Quantitative Analysis of erythroblast cells Transcriptomes treated with cholesterol or not
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE166153 | GEO | 2021/12/05
REPOSITORIES: GEO
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Project description:Tropomodulins (Tmods) cap the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse Tmod3 leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. BFU-E and CFU-E colony numbers are greatly reduced, indicating defects in progenitor populations. Flow-cytometry of fetal liver erythroblasts shows late stage populations are also decreased, including reduced percentages of enucleated cells. AnnexinV staining indicates increased apoptosis of Tmod3-/- erythroblasts, and cell cycle analysis reveals that there are more Ter119hi cells in S-phase in Tmod3-/- embryos. Notably, enucleating Tmod3-/- erythroblasts are still in the process of proliferation, suggesting impaired cell cycle exit during terminal differentiation. Tmod3-/- late erythroblasts often exhibit multi-lobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3-/- fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis, due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation. Total RNAs from Tmod3+/+ and Tmod3-/- fetal livers at E14.5 were extracted and prepared for microarray analysis using the MoGene-1_0-st-v1 Affymetrix chip in the Scripps Research Microarray Core Facility. Each experiment was repeated with three independent embryos.
2013-10-31 | E-GEOD-51960 | biostudies-arrayexpress
Project description:Tropomodulins (Tmods) cap the pointed ends of actin filaments in erythroid and nonerythoid cell types. Targeted deletion of mouse Tmod3 leads to embryonic lethality at E14.5-E18.5, with anemia due to defects in definitive erythropoiesis in the fetal liver. BFU-E and CFU-E colony numbers are greatly reduced, indicating defects in progenitor populations. Flow-cytometry of fetal liver erythroblasts shows late stage populations are also decreased, including reduced percentages of enucleated cells. AnnexinV staining indicates increased apoptosis of Tmod3-/- erythroblasts, and cell cycle analysis reveals that there are more Ter119hi cells in S-phase in Tmod3-/- embryos. Notably, enucleating Tmod3-/- erythroblasts are still in the process of proliferation, suggesting impaired cell cycle exit during terminal differentiation. Tmod3-/- late erythroblasts often exhibit multi-lobular nuclear morphologies and aberrant F-actin assembly during enucleation. Furthermore, native erythroblastic island formation was impaired in Tmod3-/- fetal livers, with Tmod3 required in both erythroblasts and macrophages. In conclusion, disruption of Tmod3 leads to impaired definitive erythropoiesis, due to reduced progenitors, impaired erythroblastic island formation, and defective erythroblast cell cycle progression and enucleation. Tmod3-mediated actin remodeling may be required for erythroblast-macrophage adhesion, coordination of cell cycle with differentiation, and F-actin assembly and remodeling during erythroblast enucleation.
2013-10-31 | GSE51960 | GEO
Project description:Understanding the pattern of gene expression and identifying the specific genes expressed during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here we have isolated four distinct populations of erythroblasts at successive erythropoietin-dependent stages of erythropoiesis including the terminal, pyknotic stage. The transcriptome has been determined using Affymetrix arrays. First, we show that cells sorted by surface expression profile express not only significantly fewer genes than unsorted cells, but also significantly more differences in the expression levels of particular genes between stages than unsorted cells, demonstrating the importance of working with defined cell populations to identify lineage and temporally-specific patterns of gene expression. Second, using standard software and matched filtering we identify eleven differentially regulated genes and one continuously expressed gene previously undetected in erythroid expression studies with unknown roles in erythropoiesis (CA3, CALB1, CTSL2, FKBP1B, GSDMB, ITLN1, LIN7B, RRAD, RUNDC3A, UNQ1887, ZNF805, MYL12B). Finally, using transcription factor binding site analysis we identify potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts are a resource for functional studies of erythropoietic protein function and gene regulation. PBMC-derived erythroblasts grown in vitro were harvested at the CFU-E, Pro-E, Int-E and Late-E stages and FACS sorted based on expression of cell surface markers. RNA was extracted from those populations which met rigorous specifications of time cultured in the presence of erythropoietin, morphology and CD36, CD71 and CD235a expression. For each stage, 3 chips were hybridised, each representing a pool of 4 different samples. In addition to these 12 chips, one chip representing a pool of RNA extracted from unsorted cells at each stages was also hybridised.
2011-03-02 | E-GEOD-22552 | biostudies-arrayexpress
Project description:Mammalian terminal erythropoiesis involves chromatin and nuclear condensation followed by enucleation. Late-stage erythroblasts undergo caspase-mediated nuclear opening formation that is important for nuclear condensation through partial release of histones. It remains unknown whether nuclear opening and histone release influence the three-dimensional (3D) genomic organization during terminal erythropoiesis. Here, we compared the genome wide 3D organization, chromatin accessibility, and transcriptome of the cultured mouse erythroid progenitors with and without the blocking of nuclear opening during differentiation. We found that terminal differentiation from the basophilic to orthochromatic stages of erythroblasts involves compaction and establishment of long-range interactions of the heterochromatin regions, which is associated with globally increased accessibility and upregulation of erythroid-related genes. Surprisingly, blocking of nuclear opening did not have a significant impact on 3D genomic organization, chromatin accessibility, or transcriptome despite the inhibition of histone release and nuclear condensation. Inhibition of nuclear opening also significantly affected enucleation. We further demonstrated this through a caspase-3 and 7 double knockout mouse model, which showed significant defects in nuclear opening and condensation with a compromise of enucleation in fetal erythroid progenitors. However, loss of these effector caspases had minimal effects on the red cell indices and survival of the recipient animals in a fetal liver cell transplantation model. Overall, these results indicate that nuclear opening and histone release may not be necessary for chromatin condensation and global transcriptome but are critical for nuclear condensation and efficient enucleation. These data also underline the robustness of enucleation despite the dysplastic or less condensed nucleus.
2022-12-16 | GSE185469 | GEO
Project description:Mammalian terminal erythropoiesis involves chromatin and nuclear condensation followed by enucleation. Late-stage erythroblasts undergo caspase-mediated nuclear opening formation that is important for nuclear condensation through partial release of histones. It remains unknown whether nuclear opening and histone release influence the three-dimensional (3D) genomic organization during terminal erythropoiesis. Here, we compared the genome wide 3D organization, chromatin accessibility, and transcriptome of the cultured mouse erythroid progenitors with and without the blocking of nuclear opening during differentiation. We found that terminal differentiation from the basophilic to orthochromatic stages of erythroblasts involves compaction and establishment of long-range interactions of the heterochromatin regions, which is associated with globally increased accessibility and upregulation of erythroid-related genes. Surprisingly, blocking of nuclear opening did not have a significant impact on 3D genomic organization, chromatin accessibility, or transcriptome despite the inhibition of histone release and nuclear condensation. Inhibition of nuclear opening also significantly affected enucleation. We further demonstrated this through a caspase-3 and 7 double knockout mouse model, which showed significant defects in nuclear opening and condensation with a compromise of enucleation in fetal erythroid progenitors. However, loss of these effector caspases had minimal effects on the red cell indices and survival of the recipient animals in a fetal liver cell transplantation model. Overall, these results indicate that nuclear opening and histone release may not be necessary for chromatin condensation and global transcriptome but are critical for nuclear condensation and efficient enucleation. These data also underline the robustness of enucleation despite the dysplastic or less condensed nucleus.
2022-12-16 | GSE185468 | GEO
Project description:Mammalian terminal erythropoiesis involves chromatin and nuclear condensation followed by enucleation. Late-stage erythroblasts undergo caspase-mediated nuclear opening formation that is important for nuclear condensation through partial release of histones. It remains unknown whether nuclear opening and histone release influence the three-dimensional (3D) genomic organization during terminal erythropoiesis. Here, we compared the genome wide 3D organization, chromatin accessibility, and transcriptome of the cultured mouse erythroid progenitors with and without the blocking of nuclear opening during differentiation. We found that terminal differentiation from the basophilic to orthochromatic stages of erythroblasts involves compaction and establishment of long-range interactions of the heterochromatin regions, which is associated with globally increased accessibility and upregulation of erythroid-related genes. Surprisingly, blocking of nuclear opening did not have a significant impact on 3D genomic organization, chromatin accessibility, or transcriptome despite the inhibition of histone release and nuclear condensation. Inhibition of nuclear opening also significantly affected enucleation. We further demonstrated this through a caspase-3 and 7 double knockout mouse model, which showed significant defects in nuclear opening and condensation with a compromise of enucleation in fetal erythroid progenitors. However, loss of these effector caspases had minimal effects on the red cell indices and survival of the recipient animals in a fetal liver cell transplantation model. Overall, these results indicate that nuclear opening and histone release may not be necessary for chromatin condensation and global transcriptome but are critical for nuclear condensation and efficient enucleation. These data also underline the robustness of enucleation despite the dysplastic or less condensed nucleus.
2022-12-16 | GSE184974 | GEO
Project description:Understanding the pattern of gene expression and identifying the specific genes expressed during erythropoiesis is crucial for a synthesis of erythroid developmental biology. Here we have isolated four distinct populations of erythroblasts at successive erythropoietin-dependent stages of erythropoiesis including the terminal, pyknotic stage. The transcriptome has been determined using Affymetrix arrays. First, we show that cells sorted by surface expression profile express not only significantly fewer genes than unsorted cells, but also significantly more differences in the expression levels of particular genes between stages than unsorted cells, demonstrating the importance of working with defined cell populations to identify lineage and temporally-specific patterns of gene expression. Second, using standard software and matched filtering we identify eleven differentially regulated genes and one continuously expressed gene previously undetected in erythroid expression studies with unknown roles in erythropoiesis (CA3, CALB1, CTSL2, FKBP1B, GSDMB, ITLN1, LIN7B, RRAD, RUNDC3A, UNQ1887, ZNF805, MYL12B). Finally, using transcription factor binding site analysis we identify potential transcription factors that may regulate gene expression during terminal erythropoiesis. Our stringent lists of differentially regulated and continuously expressed transcripts are a resource for functional studies of erythropoietic protein function and gene regulation.
2011-03-02 | GSE22552 | GEO
Project description:In malaria infection, Plasmodium spp. parasites accumulate in the bone marrow near sites of erythroid development. While it has been observed that Plasmodium falciparum infection of late-stage erythroblasts can delay terminal erythroid differentiation and enucleation, the mechanism(s) underlying this phenomenon are unknown. Here, we apply RNA-seq after fluorescence-activated cell sorting (FACS) of infected erythroblasts to identify transcriptional responses to direct and indirect interaction with P. falciparum.
2023-07-25 | GSE233739 | GEO
Project description:We identified a role for E2F-2 in the regulation of erythroblast nuclear condensation and enucleation. To help define the mechanism by which E2F-2 regulates these processes, we performed RNA-sequencing on undifferentiated hematopoietic cells and sorted, orthochromatic erythroblasts obtained from wildtype and E2F-2 knockout animals. In undifferentiated progenitor cells we find a limited number of differentially expressed genes associated with E2F-2-loss, likely due to compensation by other E2F family members. However, in late-stage erythroblasts, loss of E2F-2 results in the down-regulation of over 1200 genes. Our subsequent analyses focused on the role of a particular mitotic kinase, Citron Rho-interacting kinase, which we find is induced in an E2F-2-dependent manner during terminal erythroid maturation and identify as a novel regulator of erythroblast enucleation.
2016-10-06 | GSE87127 | GEO
Project description:It is unclear how epigenetic changes regulate the induction of erythroid-specific genes during terminal erythropoiesis. Here we use global mRNA sequencing (mRNA-seq) and chromatin immunoprecipitation coupled to high-throughput sequencing (CHIP-seq) to investigate the changes that occur in mRNA levels, RNA Polymerase II (Pol II) occupancy and multiple post-translational histone modifications when erythroid progenitors differentiate into late erythroblasts. Among genes induced during this developmental transition, there was an increase in the occupancy of Pol II, the activation marks H3K4me2, H3K4me3, H3K9Ac and H4K16Ac, and the elongation methylation mark H3K79me2. In contrast, genes that were repressed during differentiation showed relative decreases in H3K79me2 levels yet had levels of Pol II binding and active histone marks similar to those in erythroid progenitors. We also found that relative changes in histone modification levels-in particular, H3K79me2 and H4K16ac-were most predictive of gene expression patterns. Our results suggest that in terminal erythropoiesis both promoter and elongation-associated marks contribute to the induction of erythroid genes, while gene repression is marked by changes in histone modifications mediating Pol II elongation. Our data maps the epigenetic landscape of terminal erythropoiesis and suggests that control of transcription elongation regulates gene expression during terminal erythroid differentiation. Mouse fetal liver cells are double-labeled for erythroid-specific TER119 and non erythroid-specific transferrin receptor (CD71) and then sorted by flow-cytometry. E14.5 fetal livers contain at least five distinct populations of cells (R1 through R5); as they progressively differentiate they gain TER119 and then gain and subsequently lose CD71. CFU-E cells and proerythroblasts make up the R1 population; R2 consists of proerythroblasts and early basophilic erythroblasts; R3 includes early and late basophilic erythroblasts; R4 is mostly polychromatophilic and orthochromatophilic erythroblasts; and R5 is comprised of late orthochromatophilic erythroblasts and reticulocytes. We have sorted for R2-R5 cells for RNA-seq experiment.
2011-09-13 | E-GEOD-32110 | biostudies-arrayexpress