binding to cell surface receptors. Disruptions of the intricate sequences of transcriptional activation and suppression of multiple genes cause hematological diseases, such as leukemias, myelodysplastic syndromes or myeloproliferative syndromes. From a clinical standpoint, deciphering the pattern of gene expression during hematopoiesis may help unravel disease-specific mechanisms in hematopoietic malignancies. Herein, we describe a human in vitro hematopoietic model system where lineage specific differentiation of CD34+ cells was accomplished using specific cytokines. Microarray and RNAseq based whole transcriptome and exome analysis was performed on the differentiated erythropoietic, granulopoietic and megakaryopoietic cells to delineate changes in expression of whole transcripts and exons. Analysis on the Human 1.0 ST exon arrays indicated differential expression of 172 genes (P< 0.0000001) and significant splicing of 86 genes during differentiation. Pathway analysis identified these genes to be involved in Rac/RhoA signaling, Wnt/B-catenin signaling and alanine/aspartate metabolism. Comparison of the microarray data to next generation RNAseq analysis during erythroid differentiation demonstrated a high degree of correlation in gene (R= 0.72) and exon (R=0. 62) expression. Our data provides a molecular portrait of events that regulate differentiation of hematopoietic cells. Knowledge of molecular processes by which the cells acquire their cell specific fate would be beneficial in developing cell-based therapies for human diseases. Comparison of gene expression patterns between CD34, E, M and G groups
2011-11-22 | E-GEOD-29989 | biostudies-arrayexpress
Project description:Epigenetic mechanisms including histone modifications have emerged as important factors influencing cell fate determination. The functional role of H3K4 methylation, however, remains largely unclear in the maintenance and differentiation of hematopoietic stem/progenitor cells (HSC/HPCs). Here we show that DPY30, a shared core subunit of the SET1/MLL family methyltransferase complexes and a facilitator of their H3K4 methylation activity, is important for ex vivo proliferation and differentiation of human CD34+ HPCs. DPY30 promotes HPC proliferation by directly regulating the expression of genes critical for cell proliferation. Interestingly, while DPY30 knockdown (KD) in HPCs impaired their differentiation into the myelomonocytic lineage, it potently promoted hemoglobin production and affected the kinetics of their differentiation into the erythroid lineage. In an in vivo model, we show that morpholino-mediated dpy30 KD resulted in severe defects in the development of the zebrafish hematopoietic system, which could be partially rescued by co-injection of dpy30 mRNA. Taken together, our results establish a critical role of DPY30 in the proliferation and appropriate differentiation of hematopoietic progenitor cells as well as in animal hematopoiesis. Finally, we also demonstrate a crucial role of DPY30 in the growth of several MLL1-fusion-mediated leukemia cell lines. Total RNAs from control (scr) or knockdown (hD2, hD5) cells before and after culturing under condition promoting myelomonocytic differentiation were subjected to Illumina microarray analyses.
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