Characterization of alpha-globin locus chromatin accessibility in erythroid cells following insertion of PP7 stem-loops at the Hba-a1 gene
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ABSTRACT:
PROVIDER: PRJNA783197 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17027017_1.fastq.gz | Fastqsanger.gz | |||
| SRR17027017_2.fastq.gz | Fastqsanger.gz | |||
| SRR17027018_1.fastq.gz | Fastqsanger.gz | |||
| SRR17027018_2.fastq.gz | Fastqsanger.gz | |||
| SRR17027019_1.fastq.gz | Fastqsanger.gz |
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Project description:PP7 stem-loop DNA was inserted into the Hba-a1 gene in order to monitor nascent transcription dynamics of alpha-globin during erythroid differentiation. To confirm that this genetic modification does not influence the chromatin accessibility of the locus in erythroid cells we performed ATAC-seq in wild-type and Hba-a1-PP7 (PP7) cells, as well as Hba-a1-PP7 clones with a stably integrated PP7 coat protein (PCP) expression construct. No differences were observed between these cell lines in terms of chromatin accessibility at the alpha-globin locus, indicating the PP7 modification likely did not disrupt normal gene activation in these cells.
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Project description:We investigate the three-dimensional conformations of the -globin locus at the single-allele level in murine ES and erythroid cells, combining polymer physics models and high-resolution Capture-C data. Model predictions are validated against independent FISH data measuring pairwise distances, and Tri-C data identifying three-way contacts. The architecture is rearranged during the transition from ES cells to erythroid cells, associated with the activation of the globin genes. We find that in ES cells, the spatial organization conforms to a highly intermingled 3D structure involving non-specific contacts, while in erythroid cells the -globin genes and their enhancers form a self-contained domain, arranged in a folded hairpin conformation, separated from intermingling flanking regions by a thermodynamic mechanism of micro-phase separation. The flanking regions are rich in convergent CTCF sites, which only marginally participate in the erythroid-specific gene-enhancer contacts, suggesting that beyond the interaction of CTCF sites multiple molecular mechanisms cooperate to form an interacting domain.
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Project description:We investigate the three-dimensional conformations of the -globin locus at the single-allele level in murine ES and erythroid cells, combining polymer physics models and high-resolution Capture-C data. Model predictions are validated against independent FISH data measuring pairwise distances, and Tri-C data identifying three-way contacts. The architecture is rearranged during the transition from ES cells to erythroid cells, associated with the activation of the globin genes. We find that in ES cells, the spatial organization conforms to a highly intermingled 3D structure involving non-specific contacts, while in erythroid cells the -globin genes and their enhancers form a self-contained domain, arranged in a folded hairpin conformation, separated from intermingling flanking regions by a thermodynamic mechanism of micro-phase separation. The flanking regions are rich in convergent CTCF sites, which only marginally participate in the erythroid-specific gene-enhancer contacts, suggesting that beyond the interaction of CTCF sites multiple molecular mechanisms cooperate to form an interacting domain.
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Project description:The fetal and adult globin genes in the human b-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal g-globin (HBG) to replace abnormal adult sickle bS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of g-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize protein expression patterns during the g-globin to b-globin (g/b) switch observed throughout in vitro erythroid differentiation. We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the g/b-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal g-globin expression at day 7 with a switch to a predominance of b-globin expression by day 28 and the g/b-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated >1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kb, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in g-and b-globin regulation were identified. The transcriptome analysis completed with erythroid progenitors grown in vitro identified groups of genes with distinct expression profiles, which function in metabolic pathways associated with cell survival, hematopoiesis, blood cells activation, and inflammatory responses. This study represents the first report of a transcriptome analysis in human primary erythroid progenitors to identify transcription factors involved in hemoglobin switching. Our results also demonstrate that the in vitro liquid culture system is a good model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation. Peripheral blood mononuclear cells were isolated from three normal donors using Histapaque-1.077. The mononuclear cells were grown in three independent cultures using the one-phase protocol as previously published. Cells were cultured in alphaMEM containing 30% fetal bovine serum, 1% deionized BSA with penicillin (100 U/mL) and streptomycin (0.1 mg/mL) at 37°C and 5% CO2. The following growth factors were added on day 0: stem cell factor (50 ng/mL), interleukin-3 (10 ng/mL) and erythropoietin (4 U/mL). Approximately 1.5 million cells were harvested from each culture (triplicate samples) on day 7, 14, 21 and 28 for microarray analysis on the whole-genome Illumina HumanWG-6 V2 Expression BeadChip.
2012-01-14 | E-GEOD-35102 | biostudies-arrayexpress
Project description:The fetal and adult globin genes in the human b-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal g-globin (HBG) to replace abnormal adult sickle bS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of g-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize protein expression patterns during the g-globin to b-globin (g/b) switch observed throughout in vitro erythroid differentiation. We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the g/b-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal g-globin expression at day 7 with a switch to a predominance of b-globin expression by day 28 and the g/b-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated >1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kb, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in g-and b-globin regulation were identified. The transcriptome analysis completed with erythroid progenitors grown in vitro identified groups of genes with distinct expression profiles, which function in metabolic pathways associated with cell survival, hematopoiesis, blood cells activation, and inflammatory responses. This study represents the first report of a transcriptome analysis in human primary erythroid progenitors to identify transcription factors involved in hemoglobin switching. Our results also demonstrate that the in vitro liquid culture system is a good model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation.
2012-01-14 | GSE35102 | GEO
Project description:Genes encoding the human β-like hemoglobin proteins undergo a developmental switch from fetal γ-globin to adult β-globin expression at around the time of birth. β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, result from mutations affecting the adult β-globin gene. The only treatment options currently available carry significant side-effects. Analyses of heritable variations in fetal hemoglobin (HbF) levels have provided evidence that reactivation of the silenced fetal γ-globin genes in adult erythroid cells is a promising therapy. The γ-globin repressor BCL11A has become the major focus with several studies investigating its regulation and function as a first step to inhibiting its expression or activity. However, a second repression mechanism has recently been shown to be mediated by the transcription factor ZBTB7A/LRF, suggesting that understanding the regulation of ZBTB7A may also be useful. Here we show that KLF1 directly drives expression of ZBTB7A in erythroid cells by binding to its proximal promoter. We have also uncovered an erythroid-specific regulation mechanism, leading to the upregulation of a novel ZBTB7A transcript in the erythroid compartment. The demonstration that ZBTB7A, like BCL11A, is a KLF1 target gene also fits with the observation that reduced KLF1 expression or activity is associated with HbF derepression.
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Project description:RNAPII-S5P ChIP e4C was performed on mouse erythroid tissue, using the alpha and beta globin genes as baits
2009-12-01 | E-TABM-822 | biostudies-arrayexpress
Project description:The Ldb1/GATA-1/TAL1/LMO2 complex mediates long range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated human NLI (Ldb1 homologue) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we find robust NLI complex occupancy at a site downstream of the Aγ-globin gene, within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members together with co-repressor ETO2 and γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is re-activated by cytokines in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. Thus, alternative NLI complexes mediate γ-globin transcription or silencing through long range LCR interactions involving an intergenic site of non-coding RNA transcription and ETO2 is critical to this process. ChIP-chip of GATA-1, NLI, and pol II in cell lines Comparison of GATA-1, NLI, and pol II binding on chromatin. 2 replicates for each protein; ChIP and input DNA for each replicate
2012-01-05 | E-GEOD-30047 | biostudies-arrayexpress