Modulation of p210BCR-ABL activity in transduced primary human hematopoietic cells controls lineage programming

Blood ◽  
2002 ◽  
Vol 99 (9) ◽  
pp. 3197-3204 ◽  
Author(s):  
Yves Chalandon ◽  
Xiaoyan Jiang ◽  
Glen Hazlewood ◽  
Slade Loutet ◽  
Eibhlin Conneally ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Kinase Inhibitor ◽  
Hematopoietic Cells ◽  
Erythroid Differentiation ◽  
Human Leukemia ◽  
Retroviral Transduction ◽  
Sti 571 ◽  
Human Cd34 ◽  
Green Fluorescent

Abstract Retroviral transduction of primary hematopoietic cells with human oncogenes provides a powerful approach to investigating the molecular mechanisms controlling the normal proliferation and differentiation of these cells. Here we show that primitive human CD34+ cord blood cells, including multipotent as well as granulopoietic- and erythroid-restricted progenitors, can be efficiently transduced with a MSCV-BCR-ABL-IRES-GFP retrovirus, resulting in the sustained expression by their progeny of very high levels of tyrosine phosphorylated p210BCR-ABL. Interestingly, even in the presence of growth factors that supported the exclusive production of granulopoietic cells from green fluorescent protein (GFP)–transduced control cells, BCR-ABL–transduced progenitor subpopulations generated large numbers of erythropoietin-independent terminally differentiating erythroid cells and reduced numbers of granulopoietic cells. Analyses of individual clones generated by single transduced cells in both semisolid and liquid cultures showed this BCR-ABL–induced erythroid differentiation response to be elicited at a high frequency from all types of transduced CD34+ cells independent of their apparent prior lineage commitment status. Additional experiments showed that this erythroid differentiation response was largely prevented when the cells were transduced and maintained in the presence of the BCR-ABL–specific tyrosine kinase inhibitor, STI-571. These findings indicate that overexpression of BCR-ABL in primary human hematopoietic cells can activate an erythroid differentiation program in apparently granulopoietic-restricted cells through a BCR-ABL kinase-dependent mechanism, thus providing a new molecular tool for elucidating mechanisms underlying lineage fate determination in human hematopoietic cells and infidelity in human leukemia.

WZsGreen/+: a new green fluorescent protein knock-in mouse model for the study of KIT-expressing cells in gut and cerebellum

2005 ◽  
Vol 22 (3) ◽  
pp. 412-421 ◽  
Author(s):  
Mira Wouters ◽  
Karine Smans ◽  
Jean-Marie Vanderwinden
Keyword(s):  
Small Intestine ◽  
Green Fluorescent Protein ◽  
Gastrointestinal Tract ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Molecular Layer ◽  
Transgenic Animals ◽  
The Novel ◽  
Suitable Alternative ◽  
Green Fluorescent

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. KIT is currently the sole practical marker for ICC. Attempts to purify living ICC have so far largely failed, due to the loss of the KIT epitope during enzymatic dissociation. Aiming to identify and isolate living ICC, we designed a knock-in strategy to express a fluorescent tag in KIT-expressing cells by inserting the sequence of the novel green fluorescent protein ZsGreen into the first exon of the c-Kit gene, creating a null allele called WZsGreen. In the gastrointestinal tract of heterozygous WZsGreen/+ mice, tiny ZsGreen fluorescent dots were observed in all KIT-expressing ICC populations, with exception of ICC at the deep muscular plexus in small intestine. During development of the gastrointestinal tract, ZsGreen expression followed KIT expression in a spatiotemporal way. Stellate and basket KIT-expressing cells in the molecular layer of the cerebellum also exhibited ZsGreen dots, whereas no ZsGreen was detected in skin, testis, and bone marrow. ZsGreen dot-containing intestinal cells could be isolated from jejunum and maintained alive in culture for at least 3 days. ZsGreen is a suitable alternative to EGFP in transgenic animals. The novel WZsGreen/+ model reported here appears to be a promising tool for live studies of KIT-expressing cells in the gastrointestinal tract and cerebellum and for the further analysis of pacemaker mechanisms.

Impairment of human terminal erythroid differentiation by histone deacetylase 5 deficiency

Blood ◽  
2021 ◽  
Author(s):  
Yaomei Wang ◽  
Wei Li ◽  
Vince Schulz ◽  
Huizhi Zhao ◽  
Xiaoli Qu ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Molecular Mechanisms ◽  
Chromatin Condensation ◽  
Erythroid Differentiation ◽  
Chromatin Accessibility ◽  
Erythroid Cell ◽  
Global Changes ◽  
Human Cd34 ◽  
Wide Range ◽  
Terminal Erythroid Differentiation

Histone deacetylases (HDACs) are a group of enzymes catalyzing the removal of acetyl groups from histone and non-histone proteins. HDACs have been shown to play diverse functions in a wide range of biological processes. However, their roles in mammalian erythropoiesis remain to be fully defined. We show here that of the eleven classic HDAC family members, six of them (HDAC 1,2,3 and HDAC 5,6,7) are expressed in human erythroid cells with HDAC5 most significantly up regulated during terminal erythroid differentiation. Knockdown of HDAC5 by either shRNA or siRNA in human CD34+ cells followed by erythroid cell culture led to increased apoptosis, decreased chromatin condensation, and impaired enucleation of erythroblasts. Biochemical analyses revealed that HDAC5 deficiency resulted in activation of p53 in association with increased acetylation of p53. Furthermore, while acetylation of histone 4 (H4) is decreased during normal terminal erythroid differentiation, HDAC5 deficiency led to increased acetylation of H4 (K12) in late stage erythroblasts. This increased acetylation was accompanied by decreased chromatin condensation, implying a role for H4 (K12) deacetylation in chromatin condensation. ATAC-seq and RNA-seq analyses revealed that HDAC5 knockdown leads to increased chromatin accessibility genome wide and global changes in gene expression. Moreover, pharmacological inhibition of HDAC5 by the inhibitor LMK235 also led to increased H4 acetylation, impaired chromatin condensation and enucleation. Taken together, our findings have uncovered previously unrecognized roles and molecular mechanisms of action for HDAC5 in human erythropoiesis. These results may provide insights into understanding the anemia associated with HDAC inhibitor treatment.

scholarly journals Gfi1aa and Gfi1b set the pace for primitive erythroblast differentiation from hemangioblasts in the zebrafish embryo

2018 ◽  
Vol 2 (20) ◽  
pp. 2589-2606 ◽  
Author(s):  
Chris Moore ◽  
Joanna L. Richens ◽  
Yasmin Hough ◽  
Deniz Ucanok ◽  
Sunir Malla ◽  
...  
Keyword(s):  
Cell Fate ◽  
Fluorescent Protein ◽  
Erythroid Differentiation ◽  
Transcriptional Repressors ◽  
Rna Seq ◽  
Promote Cell Proliferation ◽  
Cell Fate Decisions ◽  
Maturation Defect ◽  
Green Fluorescent

Abstract The transcriptional repressors Gfi1(a) and Gfi1b are epigenetic regulators with unique and overlapping roles in hematopoiesis. In different contexts, Gfi1 and Gfi1b restrict or promote cell proliferation, prevent apoptosis, influence cell fate decisions, and are essential for terminal differentiation. Here, we show in primitive red blood cells (prRBCs) that they can also set the pace for cellular differentiation. In zebrafish, prRBCs express 2 of 3 zebrafish Gfi1/1b paralogs, Gfi1aa and Gfi1b. The recently identified zebrafish gfi1aa gene trap allele qmc551 drives erythroid green fluorescent protein (GFP) instead of Gfi1aa expression, yet homozygous carriers have normal prRBCs. prRBCs display a maturation defect only after splice morpholino-mediated knockdown of Gfi1b in gfi1aaqmc551 homozygous embryos. To study the transcriptome of the Gfi1aa/1b double-depleted cells, we performed an RNA-Seq experiment on GFP-positive prRBCs sorted from 20-hour-old embryos that were heterozygous or homozygous for gfi1aaqmc551, as well as wt or morphant for gfi1b. We subsequently confirmed and extended these data in whole-mount in situ hybridization experiments on newly generated single- and double-mutant embryos. Combined, the data showed that in the absence of Gfi1aa, the synchronously developing prRBCs were delayed in activating late erythroid differentiation, as they struggled to suppress early erythroid and endothelial transcription programs. The latter highlighted the bipotent nature of the progenitors from which prRBCs arise. In the absence of Gfi1aa, Gfi1b promoted erythroid differentiation as stepwise loss of wt gfi1b copies progressively delayed Gfi1aa-depleted prRBCs even further, showing that Gfi1aa and Gfi1b together set the pace for prRBC differentiation from hemangioblasts.

Induced Expression of the Heat Shock Protein Genes uspA and grpE during Starvation at Low Temperatures and Their Influence on Thermal Resistance of Escherichia coli O157:H7

2003 ◽  
Vol 66 (11) ◽  
pp. 2045-2050 ◽  
Author(s):  
YI ZHANG ◽  
MANSEL W. GRIFFITHS
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Stress Responses ◽  
Thermal Tolerance ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Bacterial Cells ◽  
Induced Expression ◽  
Green Fluorescent ◽  
Shock Proteins

Heat shock proteins play an important role in protecting bacterial cells against several stresses, including starvation. In this study, the promoters for two genes encoding heat shock proteins involved in many stress responses, UspA and GrpE, were fused with the green fluorescent protein (gfp) gene. Thus, the expression of the two genes could be quantified by measuring the fluorescence emitted by the cells under different environmental conditions. The heat resistance levels of starved and nonstarved cells during storage at 5, 10, and 37°C were compared with the levels of expression of the uspA and grpE genes. D52-values (times required for decimal reductions in count at 52°C) increased by 11.5, 14.6, and 18.5 min when cells were starved for 3 h at 37°C, for 24 h at 10°C, and for 2 days at 5°C, respectively. In all cases, these increases were significant (P < 0.01), indicating that the stress imposed by starvation altered the ability of E. coli O157:H7 to survive subsequent heat treatments. Thermal tolerance was correlative with the induction of UspA and GrpE. At 5°C, the change in the thermal tolerance of the pathogen was positively linked to the induced expression of the grpE gene but negatively related to the expression of the uspA gene. The results obtained in this study indicate that UspA plays an important role in starvation-induced thermal tolerance at 37°C but that GrpE may be more involved in regulating this response at lower temperatures. An improvement in our understanding of the molecular mechanisms involved in these cross-protection responses may make it possible to devise strategies to limit their effects.

The Cdk and PCNA domains on p21Cip1 both function to inhibit G1/S progression during hyperoxia

2004 ◽  
Vol 286 (3) ◽  
pp. L506-L513 ◽  
Author(s):  
Christopher E. Helt ◽  
Rhonda J. Staversky ◽  
Yi-Jang Lee ◽  
Robert A. Bambara ◽  
Peter C. Keng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Fluorescent Protein ◽  
Nuclear Antigen ◽  
Cell Cycle Regulators ◽  
Amino Terminal ◽  
Binding Domains ◽  
Green Fluorescent

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G1 when exposed to 95% oxygen. Instead, cells arrested in S and G2. Stable expression of p53 restored induction of p21 and G1 arrest without affecting mRNA expression of the other Cip or INK4 G1 kinase inhibitors. To confirm the role of p21 in G1 arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G1 arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G1 during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G1 arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.

scholarly journals Retroviral-Mediated Transfer of the Green Fluorescent Protein Gene Into Murine Hematopoietic Cells Facilitates Scoring and Selection of Transduced Progenitors In Vitro and Identification of Genetically Modified Cells In Vivo

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 1777-1786 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Esther R. Allay ◽  
Richard J. Smeyne ◽  
Richard A. Ashmun ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
High Titer ◽  
Hematopoietic Cells ◽  
Green Fluorescent Protein Gene ◽  
Retroviral Gene Transfer ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-titer, ecotropic retroviral producer cells free of replication competent virus were generated and used to transduce murine bone marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were intensely fluorescent compared to mock-transduced cells or cells transduced with a control retrovirus. Erythroid and myeloid hematopoietic colonies derived from GFP-transduced marrow were easily scored for retroviral gene transfer by direct in situ fluorescence microscopy. Clonogenic progenitors expressing increased levels of antifolate drug resistance could be enriched from the GFP-transduced marrow population by fluorescence activated cell sorting of cells expressing high levels of GFP. In vivo, splenic hematopoietic colonies and peripheral blood cells from animals transplanted with GFP-transduced marrow displayed intense fluorescence. These results show that GFP is an excellent marker for scoring and tracking gene-modified hematopoietic cells and for allowing rapid selection and enrichment of transduced cells expressing high levels of the transgene.

Targeting gene-modified hematopoietic cells to the central nervous system: Use of green fluorescent protein uncovers microglial engraftment

2001 ◽  
Vol 7 (12) ◽  
pp. 1356-1361 ◽  
Author(s):  
Josef Priller ◽  
Alexander Flügel ◽  
Tim Wehner ◽  
Matthias Boentert ◽  
Carola A. Haas ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Hematopoietic Cells ◽  
System Use ◽  
The Central Nervous System ◽  
Green Fluorescent
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