Project description:To uncover how CHD8 regulates terminal erythropoiesis, we performed CUT&RUN assays to check the direct binding of CHD8 on the target genes in terminal erythroblasts. In addition, we perfromed RNA sequence in sorted ProEs from WT, P53 KO and Chd8 P53 DKO mice to identify the differentially expressed genes due to loss of CHD8.
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.
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.
Project description:This SuperSeries is composed of the following subset Series: GSE27893: Genome-wide maps of chromatin state in early erythroid precursors versus later, more differentiated erythroblasts. GSE32110: RNA-seq expression profiles during terminal erythropoiesis Refer to individual Series
Project description:The erythroblastic island (EBI), composed of a central macrophage and surrounding maturing erythroblasts, is the erythroid precursor niche. Despite numerous studies, its precise composition is still unclear. Using multispectral imaging flow cytometry (IFC), in vitro island reconstitution, and single cell RNA-seq of EBI-component cells enriched by gradient sedimentation, we present evidence that CD11b+ cells present in the EBIs are neutrophil precursors specifically associated with bone marrow EBI macrophages, indicating that erythro-(myelo)-blastic islands are a site for terminal granulopoiesis as well as erythropoiesis and production of these lineages is dynamically regulated within this niche. We further demonstrate that the balance between these lineages is determined by pathophysiological conditions, favoring granulopoiesis during anemia of inflammation, or erythropoiesis after erythropoietin (Epo) stimulation. Finally, we provide the heterogeneous molecular profiling of EBI macrophages as revealed by Cellular Indexing of Transcriptome and Epitopes (CITE)-sequencing of mouse bone marrow EBIs at baseline and after Epo-stimulation in vivo. Altogether, these data demonstrate that EBIs serve a dual role as terminal erythropoiesis and granulopoiesis niches and the central macrophages adapt to the needs of stress erythropoiesis as well as granulopoiesis.
Project description:To gain the whole picture of terminal erythroid differentiation, bone marrow erythroblasts of different maturation stages including proerythroblasts (Pro),basophilic erythroblasts (Baso), polychromatic erythroblasts (Poly) and orthochromatic erythroblasts (Ortho) were isolated and sorted.Transcriptomic analysis of these four stages of cells was performed.