Search

Objective To explore potential function of IKAROS family zinc finger 1 (Ikzf1) in terminal erythroid differentiation. Methods Single-cell regulatory network inference and clustering (SCENIC) was used to predict transcription factors of terminal erythroid differentiation based on single cell transcriptomes of human and mouse bone marrow erythroid cells. Expression of the predicted Ikzf1 during terminal erythroid differentiation was acquired from public RNA-seq data and validated by RT-qPCR. Short hairpin RNA (shRNA) interference was performed to knock down Ikzf1 in mouse erythroid cells from fetal liver with a GFP cassette to mark successfully transfected cells. The knockdown efficiency in GFP+ cells was verified by RT-qPCR. The effect of Ikzf1 knockdown on erythroid differentiation and enucleation was analyzed by flow cytometry. Results SCENIC predicted that the transcription factor Ikzf1 regulated both human and mouse erythroid differentiation. RNA-seq data and RT-qPCR showed highly expressed Ikzf1 at proerythroblast and basophilic erythroblast stages of terminal erythroid differentiation. Ikzf1 expression was indeed down-regulated by shRNA in erythroid cells from mouse fetal liver. Flow cytometry analysis showed that knockdown of Ikzf1 affected terminal differentiation and significantly reduced the ratio of erythroid enucleation (P＜ 0.05). Conclusions Transcription factor Ikzf1 is highly expressed in early stage erythroblasts and involved in terminal erythroid differentiation as well as enucleation.

Objective To explore potential function of IKAROS family zinc finger 1 (Ikzf1) in terminal erythroid differentiation. Methods Single-cell regulatory network inference and clustering (SCENIC) was used to predict transcription factors of terminal erythroid differentiation based on single cell transcriptomes of human and mouse bone marrow erythroid cells. Expression of the predicted Ikzf1 during terminal erythroid differentiation was acquired from public RNA-seq data and validated by RT-qPCR. Short hairpin RNA (shRNA) interference was performed to knock down Ikzf1 in mouse erythroid cells from fetal liver with a GFP cassette to mark successfully transfected cells. The knockdown efficiency in GFP+ cells was verified by RT-qPCR. The effect of Ikzf1 knockdown on erythroid differentiation and enucleation was analyzed by flow cytometry. Results SCENIC predicted that the transcription factor Ikzf1 regulated both human and mouse erythroid differentiation. RNA-seq data and RT-qPCR showed highly expressed Ikzf1 at proerythroblast and basophilic erythroblast stages of terminal erythroid differentiation. Ikzf1 expression was indeed down-regulated by shRNA in erythroid cells from mouse fetal liver. Flow cytometry analysis showed that knockdown of Ikzf1 affected terminal differentiation and significantly reduced the ratio of erythroid enucleation (P＜ 0.05). Conclusions Transcription factor Ikzf1 is highly expressed in early stage erythroblasts and involved in terminal erythroid differentiation as well as enucleation. [ABSTRACT FROM AUTHOR]

Objective To investigate the expression and function of protein tyrosine phosphatase 4A3(PTP4A3) in terminal erythroid differentiation and underlying molecular mechanism. Methods RT-qPCR was used to detect the expression of Ptp4a3 during terminal differentiation. Knockdown of Ptp4a3 expression in mouse fetal liver induced erythroid differentiation system by short hairpin RNA(shRNA) interference, the knockdown efficiency was verified by RT-qPCR after GFP positive cells were sorted by flow cytometry, and the effect of Ptp4a3 knockdown on erythroid differentiation and enucleation was detected by flow cytometry. Transcriptional profiling was performed to explore the potential downstream pathways of Ptp4a3. Results Ptp4a3 was among the top 100 ranked expressing genes during mouse terminal erythroid differentiation, and its expression gradually increased following the differentiation process. In mouse fetal liver-derived erythroid cells, Ptp4a3 was successfully knocked down by shRNA interference. Flow cytometry analysis showed that Ptp4a3 knockdown significantly decreased the proportion of enucleated erythrocytes(P＜0.05). Transcriptome analysis revealed that Ptp4a3 knockdown resulted in upregulation of cell cycle-related genes. Conclusions PTP4A3 is highly expressed in late stage of terminal erythroid differentiation. Knockdown Ptp4a3 significantly reduces enucleation erythroid of cells.

Anemia is the most prevalent nutrition-related disorder worldwide. Zinc is an essential trace element for various biological processes in the body, and zinc deficiency has been associated with anemia in humans. However, the molecular mechanisms by which zinc availability alters red blood cell development remain uncertain. The present study identifies the essentiality of zinc during erythroid development, particularly for normal heme biosynthesis. G1E-ER4 mouse cells were used as an in vitro model of terminal erythroid differentiation, which featured elevated cellular zinc content by development. Restriction of zinc import compromised the rate of heme and α-globin production and, thus, the hemoglobinization of the erythroid progenitors. Heme is synthesized by the incorporation of iron into protoporphyrin. The lower heme production under zinc restriction was not due to changes in iron but was attributable to less porphyrin synthesis. The requirement of adequate zinc for erythroid heme metabolism was confirmed in another erythropoietic cell model, MEL-DS19. Additionally, we found that a conventional marker of iron deficiency anemia, the ZnPP-to-heme ratio, responded to zinc restriction differently from iron deficiency. Collectively, our findings define zinc as an essential nutrient integral to erythroid heme biosynthesis and, thus, a potential therapeutic target for treating anemia and other erythrocyte-related disorders. [ABSTRACT FROM AUTHOR]

Anemia is the most prevalent nutrition-related disorder worldwide. Zinc is an essential trace element for various biological processes in the body, and zinc deficiency has been associated with anemia in humans. However, the molecular mechanisms by which zinc availability alters red blood cell development remain uncertain. The present study identifies the essentiality of zinc during erythroid development, particularly for normal heme biosynthesis. G1E-ER4 mouse cells were used as an in vitro model of terminal erythroid differentiation, which featured elevated cellular zinc content by development. Restriction of zinc import compromised the rate of heme and α-globin production and, thus, the hemoglobinization of the erythroid progenitors. Heme is synthesized by the incorporation of iron into protoporphyrin. The lower heme production under zinc restriction was not due to changes in iron but was attributable to less porphyrin synthesis. The requirement of adequate zinc for erythroid heme metabolism was confirmed in another erythropoietic cell model, MEL-DS19. Additionally, we found that a conventional marker of iron deficiency anemia, the ZnPP-to-heme ratio, responded to zinc restriction differently from iron deficiency. Collectively, our findings define zinc as an essential nutrient integral to erythroid heme biosynthesis and, thus, a potential therapeutic target for treating anemia and other erythrocyte-related disorders.

Fine-resolution differentiation trajectories of adult human hematopoietic stem cells (HSCs) involved in the generation of red cells is critical for understanding dynamic developmental changes that accompany human erythropoiesis. Using single-cell RNA sequencing (scRNA-seq) of primary human terminal erythroid cells (CD34-CD235a+) isolated directly from adult bone marrow (BM) and umbilical cord blood (UCB), we documented the transcriptome of terminally differentiated human erythroblasts at unprecedented resolution. The insights enabled us to distinguish polychromatic erythroblasts (PolyEs) at the early and late stages of development as well as the different development stages of orthochromatic erythroblasts (OrthoEs). We further identified a set of putative regulators of terminal erythroid differentiation and functionally validated three of the identified genes, AKAP8L, TERF2IP, and RNF10, by monitoring cell differentiation and apoptosis. We documented that knockdown of AKAP8L suppressed the commitment of HSCs to erythroid lineage and cell proliferation and delayed differentiation of colony-forming unit-erythroid (CFU-E) to the proerythroblast stage (ProE). In contrast, the knockdown of TERF2IP and RNF10 delayed differentiation of PolyE to OrthoE stage. Taken together, the convergence and divergence of the transcriptional continuums at single-cell resolution underscore the transcriptional regulatory networks that underlie human fetal and adult terminal erythroid differentiation. [ABSTRACT FROM AUTHOR]

Fine-resolution differentiation trajectories of adult human hematopoietic stem cells (HSCs) involved in the generation of red cells is critical for understanding dynamic developmental changes that accompany human erythropoiesis. Using single-cell RNA sequencing (scRNA-seq) of primary human terminal erythroid cells (CD34 - CD235a + ) isolated directly from adult bone marrow (BM) and umbilical cord blood (UCB), we documented the transcriptome of terminally differentiated human erythroblasts at unprecedented resolution. The insights enabled us to distinguish polychromatic erythroblasts (PolyEs) at the early and late stages of development as well as the different development stages of orthochromatic erythroblasts (OrthoEs). We further identified a set of putative regulators of terminal erythroid differentiation and functionally validated three of the identified genes, AKAP8L , TERF2IP , and RNF10 , by monitoring cell differentiation and apoptosis. We documented that knockdown of AKAP8L suppressed the commitment of HSCs to erythroid lineage and cell proliferation and delayed differentiation of colony-forming unit-erythroid (CFU-E) to the proerythroblast stage (ProE). In contrast, the knockdown of TERF2IP and RNF10 delayed differentiation of PolyE to OrthoE stage. Taken together, the convergence and divergence of the transcriptional continuums at single-cell resolution underscore the transcriptional regulatory networks that underlie human fetal and adult terminal erythroid differentiation., Competing Interests: The authors declare no competing interest.