Differentiating Mouse Erythroleukemia Cells Research Articles

Abcb10 physically interacts with mitoferrin-1 (Slc25a37) to enhance its stability and function in the erythroid mitochondria

Mitoferrin-1 (Mfrn1; Slc25a37), a member of the solute carrier family localized in the mitochondrial inner membrane, functions as an essential iron importer for the synthesis of mitochondrial heme and iron-sulfur clusters in erythroblasts. The biochemistry of Mfrn1-mediated iron transport into the mitochondria, however, is poorly understood. Here, we used the strategy of in vivo epitope-tagging affinity purification and mass spectrometry to investigate Mfrn1-mediated mitochondrial iron homeostasis. Abcb10, a mitochondrial inner membrane ATP-binding cassette transporter highly induced during erythroid maturation in hematopoietic tissues, was found as one key protein that physically interacts with Mfrn1 during mouse erythroleukemia (MEL) cell differentiation. Mfrn1 was shown previously to have a longer protein half-life in differentiated MEL cells compared with undifferentiated cells. In this study, Abcb10 was found to enhance the stabilization of Mfrn1 protein in MEL cells and transfected heterologous COS7 cells. In undifferentiated MEL cells, cotransfected Abcb10 specifically interacts with Mfrn1 to enhance its protein stability and promote Mfrn1-dependent mitochondrial iron importation. The structural stabilization of the Mfrn1-Abcb10 complex demonstrates a previously uncharacterized function for Abcb10 in mitochondria. Furthermore, the binding domain of Mfrn1-Abcb10 interaction maps to the N terminus of Mfrn1. These results suggest the tight regulation of mitochondrial iron acquisition and heme synthesis in erythroblasts is mediated by both transcriptional and posttranslational mechanisms, whereby the high level of Mfrn1 is stabilized by oligomeric protein complexes.

Abcb10 Physically Interacts with Mitoferrin1 to Enhance Its Stability for Heme Synthesis in the Erythroid Mitochondria

We previously described a zebrafish mutant, frascati (frs), with hypochromic anemia and erythroid maturation arrest. Using positional cloning, we identified mitoferrin1 (mfrn1, slc25a37) as the gene disrupted in frs mutants (Shaw GC, et al. 2006 Nature 440:96–100). Mfrn1, the principle iron-importer in the mitochondria, is essential for heme and iron-sulphur (Fe/S) cluster syntheses in erythroblasts, and is required for primitive and definitive erythropoiesis. The biochemistry for Mfrn1-mediated iron acquisition into the mitochondria of developing erythroblasts, however, is poorly understood. In order to identify Mfrn1-associated proteins involved in mitochondrial iron homeostasis, we employed the strategy of in vivo epitope-tagged affinity purification and mass spectrometry (MS). A “bait protein,” Biotag-mouse Mfrn1 (BT-Mfrn1), was engineered to affinity-purify the associated proteins. A series of control experiments were first conducted to demonstrate that the BT-Mfrn1 protein properly targeted to the mitochondria and functionally complemented the anemia in frs embryos. A two-dimensional Blue Native gel followed by SDS-PAGE and western blot showed Mfrn1 forms higher-order protein complexes with interacting proteins in the mitochondrial fraction. A stable mouse erythroleukemia (MEL) clone expressing BT-Mfrn1 protein was derived as a source for protein purification. Affinity-purified BT-Mfrn1 protein complexes were analyzed by MS in triplicate runs in comparison to control MEL cells. Abcb10, a GATA-1 induced ATP-binding cassette transporter highly expressed in hematopoietic tissues and involved in heme biosynthesis (Shirihai OS, et al. 2000 EMBO J. 19:2492–2502), was found as one of the proteins which physically associated with Mfrn1 during MEL differentiation. The Abcb10:Mfrn1 interaction was confirmed by immunoprecipitation (IP) and western analysis with endogenously expressed proteins in MEL cells and proteins expressed by transient transfection in heterologous cells. Mfrn1 was previously shown to have longer protein half-life in differentiated MEL cells compared to undifferentiated cells (Paradkar PN, et al. submitted). To test our hypothesis that the Abcb10:Mfrn1 interaction may enhance Mfrn1 protein stability, a pulse-chase assay using 35S-methionine labeled cells was performed, followed by IP. Abcb10 was found to stabilize Mfrn1 protein in COS7 cells co-transfected with Abcb10 and Mfrn1 compared to control cells transfected with Mfrn1 alone. Similar results were obtained when total steady-state Mfrn1 protein was evaluated by western analysis comparing lysates from Abcb10:Mfrn1 cotransfected and Mfrn1 transfected cells. Our data suggest that Abcb10 physically interacts with Mfrn1 to enhance its stability and promote Mfrn1-dependent mitochondrial heme biogenesis.

467. Lentiviral Vector (LV) Non-Coding Elements That Impart High Titers and Expression to β-Globin (hβ) Cassettes

Top of pageAbstract Thalassemias are the most common monogenic defects that result from absent/reduced h|[beta]| in RBC. However, the complexities of the h|[beta]| gene and its regulatory elements have been significant obstacles for optimization of gene therapy approaches using |[gamma]|-oncoretroviral vectors. Recently, LV have been shown to stably transfer and express h|[beta]| at high levels. The goal of our study was to identify non-coding elements in LV required for this effect. A series of LV carrying different elements from the gag and env fragments were made. All vectors encoded h|[beta]| gene driven by |[beta]|-regulatory elements (|[beta]|-promoter, HS234 and |[beta]| 3' enhancer), contained the same 5' and 3' SIN-LTR: sBG-0 was a gutted LV vector containing only the packaging (|[Psi]| region including 40bp of the 5' gag).In sBG-1 the central polypurine tract (cPPT) element was added to sBG-0.In sBG-2, the rev response element (RRE) was added to sBG-1. In sBG-3 and sBG-4, the wt or the mutated splice acceptor (SA) in the env sequence was added to sBG-2, respectively. In sBG-5, |[sim]|600bp of gag and RRE was added to sBG-1. In sBG-6, the |[sim]|400bp of gag was added to sBG-3. All vectors were packaged using the standard transient co-transfection system in 293T cells, concentrated 1400-fold and assayed in differentiated mouse erythroleukemia (MEL) cells. The level of h|[beta]| expression in MEL cells was analyzed by RNase protection assay (RPA) and FACS analysis with 3 independent viral lots. sBG-6 had the highest titers (6|[times]|10E8 TU/mL) while the gutted LV vector sBG-0 and sBG-1 had very poor titers (Fig. 1A). Addition of RRE (sBG-2) increased the titers very significantly. Addition of the env fragment (sBG-3) further increased titers nearly 2-3 fold. However, when the SA in the env gene was mutated (sBG-4), the titers of sBG-4 vectors dropped to levels lower than sBG-2, despite sBG-3 and sBG-4 containing RRE, suggesting that some contribution of titers by the env fragment in sBG-3 reside in the SA, and its mutation has a negative effect on RRE. Replacement of a |[sim]|600bp of gag in place of the env (+SA) in sBG-5 was sufficient to improve titers to 84% of sBG-6. These results were confirmed at the RNA level (RPA in Fig. 1B). When probed with an LCR fragment, Northern blot analysis showed stable transmission of full-length LV mRNA in 293T cells, with varying ratio of spliced and unspliced LTR transcripts in different vectors. The mean fluorescence intensity of h|[beta]| expression in differentiated MEL cells was similar with all vectors, suggesting that upon integration, viral cis elements probably play a minimal role in improving expression of the h|[beta]| transcripts, although this is under investigation; as is RNA export and stable transmission from these vectors. In conclusion, while the rev/RRE in HIV is likely the most important element imparting high titers to h|[beta]| cassettes, the env SA and regions of gag appear to contribute to the titers from SIN-LV.

Leukemia-related transcription factor TEL accelerates differentiation of Friend erythroleukemia cells.

TEL belongs to a member of the ETS family transcription factors that represses transcription of target genes such as FLI-1. Although TEL is essential for establishing hematopoiesis in neonatal bone marrow, its role in erythroid lineage is not understood. To investigate a role for TEL in erythroid differentiation, we introduced TEL into mouse erythroleukemia (MEL) cells. Overexpressing wild-type-TEL in MEL cells enhanced differentiation induced by hexamethylene bisacetamide or dimethylsulfoxide, as judged by the increased levels of erythroid-specific delta-aminolevulinate synthase and beta-globin mRNAs. TEL bound to a corepressor mSin3A through the helix-loop-helix domain. A TEL mutant lacking this domain still bound to the ETS binding site, but lost its transrepressional effect. This mutant completely blocked erythroid differentiation in MEL cells. Moreover, it showed dominant-negative effects over TEL-mediated transcriptional repression and acceleration of erythroid differentiation. Endogenous TEL mRNA was found to increase during the first 3 days in differentiating MEL cells and drastically decrease thereafter. All these data suggest that TEL might play some role in erythroid cell differentiation.

Erythroid 5-Aminolevulinate Synthase Is Required for Erythroid Differentiation in Mouse Embryonic Stem Cells

ABSTRACT: We have examined the induction of the enzymes of the heme biosynthetic pathway during erythroid differentiation of mouse embryonic stem (ES) cells. Following transfer to appropriate medium all of the pathway enzymes are induced within three days. Unlike differentiating mouse erythroleukemia cells (Lake-Bullock, H. and Dailey, H.A.Mol Cell Biol13:7122-7132, 1993), all of the enzymes appear to be induced simultaneously and not sequentially in differentiating ES cells. The role of erythroid 5-aminolevulinate synthase (ALAS-2) in this differentiation process was examined by disruption of the ALAS-2 gene. The targeting vector used for disruption replaced all of exons 4 to 6 with a selectable neomycin resistance gene. The resulting genetically modified (ALAS-2 knockout) cells, as well as normal ES cells were used to study induction of heme biosynthesis. Following 10 days of culture in methylcellulose media significant morphological differences between the embryoid bodies (EBs) of the two cell lines were observed. ES cells exhibited morphology of typical EBs with a dark field (blood island) in the center, while ALAS-2 knockout ES cells developed very poorly both in size and shape. At 8 days of differentiation, only 3% of all EBs contained visible erythropoietic cells (i.e., stained positively for hemoglobin) in the ALAS-2 knockout cell line, compared with 50% in ES cells. Most of the genes in the heme synthetic pathway were expressed to a stable level within 3 to 6 days after induction in normal ES cells, while the ALAS-2 knockout cell line failed to significantly increase the level of expression of these genes. Fetal β-globin mRNA was not detectable in the differentiating ALAS-2 knockout cells, whereas mRNA for this gene was detected in normal ES cells within 3 days of differentiation. These results suggest that ALAS-2 is necessary for ES cell erythroid differentiation and that there is an interrelationship between heme and globin synthesis in differentiating ES cells.

Characterization of a protein tyrosine phosphatase (RIP) expressed at a very early stage of differentiation in both mouse erythroleukemia and embryonal carcinoma cells

From our previous studies, several protein tyrosine phosphatases (PTPase) are implicated in the early events leading to in vitro differentiation of both mouse erythroleukemia (MEL) and embryonal carcinoma (F9) cells. Among the PTPases, recent experiments suggest that a new PTPase (RIP) plays a critical role in differentiation processes, particularly at their early stages. We isolated cDNA clones for RIP from a RNA preparation isolated from differentiating MEL cells, and determined the total 7932 bp base sequence for RIP cDNA. The cDNA codes for a putative 269.8 kDa (2450 amino acids) protein with a PTPase catalytic domain. We have demonstrated that the transcripts exist in multiple forms, and among mouse tissues they were found predominantly in kidney and, to a lesser extent, in lung, heart, brain and testis. The RIP gene was mapped between D5Mit90 and D5Mit25 on mouse chromosome 5.

Purification and characterization of a nuclear DNA-binding factor complex containing topoisomerase II and chromosome scaffold protein 2.

In a search for factors that influence the process of erythroid differentiation at the molecular level, we have identified UB2, a nuclear protein factor that was originally observed for its ability to bind to a very specific and highly conserved sequence motif present in human, mouse, rabbit, and chicken beta-globin genes, as well as carbonic anhydrase I, c-myb, and the immunoglobulin heavy chain enhancer region. It was also observed for its appearance in undifferentiated but not differentiated mouse erythroleukemia cells. Purification of UB2 by DEAE-cellulose chromatography and repeated passages through a DNA affinity column, revealed a complex pattern with three major components of 170, 116, and 48 kDa, respectively. The 170-kDa protein was identified as topoisomerase (topo) II by Western blot analysis, catalytic assays, and antibody interference with UB2 binding. The complex topo II in UB2, however, has a more stringent sequence requirement for DNA binding than does topo II. The 116-kDa protein has been determined to be a proteolytic product of topo II. The chromosome scaffold protein 2 (135 kDa) copurified with UB2, and anti-scaffold protein 2 serum inhibited UB2 binding to DNA.

Increased histone H1 ° expression in differentiating mouse erythroleukemia cells is related to decreased cell proliferation

The amount of histone H1 ° increases relative to other H1 subtypes in terminally differentiated cells, and its expression has been associated with the onset of differentiation. We have studied the kinetics of H1 ° accumulation in mouse erythroleukemia (MEL) cells and found that the levels of H1 ° reflect the rate of cell proliferation rather than the state of differentiation. This suggests that changes in the relative amount of H1 ° during MEL cell differentiation are primarily a consequence of cell cycle arrest.

In vitro characterization of tissue-specific nuclear proteins preferentially bound to the mouse beta-globin gene during MEL cell terminal differentiation.

Using DNA restriction fragments of the mouse beta-globin gene and other promoter-containing DNA fragments (LTR-MMTV and pBR322) as controls, we have characterized by protein blotting, in extracts of mouse erythroleukemia (MEL) cells, specific nuclear DNA binding proteins with a preferential affinity for the beta-globin DNA. Some proteins (110 and 75 kDa) appear in differentiated MEL cells while others (100, 95, and 35 kDa) are present in immature MEL and normal erythroblast cells and bind selectively to the far-upstream region of the gene. These proteins could modulate either positively or negatively the expression of the beta-globin gene and maybe, of other genes, during the terminal differentiation of erythroid cells.

Expression of the c-myb and c-myc genes is regulated independently in differentiating mouse erythroleukemia cells by common processes of premature transcription arrest and increased mRNA turnover.

The mechanisms that modulate c-myb mRNA levels in mouse erythroleukemia cells induced toward erythroid differentiation were compared with those that act on c-myc. Both genes exhibited regulation at the levels of premature transcription arrest and RNA turnover. However, these common processes allowed temporally distinct control of gene expression.

Expression of the c-myb and c-myc genes is regulated independently in differentiating mouse erythroleukemia cells by common processes of premature transcription arrest and increased mRNA turnover.

The mechanisms that modulate c-myb mRNA levels in mouse erythroleukemia cells induced toward erythroid differentiation were compared with those that act on c-myc. Both genes exhibited regulation at the levels of premature transcription arrest and RNA turnover. However, these common processes allowed temporally distinct control of gene expression.

Synthesis of globin RNA in enucleated differentiating murine erythroleukemia cells.

In an earlier report (Volloch, V., 1986, Proc. Natl. Acad. Sci. USA., 83:1208-1212) we had presented evidence for the occurrence of the cytoplasmic synthesis of globin mRNA and of RNA complementary to globin mRNA which differed from DNA-dependent transcription by its insensitivity to actinomycin D. In this paper, we describe the use of enucleated differentiating mouse erythroleukemia cells to demonstrate directly the occurrence of cytoplasmic synthesis of both positive- and negative-strand globin RNA. For this purpose, we developed an enucleation procedure which yielded pure cytoplasts from differentiated mouse erythroleukemia cells in the absence of cytochalasin B and selectively permeabilized the cytoplasts to small molecules by treatment with dextran sulfate and saponin. The permeabilized cytoplasts incorporated [3H]dUTP into positive- and negative-strand globin RNA and experiments with mercurated nucleotide substrate suggested that this process involved de novo RNA synthesis rather than limited terminal nucleotide addition. Globin RNA synthesis required Mg++, was inhibited by Mn++, and was unaffected by the addition of Zn++. Studies of its response to inhibitors of DNA-dependent RNA synthesis showed that it differed from that process in its insensitivity to actinomycin D and alpha-amanitin, but that like many other macromolecular biosynthetic reactions it was inhibited by rifamycin AF/ABDP and aurintricarboxylic acid. These observations provide additional evidence for the occurrence of cytoplasmic RNA-dependent RNA synthesis in differentiated cells and show permeabilized enucleated cells to be a useful experimental system for further studies of the characteristics of that process.

Effects of hematoporphyrin-derivative on mouse erythroleukemia cells in the absence of light irradiation

This paper concerns a general study on the effects of hematoporphyrin-derivative (HpD) on mouse erythroleukemia (MEL) cells, in the absence of light irradiation. In particular, HpD intrinsic cytotoxicity was evaluated at different doses and the results correlated with those referring to membrane functional and morphological changes. HpD uptake and release processes were also studied and compared with the above-mentioned results. In order to have an overall picture of HpD-cell interactions, time-resolved fluorescence measurements were performed on both undifferentiated and differentiated MEL cells. The results obtained indicate that, even at HpD doses exhibiting neither any cytotoxicity nor any morphological damage (1–10 μg/ml), membrane permeability alterations are observed. Thirty minutes of treatment are sufficient for HpD to develop its toxic effect: indeed, no differences in HpD influence on cell viability can be observed after 30 min, 60 min or 5 days of treatment. HpD cytotoxicity is reduced by high protein content in the incubation culture medium. The presence of both monomeric species and 580 nm emitting species was observed at cellular level. The latter is likelier in undifferentiated MEL cells, which also exhibit higher overall HpD uptake, as compared with differentiated MEL cells.