RNA-seq analysis of HUDEP-2 cells after erythroid differentiation
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE280894 | GEO | 2025/10/26
REPOSITORIES: GEO
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Project description:RNA-seq analysis of HUDEP-2 cells after erythroid differentiation
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Project description:MicroRNAs (miRNAs) are small non-coding RNAs (sncRNAs) crucial for post-transcriptional and translational regulation of cellular and developmental pathways. Study of miRNAs in erythropoiesis thus elucidates the process and facilitates diagnosis and therapy development for related disorders. Here, we used high-throughput DNA Nanoball small RNA sequencing to characterise sncRNAs, and in particular miRNAs, comprehensively in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and on two influential erythroid cell lines with adult and fetal expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential expression (DE) to erythroid differentiation, cell type, and fetal and adult hemoglobin expression patterns. Sequencing results were validated by reverse-transcription quantitative PCR (RT-qPCR) of selected DE miRNAs (miR-451a, miR-182-5p, miR-146a-5p, miR-221-3p, miR-222-3p) and indicated overall common erythroid differentiation signatures in primary cells and cell lines, in terminal stages of erythropoiesis characterized by decreased complexity of global miRNA expression and reciprocally increased expression of individual lineage-specific miRNAs. Interestingly, against a backdrop of evident clustering and thus similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted readily interpretable discrepancies between both cell types. Moreover, differential expression analysis between the closely related HUDEP-2 and HUDEP-1 cells provided pointers to miRNAs, transcription factors (TFs), target genes, and pathways mechanistically linked to globin switching. In resulting TF-miRNA co-regulatory networks, TFs known to be implicated in hemoglobin switching, including the major γ-globin repressors BCL11A and ZBTB7A, were targeted by many co-regulated miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.
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Project description:We examined the chromatin occupancy of ZNF410 by conducting CUT&RUN. We used an HA antibody to probe for epitope tagged ZNF410 in HUDEP-2 cells. We used a ZNF410 antibody to probe for endogenous ZNF410 in HUDEP-2 cells, human CD34+ hematopoietic stem and progenitor cell (HSPC) derived erythroid precursors and mouse erythroleukemia (MEL) cells.
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Project description:Terminal differentiation is characterized by a respecification of the global protein complement, as epitomized by erythrocytes, whose cytosol is ~98% globin. Remodeling of the proteome involves programmed elimination of generic cellular constituents in parallel with synthesis of cell-type-specific proteins. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving elimination of preexisting cytosolic proteins are unidentified. UBE2O is a ubiquitin-conjugating enzyme expressed at elevated levels during late stage erythropoiesis. A Ube2o null mutation in mice results in anemia. Proteomic analysis of this mutant suggests that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, a hallmark of the reticulocyte to mature erythrocyte transition—ribosome elimination—is defective in Ube2o-/- mutants. UBE2O recognizes ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, and perhaps other highly differentiated cells, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.
2017-08-08 | PXD005904 | Pride
Project description:Purpose: The goal of this study is to compare the altered transcriptional program (RNA-seq) and cellular adaptation to stress caused by excess a-globin synthesis. Results: 100bp paired-end polyA-enriched RNA sequencing were performed with a depth of minimum of 20 million read counts per sample. The sequencing library was sequenced on the Illumina HiSeq2500 at Azenta. Raw data then went through read quality assessment by FastQC (version 0.11.5), mapping to the human genome (GRch38) by the STAR aligner and gene-level reads quantification using the Rsubread package. RNA-seq data analysis identified 1870 upregulated and 1549 down regulated genes on Day 0 (FDR <=0.05) and 1924 upregulated and 1960 down regulated genes on Day 8 (FDR <=0.05). MA plot analysis of differentially expressed genes uncovered an altered transcriptional program favouring γ-globin expression not solely dependent upon the HRI ATF4-BCL11A axis. Instead, we noted attenuated expression of several key γ-globin co-repressors, including SOX6, ZBTB7A, KLF1 and GATA1, which coincided with increased expression of co-activators such as CBFB/NY-FB and GATA2 following erythroid differentiation. Gene Set Enrichment Analysis (GSEA) software was used to determine significantly enriched gene sets. Downregulated DEGs in β0 HUDEP 2 cells in pathways related to haeme metabolism, hypoxia, MTORC1, unfolded protein response and TNFA/NFkB signalling were enriched. Meanwhile, upregulated DEGs in β0 HUDEP-2 cells related to G-protein-coupled receptors (GPCR) signalling, IL-2/STAT5 and hypoxia signalling pathway were also retrieved. Pathways shown to be significantly altered in both β0 HUDEP-2 cells and patient group include apoptosis, MAPK signalling and NFKB pathway thus provided complementary insights in transcriptome profiling. Conclusions: Our study represents analysis of HUDEP2 transcriptomes, with biologic replicates, generated by RNA-seq technology. The data analysis reported here should provide a framework for comparative investigations of expression profiles. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of the cellular adaptation to intrinsic stress caused by excess a-globin synthesis.
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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:To explore the mechanisms controlling erythroid differentiation and development in human, we analyzed the genome-wide transcription dynamics that occurs during the differentiation of HESCs into the erythroid lineage and development of embryonic to adult erythropoiesis using high throughput sequencing technology. Undifferentiated HESCs as well as erythroid cells at three developmental stages-ESER (embryonic stage), FLER (fetal stage) and PBER (adult stage)-were analyzed. Our findings revealed that the number of expressed genes decreased during the differentiation, while the total expression intensity increased. At the 3 transitions (HESC-ESER, ESER-FLER and FLER-PBER), differential expression of many types of genes was observed at every transition. These differentially expressed genes were involved in maintaining the pluripotency of stem cells, early erythroid specification, rapid cell growth and enucleation potential. In addition, differentially expressed genes were found to constitute networks and central nodes at each transition. Clusters of genes in some chromosomal regions switched between expression and silence. We also discovered that differentially expressed genes constituted networks and central nodes of them in each transition. Our studies provide a fundamental basis for further investigation of erythroid differentiation and development.
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