Deep proteomic analysis of murine erythroid differentiation
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ABSTRACT:
PROVIDER: PRJNA492908 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7896160_1.fastq.gz | Fastqsanger.gz | |||
| SRR7896160_2.fastq.gz | Fastqsanger.gz | |||
| SRR7896170_1.fastq.gz | Fastqsanger.gz | |||
| SRR7896170_2.fastq.gz | Fastqsanger.gz | |||
| SRR7896161_1.fastq.gz | Fastqsanger.gz |
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Project description:Deciphering cellular proteomes is critical for our understanding of erythropoiesis. To better comprehend commonly used models of erythropoiesis, we obtained absolute quantification of proteins expressed in cultured primary murine bone-marrow derived erythroblasts throughout erythroid differentiation. These data were compared to proteomes from Friend murine erythroleukemia (MEL), G1ER, and MEDEP cells. Overall, more than 7200 proteins were quantified in at least one of these models of murine erythropoiesis. Protein expression during differentiation of MEDEP cells was most similar to that of differentiating primary murine erythroid cells, while proteomes of Friend MEL and G1ER cells were more distantly related. Comparison of the proteomes of murine and human erythroblasts revealed species-linked variability, but these differences were not as dramatic as those observed comparing human and murine transcriptomes during erythroid differentiation. To functionally validate the differences between transcriptional and translational control, heme synthesis was inhibited in MEDEP cells and the effects on the transcriptome and the proteome examined. While heme deficiency had minimal global effect on the cellular proteome, globin proteins were significantly down regulated, supporting observations heme regulation of globin synthesis is tightly regulated at the protein level to avoid deleterious over production of globin chains. As predicted, heme deficiency led to up regulation of -aminolevulinic acid synthase 2 (Alas2) protein. Interestingly, Alas1 was upregulated at both the transcriptional and protein levels. Characterization of cellular proteomes during erythropoiesis has potential to yield insight into mechanistic principles of human disease, as well as reveal novel therapeutic targets.
2020-04-20 | GSE120379 | GEO
Project description:Erythropoiesis is a tightly controlled process. Although human and murine erythropoiesis are grossly similar, several aspects of these processes present significant differences. We have performed a label free quantification of proteins expressed during murine erythroid differentiation of primary cells with the aim to compare human and murine differentiations. Furthermore, we also analyzed the proteome of three murine cell lines, MEDEP, G1ER and Friend cells, to determine their similarities with the proteome of primary cells.
2020-05-26 | PXD009384 | Pride
Project description:Purpose:The purpose of this study is to create unbiased, stage-specific transcriptomes by RNA-seq analyses of pure populations of both murine and human erythroblasts at distinct developmental stages. Methods: Recently developed FACS-based methods (Chen et al, PNAS, Liu et al, Blood, Hu et al Blood) were employed to purify morphologically and functionally discrete populations of cells, each representing specific stages of terminal erythroid differentiation. RNA was prepared from these cells and subjected to RNA-seq analyses. Results: There were vast temporal changes in gene expression across the differentiation stages, with each stage exhibiting unique transcriptomes.Clustering and network analyses revealed that differing stage-specific patterns of expression across differentiation were transcriptionally enriched for genes of differing function. Numerous differences were present between human and murine transcriptomes, with significant variation in the global patterns of gene expression. Conclusions: These data provide a significant resource for studies of normal and perturbed erythropoiesis, allowing a deeper understanding of mechanisms of erythroid development, differentiation, and inherited and acquired disease. Both murine and human erythroblasts at distinct developmental stage mRNA profiles were generated by deep sequencing, in triplicate, using IlluminaHiSeq 2000.
2014-03-19 | E-GEOD-53983 | biostudies-arrayexpress
Project description:Murine-based cellular models have provided and continue to provide many useful insights into the fundamental mechanisms of erythropoiesis, as well as insights into the pathophysiology of inherited and acquired red cell disorders. Although detailed information on many aspects of these cell models is available, comprehensive proteomic data are lacking. This is a critical knowledge gap, as proteins are effectors of most biologic processes. To address this critical unmet need, proteomes of the murine cell lines Friend erythroleukemia (MEL), GATA1 erythroid (G1ER), and embryonic stem cell-derived erythroid progenitor (MEDEP) and proteomes of cultured murine marrow-derived erythroblasts at different stages of terminal erythroid differentiation were analyzed. The proteomes of MEDEP cells and primary murine erythroid cells were most similar, whereas those of MEL and G1ER cells were more distantly related. We demonstrated that the overall cellular content of histones does not decrease during terminal differentiation, despite strong chromatin condensation. Comparison of murine and human proteomes throughout terminal erythroid differentiation revealed that many noted transcriptomic changes were significantly dampened at the proteome level, especially at the end of the terminal differentiation process. Analysis of the early events associated with induction of terminal differentiation in MEDEP cells revealed divergent alterations in associated transcriptomes and proteomes. These proteomic data are powerful and valuable tools for the study of fundamental mechanisms of normal and disordered erythropoiesis and will be of broad interest to a wide range of investigators for making the appropriate choice of various cell lines to study inherited and acquired diseases of the erythrocyte.
| S-EPMC7160260 | biostudies-literature
Project description:Schneider RK, Schenone M, Kramann R, Ferreira MV, Joyce CE, Hartigan C, Beier F, Brümmendorf TH, Gehrming U, Platzbecker U, Buesche G, Chen MC, Waters CS, Chen E, Chu LP, Novina CD, Lindsley RC, Carr SA, Ebert BL. Nat Med, 2016.
Heterozygous deletion of RPS14 occurs in del(5q) MDS and has been linked to impaired erythropoiesis, characteristic of this disease subtype. We generated a murine model with conditional inactivation of Rps14 and demonstrated a p53-dependent erythroid differentiation defect with apoptosis at the transition from polychromatic to orthochromatic erythroblasts resulting in age-dependent progressive anemia, megakaryocyte dysplasia, and loss of hematopoietic stem cell (HSC) quiescence. Using quantitative proteomics, we identified significantly increased expression of proteins involved in innate immune signaling, particularly the heterodimeric S100A8/S100A9 proteins in purified erythroblasts. S100A8 expression was significantly increased in erythroblasts, monocytes and macrophages and recombinant S100A8 was sufficient to induce an erythroid differentiation defect in wild-type cells. We rescued the erythroid differentiation defect in Rps14 haploinsufficient HSCs by genetic inactivation of S100a8 expression. Our data link Rps14 haploinsufficiency to activation of the innate immune system via induction of S100A8/A9 and the p53-dependant erythroid differentiation defect in del(5q) MDS.
2016-01-26 | MSV000079482 | MassIVE
Project description:Transcriptional profiling of murine erythroleukemia cells induced to differentiate Murine erythroleukemia (MEL) cells are derived from mice infected with the Friend erythroleukemia viral complex, which causes a lethal expansion of erythroid cells. The transformed, immortalized cells (MEL cells) isolated from infected mice are committed to erythroid differentiation, but are blocked roughly at the proerythoblast stage. Treatment with small, oxidized organic compounds like dimethyl sulfoxide or hydroxymethyl-bis-acetamide (HMBA) induce the cells to mature further, to roughly the erythroblast stage or beyond in some lines (Friend et al. 1971). During this induction, the amount of hemoglobin, some heme biosynthetic enzymes and other proteins characteristic of mature erythroid cells increases, largely as a result of changes in transcription (Aviv et al. 1976). A full description of changes in transcription of all mouse genes during this process is not available. To improve this situation, we have conducted transcriptional profiling of the RL5 line of MEL cells, using microarrays containing almost 7000 mouse genes. The RL5 line has a "random locus" containing a positive-negative selectable marker (HyTK) flanked by loxP sites, thus facilitating site-directed integration into the cells (Bouhassira et al. 1997; Feng et al. 1999; Molete et al. 2001). This particular line is not highly inducible, with less than half the cells accumulating large amounts of hemoglobin. However, they are advantageous for integration of expression constructs, which is our particular interest. MEL_RL5 cells were treated with 4 millimolar HMBA for 6 days to induce maturation, and RNA was isolated each day. Complementary DNA was synthesized and labeled with either Cy5 (red fluorescence) for the induced samples or Cy3 (green fluorescence) the untreated cells grown in parallel. For most time points, at least two independent experiments were run, some with dyes switched (SD). The microarrays were spotted at the PSU Microarray Facility. Hybridization was carried out in our laboratory, and hybridized chips were scanned on a GenePix scanner and quantified using GenePix software. The signals from each channel (red and green) were normalized so that the total red intensity equals the total green intensity. Data were filtered to remove "bad" spots, which are those in which the noise exceeded the signal. We calculated the median of the pixel intensities for each spot and standard deviation around the median, and with these values we computed a "modified Z-score" for each channel on each spot. A lenient threshold for the "modified Z-score" was applied to remove "bad" spots. After filtering, we performed global mean normalization for each chip, followed by lowess normalization for each chip to remove intensity-dependent effects.
2005-01-31 | E-GEOD-2217 | biostudies-arrayexpress
Project description:Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71+ erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation. siRNA knockdown of RBM38 was perfomed in human MCF-7 breast cancer cells. The efficiency of RBM38 knockdown was monitored by western blot using an RBM38 antibody (Santa Cruz Biotechnology, SC-85873). We conducted HJAY exon and exon junction array profiling on RNAs from four siRBM38 treated MCF-7 samples vs. four sicontrol treated MCF-7 samples Control / knockdown comparison.
2013-07-29 | E-GEOD-49293 | biostudies-arrayexpress
Project description:Ter-119 is a better murine erythroid selection marker than CD71 for bulk immunomics, murine bone marrow and fetal liver erythroid cells have different antimicrobial immune transcriptome signatures, murine bone marrow erythropoiesis is comprised of two branches In this study, we decided to benchmark erythroid cells’ selection and enrichment markers – CD71 and Ter-119 via bulk immune transcriptomics by Nanostring and perform bulk immune transcriptome profiling of Ter-119+ erythroid cells from the bone marrow and the fetal liver by Nanostring.
2023-07-31 | GSE238190 | GEO
Project description:Transcriptional profiling of murine erythroleukemia cells induced to differentiate Murine erythroleukemia (MEL) cells are derived from mice infected with the Friend erythroleukemia viral complex, which causes a lethal expansion of erythroid cells. The transformed, immortalized cells (MEL cells) isolated from infected mice are committed to erythroid differentiation, but are blocked roughly at the proerythoblast stage. Treatment with small, oxidized organic compounds like dimethyl sulfoxide or hydroxymethyl-bis-acetamide (HMBA) induce the cells to mature further, to roughly the erythroblast stage or beyond in some lines (Friend et al. 1971). During this induction, the amount of hemoglobin, some heme biosynthetic enzymes and other proteins characteristic of mature erythroid cells increases, largely as a result of changes in transcription (Aviv et al. 1976). A full description of changes in transcription of all mouse genes during this process is not available. To improve this situation, we have conducted transcriptional profiling of the RL5 line of MEL cells, using microarrays containing almost 7000 mouse genes. The RL5 line has a "random locus" containing a positive-negative selectable marker (HyTK) flanked by loxP sites, thus facilitating site-directed integration into the cells (Bouhassira et al. 1997; Feng et al. 1999; Molete et al. 2001). This particular line is not highly inducible, with less than half the cells accumulating large amounts of hemoglobin. However, they are advantageous for integration of expression constructs, which is our particular interest. MEL_RL5 cells were treated with 4 millimolar HMBA for 6 days to induce maturation, and RNA was isolated each day. Complementary DNA was synthesized and labeled with either Cy5 (red fluorescence) for the induced samples or Cy3 (green fluorescence) the untreated cells grown in parallel. For most time points, at least two independent experiments were run, some with dyes switched (SD). The microarrays were spotted at the PSU Microarray Facility. Hybridization was carried out in our laboratory, and hybridized chips were scanned on a GenePix scanner and quantified using GenePix software. The signals from each channel (red and green) were normalized so that the total red intensity equals the total green intensity. Data were filtered to remove "bad" spots, which are those in which the noise exceeded the signal. We calculated the median of the pixel intensities for each spot and standard deviation around the median, and with these values we computed a "modified Z-score" for each channel on each spot. A lenient threshold for the "modified Z-score" was applied to remove "bad" spots. After filtering, we performed global mean normalization for each chip, followed by lowess normalization for each chip to remove intensity-dependent effects. Keywords: time-course
2005-02-01 | GSE2217 | GEO
Project description:Metabolic programs contribute to hematopoietic stem and progenitor cell (HSPC) fate, but it is not known whether the metabolic regulation of protein synthesis controls HSPC differentiation. Here, we show that SLC7A1/CAT1-dependent arginine uptake and its catabolism to the polyamine spermidine control human erythroid specification of HSPCs via activation of the eukaryotic translation initiation factor 5A (eIF5A). eIF5A activity is dependent on its hypusination, a post-translational modification resulting from the conjugation of the aminobutyl moiety of spermidine to lysine. Notably, attenuation of hypusine synthesis in erythroid progenitors by inhibition of deoxyhypusine synthase abrogates erythropoiesis but not myeloid cell differentiation. Proteomic profiling reveals mitochondrial translation to be a critical target of hypusinated eIF5A and accordingly, progenitors with decreased hypusine activity exhibit diminished oxidative phosphorylation. This impacted pathway is critical for eIF5A-regulated erythropoiesis as interventions augmenting mitochondrial function partially rescue human erythropoiesis under conditions of attenuated hypusination. Levels of mitochondrial ribosomal proteins were especially sensitive to the loss of hypusine and we find that the ineffective erythropoiesis linked to haploinsufficiency of RPS14 in del(5q) myelodysplastic syndrome is associated with a diminished pool of hypusinated eIF5A. Moreover, patients with RPL11-haploinsufficient Diamond-Blackfan anemia as well as CD34+ progenitors with downregulated RPL11 exhibit a markedly decreased hypusination in erythroid progenitors, concomitant with a loss of mitochondrial metabolism. Thus, eIF5A-dependent protein synthesis regulates human erythropoiesis and our data reveal a novel role for RPs in controlling eIF5A hypusination in HSPC, synchronizing mitochondrial metabolism with erythroid differentiation.
2023-01-25 | MSV000091139 | MassIVE