scholarly journals KU 812: a pluripotent human cell line with spontaneous erythroid terminal maturation

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 2003-2013
Author(s):  
M Nakazawa ◽  
MT Mitjavila ◽  
N Debili ◽  
N Casadevall ◽  
P Mayeux ◽  
...  
Keyword(s):  
Cell Line ◽  
Messenger Rna ◽  
Minor Component ◽  
Erythroid Cell ◽  
Adherent Cell ◽  
Erythroid Cells ◽  
Gamma Chain ◽  
Single Class ◽  
Semisolid Medium ◽  
Cell Commitment

A human leukemic cell line KU 812 was recently established and described as a basophilic cell line. In the present study we show that KU 812 and two of its clones are at least bipotent: in addition to a minor component of basophils, the majority of KU 812 cells belongs to the erythroid cell lineage with a significant percentage (about 15%) of mature hemoglobinized erythroblasts. This terminal differentiation is associated with the synchronized synthesis of the main erythroid proteins, including glycophorins, spectrin beta chain, band 3, and hemoglobin. The predominant hemoglobins are adult, fetal, and Bart's hemoglobin. Adult hemoglobin represented up to 75% of all hemoglobins in the KU 812 F clone in passages containing a high number of mature erythroblasts. Transcripts of all human globin chains were present with ten times less embryonic chain messenger RNA (mRNA) than alpha-, beta- or gamma-chain mRNA. Hemin slightly increased the total hemoglobin production of the cell line, especially gamma-globin chain synthesis, but did not modify the percentage of hemoglobinized cells. Phorbol myristate acetate (PMA) had a complex effect, inducing a proportion of KU 812 cells to adhere to the plastic culture flask. The adherent cell fraction expressed a very low level of specific erythroid proteins, but their ultrastructure was consistent with immature erythroid cells. In contrast, approximately 40% of the nonadherent cells were mature erythroid cells. Cell-sorting experiments showed that this paradoxic effect of PMA is mostly due to cell selection, the more mature cells being unable to adhere. In addition, KU 812 F was found to be sensitive to erythropoietin, which slightly increased its plating efficiency range (from 0% to 50%) in semisolid medium and enhanced hemoglobin accumulation twofold. In binding experiments using 125I erythropoietin, a single class of high-affinity Epo receptors (Kd: 250 pM) was detected by binding with a density of 205 receptors per cell. The KU 812 cell line is therefore a unique model for studying cell commitment toward different hematopoietic lineages and erythroid differentiation.

scholarly journals KU 812: a pluripotent human cell line with spontaneous erythroid terminal maturation

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 2003-2013 ◽  
Author(s):  
M Nakazawa ◽  
MT Mitjavila ◽  
N Debili ◽  
N Casadevall ◽  
P Mayeux ◽  
...  
Keyword(s):  
Cell Line ◽  
Messenger Rna ◽  
Minor Component ◽  
Erythroid Cell ◽  
Adherent Cell ◽  
Erythroid Cells ◽  
Gamma Chain ◽  
Single Class ◽  
Semisolid Medium ◽  
Cell Commitment

Abstract A human leukemic cell line KU 812 was recently established and described as a basophilic cell line. In the present study we show that KU 812 and two of its clones are at least bipotent: in addition to a minor component of basophils, the majority of KU 812 cells belongs to the erythroid cell lineage with a significant percentage (about 15%) of mature hemoglobinized erythroblasts. This terminal differentiation is associated with the synchronized synthesis of the main erythroid proteins, including glycophorins, spectrin beta chain, band 3, and hemoglobin. The predominant hemoglobins are adult, fetal, and Bart's hemoglobin. Adult hemoglobin represented up to 75% of all hemoglobins in the KU 812 F clone in passages containing a high number of mature erythroblasts. Transcripts of all human globin chains were present with ten times less embryonic chain messenger RNA (mRNA) than alpha-, beta- or gamma-chain mRNA. Hemin slightly increased the total hemoglobin production of the cell line, especially gamma-globin chain synthesis, but did not modify the percentage of hemoglobinized cells. Phorbol myristate acetate (PMA) had a complex effect, inducing a proportion of KU 812 cells to adhere to the plastic culture flask. The adherent cell fraction expressed a very low level of specific erythroid proteins, but their ultrastructure was consistent with immature erythroid cells. In contrast, approximately 40% of the nonadherent cells were mature erythroid cells. Cell-sorting experiments showed that this paradoxic effect of PMA is mostly due to cell selection, the more mature cells being unable to adhere. In addition, KU 812 F was found to be sensitive to erythropoietin, which slightly increased its plating efficiency range (from 0% to 50%) in semisolid medium and enhanced hemoglobin accumulation twofold. In binding experiments using 125I erythropoietin, a single class of high-affinity Epo receptors (Kd: 250 pM) was detected by binding with a density of 205 receptors per cell. The KU 812 cell line is therefore a unique model for studying cell commitment toward different hematopoietic lineages and erythroid differentiation.

Heme-Regulated Inhibitor (HRI) Activates Transcription Factor ATF4 to Promote BCL11A Transcription and Fetal Hemoglobin Silencing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 814-814
Author(s):  
Peng Huang ◽  
Scott A. Peslak ◽  
Xianjiang Lan ◽  
Eugene Khandros ◽  
Malini Sharma ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Research Funding ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Mrna Levels ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Major Pathway ◽  
Specific Regulation

Reactivation of fetal hemoglobin in adult red blood cells benefits patients with sickle cell disease and β-thalassemia. BCL11A is one of the predominant repressors of fetal γ-globin transcription and stands as an appealing target for therapeutic genome manipulation. However, pharmacologic perturbation of BCL11A function or its co-regulators remains an unmet challenge. Previously, we reported the discovery of the erythroid-enriched protein kinase HRI as a novel regulator of γ-globin transcription and found that HRI functions in large part via controlling the levels of BCL11A transcription (Grevet et al., Science, 2018). However, the specific mechanisms underlying HRI-mediated modulation of BCL11A levels remain unknown. To identify potential HRI-controlled transcription factors that regulate BCL11A, we performed a domain-focused CRISPR screen that targeted the DNA binding domains of 1,447 genes in the human erythroid cell line HUDEP2. Activating transcription factor 4 (ATF4) emerged as a novel γ-globin repressor. Prior studies reported that ATF4 production is under positive influence of HRI. Specifically, HRI phosphorylates translation factor EIF2α which in turn augments translation of ATF4 mRNA. As expected, HRI deficiency reduced ATF4 protein amounts in HUDEP2 and primary erythroid cells. We further found that the degree of γ-globin reactivation was similar in ATF4 and HRI-depleted cells. ATF4 ChIP-seq in both HUDEP2 and primary erythroblast identified 4,547 and 3,614 high confidence binding sites, respectively. Notably, we did not observe significant enrichment of ATF4 binding or even the presence of an ATF4 consensus motif at the γ-globin promoters, suggesting that ATF4 regulates the γ-globin genes indirectly. However, ATF4 specifically bound to one of the three major BCL11A erythroid enhancers (+55) in both cell types. This was the sole binding site within the ~0.5Mb topologically associating domain that contains the BCL11A gene. Eliminating this ATF4 motif via CRISPR guided genome editing lowered BCL11A mRNA levels and increased γ-globin transcription. Capture-C showed that ATF4 knock-out or removal of the ATF4 site at the BCL11A (+55) enhancer decreased chromatin contacts with the BCL11A promoter. Forced expression of BCL11A largely restored γ-globin silencing in cells deficient for ATF4 or lacking the ATF4 motif in the BCL11A (+55) enhancer. An unexplained observation from our prior study was that HRI loss did not significantly lower Bcl11a levels in murine erythroid cells. Therefore, we mutated the analogous ATF4 motif in the Bcl11a enhancer in the murine erythroid cell line G1E. Unlike in human cells, Bcl11a mRNA synthesis was decreased only very modestly, and there was no effect on the murine embryonic globin genes whose silencing requires Bcl11a. This suggests that the species specific regulation of BCL11A by HRI results from divergent functional roles of ATF4 binding at the BCL11A (+55) enhancer. In sum, our studies uncover a major pathway that extends linearly from HRI to ATF4 to BCL11A to γ-globin. Moreover, these results further support HRI as a pharmacologic target for the selective regulation of BCL11A and γ-globin. Disclosures Blobel: Pfizer: Research Funding; Bioverativ: Research Funding.

scholarly journals Erythropoietin-induced stimulation of differentiation and proliferation in J2E cells is not mimicked by chemical induction

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 412-419 ◽  
Author(s):  
SJ Busfield ◽  
SP Klinken
Keyword(s):  
Cell Line ◽  
Messenger Rna ◽  
Cellular Proliferation ◽  
Erythroid Cell ◽  
Chemical Induction ◽  
Chemically Induced ◽  
Proliferation And Differentiation ◽  
Differentiation And Proliferation ◽  
Sudden Decrease ◽  
Physiologic Stimulus

The J2E cell line is a novel erythroid cell line that differentiates in response to erythropoietin (Epo), the physiologic stimulus for erythropoiesis. After exposure to Epo, the cells synthesize hemoglobin, and we show here that this process is tightly linked to increases in cellular proliferation and DNA synthesis. The hormone-induced terminal differentiation also results in morphologic alterations and the accumulation of transcripts for alpha, beta maj, and beta min globins. c-myc messenger RNA levels increase rapidly after exposure to Epo and precede the increase in cell division, while c-myb undergoes a transient decrease. Differentiation of J2E cells can also be achieved with sodium butyrate, but, in contrast with Epo, this is associated with a retardation of replication and a sudden decrease in c-myc levels. These results show that, in this system, chemically induced differentiation differs from terminal maturation promoted by Epo and that the processes of proliferation and differentiation in J2E cells can be uncoupled.

KLF1 Modulates Delta-Aminolevulinic Acid Dehydratase (ALAD) Gene Transcription in a Context-Specific Manner

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2094-2094
Author(s):  
Aurelie Desgardin ◽  
Tatiana Abramova ◽  
Jenny Lin ◽  
Eun-Hee Shim ◽  
John M Cunningham
Keyword(s):  
Cell Line ◽  
Gene Transcription ◽  
Heme Biosynthesis ◽  
Erythroid Cell ◽  
Specific Gene ◽  
Regulatory Sequences ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Molecular Events ◽  
In Erythroid Cells

Abstract Abstract 2094 Krüppel-like factor 1 (KLF1) is a zinc finger-encoding transcription factor that recognizes CACC elements, and is essential for maximal erythroid-specific gene transcription. Several critical mechanisms dependent on KLF1 and required for gene activation have been elucidated, predominantly using the beta-globin locus. KLF1 has been associated with the ordered recruitment of SWI/SNF and RNA polymerase-II complexes, necessary for chromatin remodeling and gene transcription respectively. KLF1 has also been reported to influence erythroid-specific heme biosynthesis. Studies in KLF1-null fetal erythroblasts and a KLF-1 deficient cell line have demonstrated that mRNA levels of the first three enzymes of the biosynthetic pathway are underrepresented. However, although in vitro studies of the rate-limiting enzymes ALAS2 and PBGD suggested a potential regulatory role for KLF1, in vivo studies failed to validate these findings. ALAD is the second enzyme of the pathway. Complete loss of ALAD expression in erythroid cells results in catastrophic events during zebrafish ontogeny. Interestingly, no human erythropoietic defect has been reported as a consequence of aberrant ALAD expression. To extend the analysis of KLF1's regulation of heme biosynthesis, we evaluated KLF1 binding of enzyme regulatory sequences by EMSA and ChIP studies, identifying a KLF1 binding CACC element in the erythroid-specific ALAD promoter. This regulatory element was transactivated specifically by a KLF1 transgene in KLF1-deficient cells. Using a unique 4-OH-Tamoxifen (4-OHT) mediated KLF1-inducible erythroid cell line (K1-ERp), we identified KLF1 as an essential, and early (within 2 hours of induction) activator of transcription of the endogenous ALAD, but not ALAS2 or PBGD genes. Further studies in K1-ERp cells, including DNAseI hypersensitivity and ChIP assays revealed that KLF1 occupancy at the erythroid-specific ALAD promoter triggers a series of molecular events including histone modifications, and enhanced recruitment of the sequence-specific transcription factors, GATA-1, NF-E2 and the TAL-1/SCL multiprotein complex. Importantly, we identified differences in the kinetics of recruitment of the closely related histone acetyltransferases proteins CBP and p300 and the SWI/SNF ATPase Brg1. The latter complex was recruited subsequent to KLF1 binding, although the ALAD promoter was already DNAseI hypersensitive. These results suggest strongly that KLF1 plays a major role in the regulation of heme biosynthesis in erythroid cells. Furthermore, our data challenges a model in which an identical temporal cascade of molecular events are required for transcription at KLF1-dependent promoters. Disclosures: No relevant conflicts of interest to declare.

PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2641-2648 ◽  
Author(s):  
Pu Zhang ◽  
Xiaobo Zhang ◽  
Atsushi Iwama ◽  
Channing Yu ◽  
Kent A. Smith ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Cell Line ◽  
Protein Interactions ◽  
Messenger Rna ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
N Terminus

Abstract The lineage-specific transcription factors GATA-1 and PU.1 can physically interact to inhibit each other's function, but the mechanism of repression of GATA-1 function by PU.1 has not been elucidated. Both the N terminus and the C terminus of PU.1 can physically interact with the C-terminal zinc finger of GATA-1. It is demonstrated that the PU.1 N terminus, but not the C terminus, is required for inhibiting GATA-1 function. Induced overexpression of PU.1 in K562 erythroleukemia cells blocks hemin-induced erythroid differentiation. In this system, PU.1 does not affect the expression of GATA-1 messenger RNA, protein, or nuclear localization. However, GATA-1 DNA binding decreases dramatically. By means of electrophoretic mobility shift assays with purified proteins, it is demonstrated that the N-terminal 70 amino acids of PU.1 can specifically block GATA-1 DNA binding. In addition, PU.1 had a similar effect in the G1ER cell line, in which the GATA-1 null erythroid cell line G1E has been transduced with a GATA-1–estrogen receptor fusion gene, which is directly dependent on induction of the GATA-1 fusion protein to effect erythroid maturation. Consistent with in vitro binding assays, overexpression of PU.1 blocked DNA binding of the GATA-1 fusion protein as well as GATA-1–mediated erythroid differentiation of these G1ER cells. These results demonstrate a novel mechanism by which function of a lineage-specific transcription factor is inhibited by another lineage-restricted factor through direct protein–protein interactions. These findings contribute to understanding how protein–protein interactions participate in hematopoietic differentiation and leukemogenesis.

scholarly journals Erythropoietin-induced stimulation of differentiation and proliferation in J2E cells is not mimicked by chemical induction

Blood ◽  
1992 ◽  
Vol 80 (2) ◽  
pp. 412-419 ◽  
Author(s):  
SJ Busfield ◽  
SP Klinken
Keyword(s):  
Cell Line ◽  
Messenger Rna ◽  
Cellular Proliferation ◽  
Erythroid Cell ◽  
Chemical Induction ◽  
Chemically Induced ◽  
Proliferation And Differentiation ◽  
Differentiation And Proliferation ◽  
Sudden Decrease ◽  
Physiologic Stimulus

Abstract The J2E cell line is a novel erythroid cell line that differentiates in response to erythropoietin (Epo), the physiologic stimulus for erythropoiesis. After exposure to Epo, the cells synthesize hemoglobin, and we show here that this process is tightly linked to increases in cellular proliferation and DNA synthesis. The hormone-induced terminal differentiation also results in morphologic alterations and the accumulation of transcripts for alpha, beta maj, and beta min globins. c-myc messenger RNA levels increase rapidly after exposure to Epo and precede the increase in cell division, while c-myb undergoes a transient decrease. Differentiation of J2E cells can also be achieved with sodium butyrate, but, in contrast with Epo, this is associated with a retardation of replication and a sudden decrease in c-myc levels. These results show that, in this system, chemically induced differentiation differs from terminal maturation promoted by Epo and that the processes of proliferation and differentiation in J2E cells can be uncoupled.

PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2641-2648 ◽  
Author(s):  
Pu Zhang ◽  
Xiaobo Zhang ◽  
Atsushi Iwama ◽  
Channing Yu ◽  
Kent A. Smith ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Cell Line ◽  
Protein Interactions ◽  
Messenger Rna ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
N Terminus

The lineage-specific transcription factors GATA-1 and PU.1 can physically interact to inhibit each other's function, but the mechanism of repression of GATA-1 function by PU.1 has not been elucidated. Both the N terminus and the C terminus of PU.1 can physically interact with the C-terminal zinc finger of GATA-1. It is demonstrated that the PU.1 N terminus, but not the C terminus, is required for inhibiting GATA-1 function. Induced overexpression of PU.1 in K562 erythroleukemia cells blocks hemin-induced erythroid differentiation. In this system, PU.1 does not affect the expression of GATA-1 messenger RNA, protein, or nuclear localization. However, GATA-1 DNA binding decreases dramatically. By means of electrophoretic mobility shift assays with purified proteins, it is demonstrated that the N-terminal 70 amino acids of PU.1 can specifically block GATA-1 DNA binding. In addition, PU.1 had a similar effect in the G1ER cell line, in which the GATA-1 null erythroid cell line G1E has been transduced with a GATA-1–estrogen receptor fusion gene, which is directly dependent on induction of the GATA-1 fusion protein to effect erythroid maturation. Consistent with in vitro binding assays, overexpression of PU.1 blocked DNA binding of the GATA-1 fusion protein as well as GATA-1–mediated erythroid differentiation of these G1ER cells. These results demonstrate a novel mechanism by which function of a lineage-specific transcription factor is inhibited by another lineage-restricted factor through direct protein–protein interactions. These findings contribute to understanding how protein–protein interactions participate in hematopoietic differentiation and leukemogenesis.

scholarly journals Changes in globin messenger RNA content during erythroid cell differentiation.

1975 ◽  
Vol 250 (15) ◽  
pp. 6054-6058
Author(s):  
F Ramirez ◽  
R Gambino ◽  
G M Maniatis ◽  
R A Rifkind ◽  
P A Marks ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Messenger Rna ◽  
Erythroid Cell ◽  
Rna Content ◽  
Erythroid Cell Differentiation

scholarly journals Tracking changes in adaptation to suspension growth for MDCK cells: cell growth correlates with levels of metabolites, enzymes and proteins

2021 ◽  
Vol 105 (5) ◽  
pp. 1861-1874
Author(s):  
Sabine Pech ◽  
Markus Rehberg ◽  
Robert Janke ◽  
Dirk Benndorf ◽  
Yvonne Genzel ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Line ◽  
Mdck Cells ◽  
Suspension Cell ◽  
Regulatory Mechanisms ◽  
Adherent Cell ◽  
Holistic View ◽  
Cellular Processes ◽  
Viral Vaccine ◽  
Analytical Approaches

Abstract Adaptations of animal cells to growth in suspension culture concern in particular viral vaccine production, where very specific aspects of virus-host cell interaction need to be taken into account to achieve high cell specific yields and overall process productivity. So far, the complexity of alterations on the metabolism, enzyme, and proteome level required for adaptation is only poorly understood. In this study, for the first time, we combined several complex analytical approaches with the aim to track cellular changes on different levels and to unravel interconnections and correlations. Therefore, a Madin-Darby canine kidney (MDCK) suspension cell line, adapted earlier to growth in suspension, was cultivated in a 1-L bioreactor. Cell concentrations and cell volumes, extracellular metabolite concentrations, and intracellular enzyme activities were determined. The experimental data set was used as the input for a segregated growth model that was already applied to describe the growth dynamics of the parental adherent cell line. In addition, the cellular proteome was analyzed by liquid chromatography coupled to tandem mass spectrometry using a label-free protein quantification method to unravel altered cellular processes for the suspension and the adherent cell line. Four regulatory mechanisms were identified as a response of the adaptation of adherent MDCK cells to growth in suspension. These regulatory mechanisms were linked to the proteins caveolin, cadherin-1, and pirin. Combining cell, metabolite, enzyme, and protein measurements with mathematical modeling generated a more holistic view on cellular processes involved in the adaptation of an adherent cell line to suspension growth. Key points • Less and more efficient glucose utilization for suspension cell growth • Concerted alteration of metabolic enzyme activity and protein expression • Protein candidates to interfere glycolytic activity in MDCK cells

scholarly journals Expression of ferrochelatase mRNA in erythroid and non-erythroid cells

1993 ◽  
Vol 292 (2) ◽  
pp. 343-349 ◽  
Author(s):  
R Y Y Chan ◽  
H M Schulman ◽  
P Ponka
Keyword(s):  
Sequence Similarity ◽  
Erythroid Differentiation ◽  
Polyadenylation Signal ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Erythroleukemia Cells ◽  
Haem Biosynthesis ◽  
Polyadenylation Sites ◽  
Dna Fragment ◽  
Polyadenylation Signals

Ferrochelatase, which catalyses the last step in haem biosynthesis, i.e. the insertion of Fe(II) into protophorphyrin IX, is present in all cells, but is particularly abundant in erythroid cells during haemoglobinization. Using mouse ferrochelatase cDNA as a probe two ferrochelatase transcripts, having lengths of 2.9 kb and 2.2 kb, were found in extracts of mouse liver, kidney, brain, muscle and spleen, the 2.9 kb transcript being more abundant in the non-erythroid tissues and the 2.2 kb transcript more predominant in spleen. In mouse erythroleukemia cells the 2.9 kb ferrochelatase transcript is also more abundant; however, following induction of erythroid differentiation by dimethyl sulphoxide there is a preferential increase in the 2.2 kb transcript, which eventually predominates. With mouse reticulocytes, the purest immature erythroid cell population available, over 90% of the total ferrochelatase mRNA is present as the 2.2 kb transcript. Since there is probably only one mouse ferrochelatase gene, the occurrence of two ferrochelatase transcripts could arise from the use of two putative polyadenylation signals in the 3′ region of ferrochelatase DNA. This possibility was explored by using a 389 bp DNA fragment produced by PCR with synthetic oligoprimers having sequence similarity with a region between the polyadenylation sites. This fragment hybridized only to the 2.9 kb ferrochelatase transcript, indicating that the two transcripts differ at their 3′ ends and suggesting that the 2.2 kb transcript results from the utilization of the upstream polyadenylation signal. The preferential utilization of the upstream polyadenylation signal may be an erythroid-specific characteristic of ferrochelatase gene expression.

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