Mouse Erythroleukemia Cell Line Research Articles

Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3-mediated regulation of actin filament assembly into the erythroblast membrane skeleton.

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament (F-actin) assembly and organization during terminal ED have revealed essential roles for the F-actin pointed-end capping proteins, tropomodulins (Tmod1 and Tmod3). Tmods bind tropomyosins (Tpms), which enhance Tmod capping and F-actin stabilization. Tmods can also nucleate F-actin assembly, independent of Tpms. Tmod1 is present in the red blood cell (RBC) membrane skeleton, and deletion of Tmod1 in mice leads to a mild compensated anemia due to mis-regulated F-actin lengths and membrane instability. Tmod3 is not present in RBCs, and global deletion of Tmod3leads to embryonic lethality in mice with impaired ED. To further decipher Tmod3's function during ED, we generated a Tmod3knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability, and increased apoptosis. Tmod3 does not assemble with Tmod1 and Tpms into the Triton X-100 insoluble membrane skeleton during ED, and loss of Tmod3 had no effect on α1,β1-spectrin and protein 4.1R assembly into the membrane skeleton. However, F-actin, Tmod1 and Tpms failed to assemble into the membrane skeleton during ED in absence of Tmod3. We propose that Tmod3 nucleation of F-actin assembly promotes incorporation of Tmod1 and Tpms into membrane skeleton F-actin, and that this is integral to morphological maturation and cell survival during erythroid terminal differentiation.

Deep Mining of Natural Genetic Variation in Erythroid Cells Reveals New Insights about In Vivo Transcription Factor Binding and Chromatin Accessibility

Erythroid transcription factors (TFs) control gene expression programs, lineage decisions, and disease outcomes. How transcription factors contact DNA has been studied extensively in vitro, but in vivo binding characteristics are less well understood as they are influenced in a reciprocal manner by chromatin accessibility and neighboring transcription factors. Here, we present a comparative analysis approach that takes advantage of non-coding sequence variation between functionally equivalent erythroid cell lines to conduct an in-depth analysis of erythroid TF binding profiles and chromatin features.Specifically, we analyzed ChIP-seq datasets to identify millions of genetic non-coding variants between the mouse erythroleukemia cell line (MEL), a GATA1-inducible erythroid progenitor cell line (G1E-ER4), and primary murine erythroblast cells. We found that while these cell lines are highly positively correlated in chromatin features, larger differences in TF binding intensity are correlated with higher degrees of genetic variation between cell lines.We next examined discriminatory genetic variants between the cell lines that are located in ChIP-seq peaks of the erythroid transcription factor GATA1. Hundreds of such variants fall within GATA1 motifs. Differential GATA1 binding intensities associated with the variants revealed nucleotide positions that contribute most to in vivo GATA1 chromatin occupancy and identified which alternative nucleotides are most likely to disrupt binding. Notably, this additional information about GATA1's in vivo nucleotide binding preferences improved prediction of GATA1 binding sites genome-wide. We applied similar approaches to determine the bp-resolution in vivo binding preferences of TAL1/SCL and CTCF.We additionally identified thousands of discriminatory genetic variants within GATA1 sites that fall outside canonical GATA elements but within binding sites of other known TFs. Association of these variants with differential GATA1 binding intensities revealed that the hematopoietic transcription factors TAL1/SCL and KLF1 positively regulate GATA1 chromatin occupancy. Strikingly, we identified a number of motifs not previously implicated in cooperating with GATA1 that positively impact GATA1 chromatin binding. Notably, we also defined motifs associated with negative regulation of GATA1 chromatin occupancy. Applying a similar analysis to TAL1/SCL and CTCF revealed additional motifs involved in regulating the chromatin occupancy of these TFs.Finally, we associated discriminatory genetic variation between erythroid cell lines with large changes in sub-kb-scale DNase hypersensitivity. We found that single base pair substitutions within or near a number of erythroid TF motifs, including that for the RUNX family of nuclear factors, are strongly associated with changes in chromatin accessibility. Our findings use novel methods in comparative ChIP-seq and DNase-seq analysis to reveal new insights about the genetic basis for erythroid TF chromatin occupancy and chromatin accessibility. DisclosuresNo relevant conflicts of interest to declare.

Genome-wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators

SummaryHematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.

Establishment of mouse erythroleukemia cell lines expressing complete Influenza C virus CM2 protein or chimeric protein consisting of CM2 and Influenza A virus M

The role of the Influenza C virus (ICV) CM2 protein in virus replication as well as its precise function as an ion channel remains to be elucidated. For this purpose, we established a CM2-expressing mouse erythroleukemia (MEL) cell line and determined the biochemical characteristics of the expressed CM2. The features of the expressed CM2 were similar to those of the viral CM2 synthesized in ICV-infected cells. Furthermore, we established MEL cell line expressing a chimeric protein consisting of characteristic regions of CM2 and Influenza A virus (IAV) M2 protein that could be helpful in elucidation of the specific ion conductance properties.

Identification of a Novel Acetyl-CoA Binding Protein (EST1) Which Epigenetically Regulates the Expression of Erythroid-Specific Genes

(Introduction) Embryonic stem (ES) cells that lack 5-aminolevulinate synthase-2 (ALAS2) gene provide a valuable model in dissecting molecular events in which heme is required during erythroid differentiation. Recently, we identified a novel acetyltransferase-like gene (EST1) through differential expression analysis between wild-type and heme-deficient erythroblasts as a potential downstream target gene of heme (BBRC 2006; 340:105–110, ASH 2007). EST1 belongs to the GNAT (GCN5-related N-acetyltransferase) superfamily since it contains the highly conserved amino acid residues (motif A), known as acetyl-CoA binding domain. Since the GNAT superfamily contains a wide variety of acetyltransferases with different substrates or with unknown functions, probing how EST1 might be required for biological events in erythroid cells, could be informative. Here, we investigated the role of EST1 during erythroid differentiation.(Methods) EST1 was constitutively expressed using Flag/HA-tagged retroviral vector into mouse erythroleukemia (MEL) cell line. EST1 protein complex was purified by affinity chromatography from nuclear extract of EST1-expessed MEL cells. To obtain dominant-negative EST1-expressing cells, both Arg-62 and Gly-65 within EST1 were substituted to glutamic acid, and similarly transduced into MEL cells. These mutations have been widely applied for abolishment of acetyl-CoA binding activity. For depletion of endogenous EST1, siRNAs specific for EST1 were introduced into Hepa1c1c7 cells(Results) Although recombinant EST1 protein did not have acetylase activity for free histones in vitro (ASH 2007), EST1 protein forms a multimeric protein complex. Western blot analysis using FLAG-eluted polypeptides revealed the presence of GCN5, TRRAP, SPT3 and GATA-1, implying that this protein complex might participate in the transcriptional regulation of erythroid-specific genes. Following EST1 depletion in Hepa1c1c cells, a significant decrease in the acetylation of H3 and a mild decrease in that of H4, were observed by Western blot analyses. Similarly, a significant decrease in the acetylation of H3 and a mild decrease in that of H4 were also observed in dominant-negative than in wild-type EST1-expressing MEL cells. Furthermore, the level of bmajor and ALAS2 mRNA were significantly lower in dominant-negative than in wild-type EST1-expressing MEL cells upon treatment with 1.5% DMSO for both 48h and 72h. We are currently exploring mechanisms of how EST1 participates in the regulation of histone modification in erythroid cells.(Conclusion) EST1 may epigenetically regulate a subset of erythroid-specific genes under control of heme. Further elucidation of the function of the EST1 gene would enhance our understanding of the transcriptional network involving erythroid differentiation.

Analyses of Heme-Regulated Genes during Erythoid Differentiation.

(Introduction) During erythroid differentiation, the level of erythroid-specific genes increases synchronizing with the intracellular heme content. In addition, heme has been shown to play a role in transcription and protein synthesis. Based on these evidences, it is possible that heme widely regulates the expression of erythroid specific genes. With this hypothesis, we compared the gene expression profile between wild-type and heme-deficient erythroblasts generated from wild-type and ALAS2 (−) ES cells in in vitro, and identified 4 heme-regulated erythroid-specific genes (UCP2, CNBP, NuSAP and unknown EST1), (BBRC 2006; 340:105–110). Among them, unknown EST1 is consisted of 110 a.a. with a conserved acetyl-CoA binding domain, which was characteristic of GNAT (GCN5-related N-acetyltransferase) superfamily. Thus, it is likely that EST1 is a novel acetyltransferase. In the present study, we focused two genes, EST1 and NuSAP, and investigated their function during erythoid differentiation.(Methods) First, the expression and regulation of NuSAP gene during erythroid differentiation was examined. For the expression analysis, in vivo erythroblasts were fractionated according to the surface expression of TER119/CD71, and the level of expression of NuSAP mRNA was examined by quantitative RT-PCR. For the promoter analysis, the promoter region of mouse NuSAP gene was cloned, and the regulatory cis-element was determined by luciferase assay and EMSA. Next, for defining the properties of EST1, EST1 was constitutively expressed using Flag/HA tagged retroviral vector into mouse erythroleukemia (MEL) cell line, and nuclear extract of EST1-expessed MEL cells was purified by affinity chromatography, which was loaded on an SDS/PAGE gel and subjected to electrophoresis. In addition, for in vitro histone acetyltransferase (HAT) assay, free histone and purified EST1 protein were incubated with [3H]acetyl-CoA, and acetyltransferase activity was measured by scintigraphy.(Results) (1) NuSAP mRNA was more significantly abundant in the subset corresponding to immature erythroblasts (TER119+CD71high) than mature erythroblasts (TER119+CD71low), and it was significantly increased in TER119+ cells from in vivo phlebotomized mice compared with control mice. Furthermore, during erythroid maturation of MEL cells by dimethylsulfoxide, NuSAP mRNA was increased at 24–72 hrs. Promoter analyses of NuSAP gene demonstrated that duplicated CCAAT boxes located at −81/−85 and −30/−34 were essential for promoter activity, which was trans-activated by NF-YA. These results suggested that NuSAP might contribute to the expansion of immature erythroblast pool under the control of NF-Y. (2) By the gel electrophoresis, it was revealed that EST1 protein forms a multimeric protein complex. Furthermore, whereas recombinant EST1 protein did not show HAT activity, EST1 complex could acetylate free histones in vitro, suggesting that EST1 might be a component of HAT complex.(Conclusion) The novel functions of EST1 and NuSAP suggest that heme regulates erythroid differentiation by controlling the expression of variety of genes.

Effects of TFAR19 gene on the in vivo biorheological properties and pathogenicity of mouse erythroleukemia cell line MEL

After injecting VP16, MEL cells and MEL-TF19 cells into the body of mice, with those injected with the same dose of saline as the control group, we observed the mice for their blood pictures, histological changes of the liver and spleen, and the hemorheological indexes within 4 weeks. The results indicated that after injecting MEL cells, the mice entered into a pathological status similar to erythroleukemia, which had the following exhibitions: the tissue structures of the liver and spleen were damaged, a mass of proerythroblasts, basophil erythroblasts and polychromatophilic erythroblasts could be observed on the smears of the bone marrow and spleen, and the deformability and orientation ability of erythrocytes were both depressed. The pathogenicity of MEL-TF19 cells carrying TFAR19 gene was obviously lower than that of MEL cells, and the MEL-TF19 cells even lost their faintish pathogenicity under the apoptosis-inducing effect of the chemotherapeutic reagent. The outcome from the animal experiments suggests that the TFAR19 gene suppresses the pathogenicity of MEL cells to the mice, and the effect may be better exerted with the synergy of the chemotherapeutic reagent.

Hemoglobin Is Expressed by Alveolar Epithelial Cells

Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the alpha- and beta-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both alpha- and beta-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung.

Ckap2 Regulates Aneuploidy, Cell Cycling, and Cell Death in a p53-Dependent Manner

We used DNA microarray screening to identify Ckap2 (cytoskeleton associated protein 2) as a novel p53 target gene in a mouse erythroleukemia cell line. DNA damage induces human and mouse CKAP2 expression in a p53-dependent manner and p53 activates the Ckap2 promoter. Overexpressed Ckap2 colocalizes with and stabilizes microtubules. In p53-null cells, overexpression of Ckap2 induces tetraploidy with aberrant centrosome numbers, suggesting disturbed mitosis and cytokinesis. In p53-competent cells, Ckap2 does not induce tetraploidy but activates p53-mediated cell cycle arrest and apoptosis. Our data suggest the existence of a functional positive feedback loop in which Ckap2 activates the G1 tetraploidy checkpoint and prevents aneuploidy.

The Human Xist Gene Promoter Prevents Silencing of an Integrated Reporter Gene.

Epigenetic silencing and position dependent expression are long-standing problems which continue to limit the development of gene replacement therapy. As a strategy to overcome this problem we have tested the ability of the human XIST (X inactivation-specific transcript) gene promoter to overcome epigenetic silencing. The XIST gene is one of a relatively small number of genes which are expressed from the inactive X chromosome. The product of this gene is an untranslated structural RNA which coats the X chromosome destined for inactivation prior to H3 Lys 9 hypoacetylation, H3 Lys 9 methylation, CpG island methylation and the subsequent silencing of most of the genes on the chromosome. Continued expression of the XIST gene in this highly repressive environment is required to maintain the chromosome in an inactive state. The region of the proximal promoter of the XIST gene on the inactive X chromosome has been shown to retain an active chromatin structure. Based on these findings we hypothesized that the XIST gene promoter would be able to resist the epigenetic changes which lead to transgene silencing. To test this idea, we subcloned a minimal XIST promoter upstream of an enhanced GFP reporter gene in a pUC-based plasmid which also contained a neomycin resistance gene. The same plasmid, with a CMV promoter, served as a control vector. The plasmids were electroporated into mouse erythroleukemia (MEL) cells and then grown in media containing G418. The MEL cell line was chosen because genes transferred into these cells are frequently silenced and because it is often used as a first screen for vectors with potential for use in therapeutic gene transfer to erythroid cells. Individual colonies were selected and G418 removed. After expansion of the clones, flow cytometry was used to determine the percentage of cells in each clonal population which were expressing GFP as determined by comparison to the untransduced MEL cell line. Silencing typically involves a gradual decrease in the proportion of cells expressing the integrated transgene. Statistical analyses of results were performed using the t-test. 16 XIST and 13 CMV clones were available for analysis at the start of the experiment (time 0). 11/13 CMV clones and 12/16 XIST clones initially expressed GFP. Of the clones which were expressing GFP, the average percentages of positive cells were higher for those with the XIST promoter (63% vs. 41%, p= 0.015). Expression was reanalyzed after 6 weeks of culture in the absence of G418 selection. At this time point, the average percentage of GFP expressing cells was much higher for the XIST clones (57% vs. 19%, p=0.00008) and when analyzed for silencing, XIST clones were expressing at an average of 90% of their time 0 levels vs. 46% for the CMV clones (p=.0008). These results indicate that the XIST promoter is resistant to silencing in our model system and is a candidate for further development and mechanistic studies.

Disruption of the Mechanism of Long Range Activation within the Human α-Globin Complex

The human alpha-globin complex lies at the tip of the short arm of chromosome 16. It comprises three functional globin genes (5'-zeta2-alpha2-alpha1-3'), the expression of which is strictly dependent on a positive regulatory element located 40-kb upstream, HS-40. This DNase I-hypersensitive site is the only known regulatory element displaying strong erythroid-specific enhancer activity within the human alpha-globin complex. How this enhancer activity is shared among different erythroid genes present in the same cluster without affecting the ubiquitous genes present within and around the complex is poorly understood. To address this issue, we used hybrid murine erythroleukemia cells containing a single copy of human chromosome 16 and targeted the insertion of different sequences downstream of HS-40 by recombinase-mediated cassette exchange. We thus demonstrate that (i). HS-40-mediated erythroid-specific activation of the alpha-globin genes is impaired solely by the insertion of a promoter sequence and not a coding sequence, unless it is methylated, and that (ii). the degree of transcriptional repression observed seems to be related directly to the transcriptional rate of the inserted promoter. Taken together, these results emphasize the importance of promoter sequences as the main targets for the activation mechanism of the human alpha-globin genes by HS-40.

PU.1 and pRB Interact and Cooperate To Repress GATA-1 and Block Erythroid Differentiation

PU.1 and GATA-1 are two hematopoietic specific transcription factors that play key roles in development of the myeloid and erythroid lineages, respectively. The two proteins bind to one another and inhibit each other's function in transcriptional activation and promotion of their respective differentiation programs. This mutual antagonism may be an important aspect of lineage commitment decisions. PU.1 can also act as an oncoprotein since deregulated expression of PU.1 in erythroid precursors causes erythroleukemias in mice. Studies of cultured mouse erythroleukemia cell lines indicate that one aspect of PU.1 function in erythroleukemogenesis is its ability to block erythroid differentiation by repressing GATA-1 (N. Rekhtman, F. Radparvar, T. Evans, and A. I. Skoultchi, Genes Dev. 13:1398-1411, 1999). We have investigated the mechanism of PU.1-mediated repression of GATA-1. We report here that PU.1 binds to GATA-1 on DNA. We localized the repression activity of PU.1 to a small acidic N-terminal domain that interacts with the C pocket of pRB, a well-known transcriptional corepressor. Repression of GATA-1 by PU.1 requires pRB, and pRB colocalizes with PU.1 and GATA-1 at repressed GATA-1 target genes. PU.1 and pRB also cooperate to block erythroid differentiation. Our results suggest that one of the mechanisms by which PU.1 antagonizes GATA-1 is by binding to it at GATA-1 target genes and tethering to these sites a corepressor that blocks transcriptional activity and thereby erythroid differentiation.

Multiple type A/B heterogeneous nuclear ribonucleoproteins (hnRNPs) can replace hnRNP A1 in mouse hepatitis virus RNA synthesis.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 has previously been shown to bind mouse hepatitis virus (MHV) RNA at the 3' end of both plus and minus strands and modulate MHV RNA synthesis. However, a mouse erythroleukemia cell line, CB3, does not express hnRNP A1 but still supports MHV replication, suggesting that alternative proteins can replace hnRNP A1 in cellular functions and viral infection. In this study, we set out to identify these proteins. UV cross-linking experiments revealed that several CB3 cellular proteins similar in size to hnRNP A1 interacted with the MHV RNA. These proteins were purified by RNA affinity column with biotinylated negative-strand MHV leader RNA and identified by mass spectrometry to be hnRNP A2/B1, hnRNP A/B, and hnRNP A3, all of which belong to the type A/B hnRNPs. All of these proteins contain amino acid sequences with strong similarity to the RNA-binding domains of hnRNP A1. Some of these hnRNPs have previously been shown to replace hnRNP A1 in regulating RNA splicing. These proteins displayed MHV RNA-binding affinity and specificity similar to those of hnRNP A1. hnRNP A2/B1, which is predominantly localized to the nucleus and shuttles between the nucleus and the cytoplasm, was shown to relocalize to the cytoplasm in MHV-infected CB3 cells. Furthermore, overexpression of hnRNP A/B in cells enhanced MHV RNA synthesis. Our findings demonstrate that the functions of hnRNP A1 in MHV RNA synthesis can be replaced by other closely related hnRNPs, further supporting the roles of cellular proteins in MHV RNA synthesis.

Effects of TFAR19 gene on the growth and biorheological properties of mouse erythroleukemia cell line MEL

Using the method of gene transfection with liposome, we obtained the mouse erythroleukemia cell line MEL-TF19, which stably carries TFAR19, a novel apoptosis-related gene. The expression of TFAR19 was detected by Western blot. Growth curve and flow cytometry analysis showed that after being transfected with TFAR19 gene, the growth of MEL-TF19 is suppressed and its apoptosis is accelerated because of the serum deprivation. Our biorheological study indicated that in the apoptotic process, compared with MEL cells, MEL-TF19 cells exhibit larger osmotic fragility, lower cell surface charge density, increased elastic modulus K (1) which is inversely proportional to cells' maximal deformation ability, obviously diminished surface viscosity mu, with elastic modulus K (2)having no distinct changes. The above results provided some bases for recognizing the function of TFAR19 completely from the viewpoint of biorheology.

Identification and Characterization of a Novel Ste20/Germinal Center Kinase-related Kinase, Polyploidy-associated Protein Kinase

A novel protein kinase, polyploidy-associated protein kinase (PAPK), was isolated using a subtraction cDNA library approach from a mouse erythroleukemia cell line that had been induced to polyploidy after serum withdrawal. PAPK shares homology with members of the Ste20/germinal center kinase family of protein kinases and is ubiquitously expressed as two spliced forms, PAPK-A and PAPK-B, that encode for proteins of 418 and 189 amino acids, respectively. The expression of endogenous PAPK-A protein increased after growth factor withdrawal in murine hematopoietic and fibroblast cells. When tested in an in vitro kinase assay, PAPK-A was activated in response to the stress-inducing agent hydrogen peroxide and slightly by fetal calf serum. Biochemical characterization of the PAPK-A-initiated pathway revealed that this novel kinase does not affect MAP kinase activity but can stimulate both c-Jun N-terminal kinase 1 (JNK1) and ERK6/p38 gamma. The kinase activity of PAPK appears to be required for the activation of ERK6/p38 gamma but not JNK1. When an inducible construct of PAPK-A was expressed in stably transfected NIH3T3 cells, the cells exhibited distinct cytoskeletal changes and became resistant to apoptotic cell death induced by serum withdrawal, effects of PAPK that require its kinase activity. These data suggest that PAPK is a new member of the Ste20/germinal center kinase family that modulates cytoskeletal organization and cell survival.

A Region Upstream of the Human δ-Globin Gene Shows a Stage-Specific Interaction with Globin Promoters in Erythroid Cell Lines

ABSTRACTWe previously showed that the 651-bp DNA fragment, located 3 kb upstream from the human |gd-globin gene (fragment F5), is able to inhibit adult, not fetal, globin promoter in mouse erythroleukemia cell lines (MEL) expressing adult globin genes. Here we show in transient analysis that fragment F5 has a strong inhibitory effect on fetal |gg-globin promoter in human erythroleukemia cell lines (HEL) expressing fetal globin genes. Since the |gb-promoter constructs were poorly expressed in fetal cells, new plasmids containing an HPFH promoter (Gγ−175, T to C), which is strongly expressed in both fetal and adult cell lines, were made. Here we report that fragment F5 in HEL cells has a strong inhibitory effect on wild-type γ-promoter only; no effect was evident on γ−175-promoter in either MEL or HEL cell lines. Altogether these results show a stage-specific interaction between fragment F5 and globin promoters during development. We also report the presence of several bindings for erythroid GATA family factors by electrophoretic mobility shift assay, using nuclear extracts from erythroid cell lines.

Cloning and expression of murine hematopoietic specific chromatin remodeling gene SMARCA5

Abstract Mouse erythroleukemia cell line was used to generate a library of genes expressed during erythroid differentiation induced by N,N′-hexamethylene-bis-acetamide (HMBA). By subtraction hybridization we detected 63 genes encoding DNA binding molecules, chromatin remodeling genes, signaling proteins, translation regulating factors, metabolic enzymes, mitochondrial genes and expressed sequence tags (EST). The differential gene expression pattern was further confirmed by northern blotting. We used mouse model of accelerated erythropoiesis induced by phenylhydrazine and carbon monoxide-induced hypoxia. The differential expression kinetics of subtracted genes was found during in vivo accelerated erythropoiesis but not during hypoxia. We studied the tissue-specific expression of the ESTs and out of them cloned a murine cDNA of a novel hematopoietic specific chromatin remodeling gene SMARCA5 from cDNA library. Moreover, SMARCA5 is expressed in two transcriptional variants: 4 and 5 kb. In MEL cells and in phenylhydrazine-stimulated mice, SMARCA5 was silenced in hematopoietic cells entering the genetic program of erythroid differentiation. We screened a murine genomic BAC library and isolated SMARCA5 gene which is being further analyzed. Our results demonstrating differentially expressed genes in erythropoiesis are supported by recent studies [Gubin et al. Genomics 59(2):168–77, (1999)] on primary erythroid cells stimulated by erythropoietin.

Beta globin gene inhibition by antisense RNA transcripts.

Sickle cell disease is caused by a mutation in the beta globin gene leading to hemoglobin S (Hb S) production. Several approaches have been explored to prevent Hb S polymerization in red blood cells and the symptoms associated with this disorder. To this end we tested a mammalian expression vector carrying a human beta globin antisense cDNA (pZeobetaAS) fragment in a mouse erythroleukemia cell line expressing the human gamma and beta globin genes. We observed a relative reduction in beta globin mRNA levels compared with gamma mRNA levels in the presence of pZeobetaAS. Moreover, analysis at the protein level showed an average 76% decrease in beta chains and a 517% increase in gamma chain biosynthesis. The inhibitory effect of the antisense vector on globin expression was maintained long term in culture. The expression vector pZeobetaAS was also transfected into primary erythroid progenitors to test its effects on globin genes undergoing normal developmental switching during differentiation. We observed a relative reduction of beta globin mRNA levels compared with gamma mRNA levels. These results support a novel role for antisense cDNA expression vectors as an alternative gene therapy strategy to inhibit betas gene expression in sickle cell disease. Gene Therapy (2000) 7, 438-444.

Cloning and Characterization of KCC3 and KCC4, New Members of the Cation-Chloride Cotransporter Gene Family

The K+-Cl- cotransporters (KCCs) belong to the gene family of electroneutral cation-chloride cotransporters, which also includes two bumetanide-sensitive Na+-K+-2Cl- cotransporters and a thiazide-sensitive Na+-Cl- cotransporter. We have cloned cDNAs encoding mouse KCC3, human KCC3, and human KCC4, three new members of this gene family. The KCC3 and KCC4 cDNAs predict proteins of 1083 and 1150 amino acids, respectively. The KCC3 and KCC4 proteins are 65-71% identical to the previously characterized transporters KCC1 and KCC2, with which they share a predicted membrane topology. The four KCC proteins differ at amino acid residues within key transmembrane domains and in the distribution of putative phosphorylation sites within the amino- and carboxyl-terminal cytoplasmic domains. The expression of mouse KCC3 in Xenopus laevis oocytes reveals the expected functional characteristics of a K+Cl- cotransporter: Cl--dependent uptake of 86Rb+ which is strongly activated by cell swelling and weakly sensitive to furosemide. A direct functional comparison of mouse KCC3 to rabbit KCC1 indicates that KCC3 has a much greater volume sensitivity. The human KCC3 and KCC4 genes are located on chromosomes 5p15 and 15q14, respectively. Although widely expressed, KCC3 transcripts are the most abundant in heart and kidney, and KCC4 is expressed in muscle, brain, lung, heart, and kidney. The unexpected molecular heterogeneity of K+-Cl- cotransport has implications for the physiology and pathophysiology of a number of tissues.

Aqp1 expression in erythroleukemia cells: genetic regulation of glucocorticoid and chemical induction.

The aquaporin-1 (AQP1) water channel protein is expressed in multiple mammalian tissues by several different developmental programs; however, the genetic regulation is undefined. The proximal promoter of mouse Aqp1 contains multiple putative cis-acting regulatory elements, and mouse erythroleukemia (MEL) cells are a well-characterized model for erythroid differentiation. Corticosteroid or dimethyl sulfoxide (DMSO) exposure induces AQP1 protein expression in MEL cells, and transcriptional regulation was investigated by transient transfections with Aqp1 promoter-reporter constructs. Dexamethasone induction is abrogated by deletion of two glucocorticoid response elements -0.5 kilobases (kb) from the transcription initiation site. Mutation of the GATA element at -0.62 kb has no effect, whereas mutation of the CACCC site at -37 bp significantly reduces DMSO-induced promoter activity. Hydroxyurea induces expression of AQP1 protein without acting through the proximal promoter. The MEL cell line is a reproducible erythroid model system for studying transcriptional regulation of the Aqp1 gene while determining the consequences on AQP1 protein biosynthesis.