scholarly journals Expression of ferrochelatase mRNA in erythroid and non-erythroid cells

1993 ◽  
Vol 292 (2) ◽  
pp. 343-349 ◽  
Author(s):  
R Y Y Chan ◽  
H M Schulman ◽  
P Ponka
Keyword(s):  
Sequence Similarity ◽  
Erythroid Differentiation ◽  
Polyadenylation Signal ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Erythroleukemia Cells ◽  
Haem Biosynthesis ◽  
Polyadenylation Sites ◽  
Dna Fragment ◽  
Polyadenylation Signals

Ferrochelatase, which catalyses the last step in haem biosynthesis, i.e. the insertion of Fe(II) into protophorphyrin IX, is present in all cells, but is particularly abundant in erythroid cells during haemoglobinization. Using mouse ferrochelatase cDNA as a probe two ferrochelatase transcripts, having lengths of 2.9 kb and 2.2 kb, were found in extracts of mouse liver, kidney, brain, muscle and spleen, the 2.9 kb transcript being more abundant in the non-erythroid tissues and the 2.2 kb transcript more predominant in spleen. In mouse erythroleukemia cells the 2.9 kb ferrochelatase transcript is also more abundant; however, following induction of erythroid differentiation by dimethyl sulphoxide there is a preferential increase in the 2.2 kb transcript, which eventually predominates. With mouse reticulocytes, the purest immature erythroid cell population available, over 90% of the total ferrochelatase mRNA is present as the 2.2 kb transcript. Since there is probably only one mouse ferrochelatase gene, the occurrence of two ferrochelatase transcripts could arise from the use of two putative polyadenylation signals in the 3′ region of ferrochelatase DNA. This possibility was explored by using a 389 bp DNA fragment produced by PCR with synthetic oligoprimers having sequence similarity with a region between the polyadenylation sites. This fragment hybridized only to the 2.9 kb ferrochelatase transcript, indicating that the two transcripts differ at their 3′ ends and suggesting that the 2.2 kb transcript results from the utilization of the upstream polyadenylation signal. The preferential utilization of the upstream polyadenylation signal may be an erythroid-specific characteristic of ferrochelatase gene expression.

Klf1 Regulatory Networks in Primary Erythroid Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1462-1462
Author(s):  
Michael Tallack ◽  
Thomas Whitington ◽  
Brooke Gardiner ◽  
Eleanor Wainwright ◽  
Janelle Keys ◽  
...  
Keyword(s):  
Regulatory Networks ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Es Cells ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Gene Promoters ◽  
Erythroid Cells ◽  
Red Cell Membrane ◽  
Sequencing Platform

Abstract Abstract 1462 Poster Board I-485 Klf1/Eklf regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which Klf1 works, we performed Klf1 ChIP-seq using the SOLiD deep sequencing platform. We mapped more than 10 million unique 35mer tags and found ∼1500 sites in the genome of primary fetal liver erythroid cells are occupied by endogenous Klf1. Many reside within well characterised erythroid gene promoters (e.g. b-globin) or enhancers (e.g. E2f2 intron 1), but some are >100kb from any known gene. We tested a number of Klf1 bound promoter and intragenic sites for activity in erythroid cell lines and zebrafish. Our data suggests Klf1 directly regulates most aspects of terminal erythroid differentiation including synthesis of the hemoglobin tetramer, construction of a deformable red cell membrane and cytoskeleton, bimodal regulation of proliferation, and co-ordination of anti-apoptosis and enucleation pathways. Additionally, we suggest new mechanisms for Klf1 co-operation with other transcription factors such as those of the gata, ets and myb families based on over-representation and spatial constraints of their binding motifs in the vicinity of Klf1-bound promoters and enhancers. Finally, we have identified a group of ∼100 Klf1-occupied sites in fetal liver which overlap with Klf4-occupied sites in ES cells defined by Klf4 ChIP-seq. These sites are associated with genes controlling the cell cycle and proliferation and are Klf4-dependent in skin, gut and ES cells, suggesting a global paradigm for Klfs as regulators of differentiation in many, if not all, cell types. Disclosures No relevant conflicts of interest to declare.

Studies on Heme Oxygenase 1 During Erythroid Differentiation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4254-4254
Author(s):  
Daniel Garcia Santos ◽  
Jesse Eisenberg ◽  
Matthias Schranzhofer ◽  
Prem Ponka
Keyword(s):  
Aminolevulinic Acid ◽  
Conflicts Of Interest ◽  
Tissue Injury ◽  
Erythroid Differentiation ◽  
The Body ◽  
Cellular Damage ◽  
Erythroid Cell ◽  
Heme Oxygenase 1 ◽  
Erythroid Cells ◽  
Murine Erythroleukemia

Abstract Abstract 4254 Heme is indispensable for the function of all aerobic cells as a prosthetic group of innumerable proteins. However, “free heme” (uncommitted) can initiate the formation of free radicals and cause lipid peroxidation, which can lead to cellular damage and tissue injury. Therefore, the rate of heme biosynthesis and catabolism must be well balanced by tight control mechanisms. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC), whose precursors synthesize heme with rates that are at least one order of magnitude higher (on the per cell basis) than those in the liver – the second most active heme producer in the body. The degradation of heme is exclusively carried out by heme oxygenases 1 and 2 (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using both a murine erythroleukemia cell line (MEL) and primary erythroid cells isolated from mouse fetal livers, we have demonstrated that during erythroid differentiation HO1 is up-regulated at both mRNA and protein levels. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase. These data suggest that in developing RBC, in addition to the continuous assembly of heme with globin chains, there is an increase in levels of uncommitted heme, which upregulates HO1 expression. Additionally, we have shown that down-regulation of HO1 via siRNA increased hemoglobinization in differentiating MEL cells. In contrast, induction of HO1 expression by NaAsO2 reduced the hemoglobinization of MEL cells. This effect could be reversed to control levels by the addition of HO1 inhibitor tin-protophorphyrin (SnPP). These results show that in differentiating erythroid cells the balance between levels of heme and HO1 have to be tightly regulated to maintain hemoglobinization at appropriate levels. Our results lead us to propose that disturbances in HO1 expression could play a role in some pathophysiological conditions such as thalassemias. Disclosures: No relevant conflicts of interest to declare.

Heme Oxygenase 1 Plays An Unexpected Role During Erythroid Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 344-344
Author(s):  
Daniel Garcia Santos ◽  
Matthias Schranzhofer ◽  
José Artur Bogo Chies ◽  
Prem Ponka
Keyword(s):  
Red Blood Cells ◽  
Heme Oxygenase ◽  
Blood Cells ◽  
Erythroid Differentiation ◽  
Heme Biosynthesis ◽  
Erythroid Cell ◽  
Heme Oxygenase 1 ◽  
Erythroid Cells ◽  
Wild Type ◽  
Protein Levels

Abstract Abstract 344 Red blood cells (RBC) are produced at a rate of 2.3 × 106 cells per second by a dynamic and exquisitely regulated process known as erythropoiesis. During this development, RBC precursors synthesize the highest amounts of total organismal heme (75–80%), which is a complex of iron with protoporphyrin IX. Heme is essential for the function of all aerobic cells, but if left unbound to protein, it can promote free radical formation and peroxidation reactions leading to cell damage and tissue injury. Therefore, in order to prevent the accumulation of ‘free' heme, it is imperative that cells maintain a balance of heme biosynthesis and catabolism. Physiologically, the only enzyme capable of degrading heme are heme oxyganase 1 & 2 (HO). Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. In contrast, virtually nothing is known about the expression of HO1 in developing RBC. Likewise, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using primary erythroid cells isolated from mouse fetal livers (FL), we have shown that HO1 mRNA and protein are expressed in undifferenetiated FL cells and that its levels, somewhat surprisingly, increase during erythropoietin-induced erythroid differentiation. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase, the second enzyme in the heme biosynthesis pathway. Moreover, we have found that down-regulation of HO1 via siRNA increases globin protein levels in DMSO-induced murine erythroleukemic (MEL) cells. Similarly, compared to wild type mice, FL cells isolated from HO1 knockout mice (FL/HO1−/−) exhibited increased globin and transferrin receptor levels and a decrease in ferritin levels when induced for differentiation with erythropoietin. Following induction, compared to wild type cells, FL/HO1−/− cells showed increased iron uptake and its incorporation into heme. We therefore conclude that the normal hemoglobinization rate appears to require HO1. On the other hand, MEL cells engineered to overexpress HO1 displayed reduced globin mRNA and protein levels when induced to differentiate. This finding suggests that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Recombinant Human Parvovirus B19 Vectors: Erythroid Cell-Specific Delivery and Expression of Transduced Genes

1998 ◽  
Vol 72 (6) ◽  
pp. 5224-5230 ◽  
Author(s):  
Selvarangan Ponnazhagan ◽  
Kirsten A. Weigel ◽  
Sudhanshu P. Raikwar ◽  
Pinku Mukherjee ◽  
Mervin C. Yoder ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Bone Marrow Cells ◽  
Parvovirus B19 ◽  
Leukemia Cell ◽  
Erythroid Differentiation ◽  
Leukemia Cell Line ◽  
Vector System ◽  
Erythroid Cell ◽  
Target Cells ◽  
Erythroid Cells

ABSTRACT A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562–566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111–1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and acquired human diseases affecting cells of erythroid lineage.

scholarly journals Lack of GDF11 Does Not Ameliorate Erythropoiesis in β-Thalassemia and Does Not Prevent the Activity of the Trap-Ligand RAP-536

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 165-165 ◽  
Author(s):  
Amaliris Guerra ◽  
Rea Oikonomidou ◽  
Sinha Gonzalez ◽  
Jianbing Zhang ◽  
Vania Lo Presti ◽  
...  
Keyword(s):  
Research Funding ◽  
Hematological Parameters ◽  
Genomic Medicine ◽  
Erythroid Differentiation ◽  
The United States ◽  
Phase Iii ◽  
Erythroid Cell ◽  
Published Data ◽  
Erythroid Cells ◽  
Cd34 Cells

Abstract Mutations in the HBB gene causes β-thalassemia (BT). Treatment for BT presents a major clinical challenge in the United States, as patients require chronic and expensive treatment for survival. A new drug in Phase III clinical trials, Luspatercept (ACE-536), has been shown to improve BT symptoms via an erythropoietin (EPO) -independent pathway. ACE-536 is a peptide drug identical to the extracellular domain of activin receptor IIB (ACVR2B). Upon administration, it competes with ACVR2B to bind members of the transforming growth factor (TGF) β superfamily. Growth differentiation factor 11 (GDF11) has been pinpointed as the primary target by which the trap ligand exerts its therapeutic efforts. Studies in murine models of BT using RAP-536 (the mouse analog of ACE-536), have suggested that Gdf11 is overexpressed in erythroblasts and that overexpression functions to inhibit erythroid differentiation. Interestingly, however, ACE-536 and RAP-536 have been shown to stimulate RBC synthesis in healthy humans and mice, where GDF11/Gdf11 overexpression has not been reported. Additionally, the expression data in mice has been questioned because of the unavailability of antibodies that can discriminate between Gdf11 and other TGF-β ligands. Due to the novelty of RAP-536 promoting erythropoiesis through an Epo-independent pathway and the lack of specific antibodies to distinguish between TGF-β ligands, we resorted to genetic tools to investigate the role of Gdf11 in erythropoiesis. For our study, we generated Hbb+/+Gdf11flox/floxand Hbbth3/+Gdf11flox/floxmice and crossed them with EpoRCre and VavCre transgenic lines, resulting in offspring harboring the Gdf11 deletion in erythroid cells and the complete hematopoietic compartment. If Gdf11 is secreted by erythroid cells, and it plays a role in inhibiting erythroid differentiation, then mice lacking Gdf11 in either erythroid cells or all hematopoietic cell lineages should show some increase in red blood cell (RBC) production, hemoglobin (Hb) and hematocrit (Hb). Furthermore, in Hbbth3/+mice, where Gdf11 has been proposed to be overexpressed, improvements in erythroid cell differentiation should be most apparent. Surprisingly, we did not detect any differences in RBC number, Hb or Hct levels of Gdf11 deficient Hbb+/+or Hbbth3/+mice compared to their Gdf11 containing controls. The discrepancy between our results and published data could be explained if Gdf11 is produced by non-hematopoietic tissues and indirectly influences erythropoiesis. Since Gdf11-/-are embryonic-lethal, we crossed Hbb+/+ Gdf11flox/floxand Hbbth3/+ Gdf11flox/flox mice with a tamoxifen (TAM) inducible Cre recombinase under the global Rosa26 promoter (RosaCre) to assess the effect of a pancellular deletion of Gdf11. No detectable differences were found in RBC, Hb or Hct levels of flthese animals after TAM treatment either acutely nor up to 5-6 months post deletion of Gdf11. Administration of RAP-536 significantly improved and increased hematopoietic parameters in the peripheral blood in all six models lacking Gdf11. In the RAP-536-treated Hbbth3/+models, amelioration of anemia was noted by a decrease in spleen size and improved ineffective erythropoiesis indicated by an increased hematological parameters and increased ratio of mature to immature erythroblasts in spleen analyzed by FACS. Therefore, lack of Gdf11 at the erythroid, hematopoietic and pancellular level did not prevent a response to the drug. Next, we investigated the effects of RAP-536 directly on erythroid cells. Since the drug causes increases in RBC and Hb of normal patients, we challenged CD34+ cells with RAP-536 at various concentrations. Results showed no increases in cell numbers, erythroid viability, hemoglobin content nor differentiation. Currently we are investigating the mRNA expression of activin receptors IIA and IIb along with TGF-β ligands in healthy and BT CD34+ cells as well as in erythroid specific progenitors of the Hbbth3/+ mouse model. Our findings suggest that Gdf11 is not the sole target of RAP-536, nor that Gdf11 is required to promote improvement of erythropoiesis. Most importantly, we show that in the absence of Gdf11, RAP-536 is effective at increasing hematological parameters in both Hbb+/+ and Hbbth3/+ mice. The results of this study demonstrate potential alternative target(s) for the action of RAP-536. Future work will focus on identifying the unknown targets of RAP-536. Disclosures Casu: Aevi Genomic Medicine, Inc: Research Funding; Ionis Pharmaceuticals, Inc.: Research Funding. Martinez:Acceleron Pharma: Employment. Suragani:Acceleron Pharma: Employment. Kumar:Acceleron Pharma: Employment. Rivella:Ionis Pharmaceuticals, Inc: Consultancy; Protagonist: Consultancy; Disc Medicine: Consultancy; MeiraGTx: Other: SAB.

Role of Atypical p50:p50:Bcl-3 NFκlB Complex in Differentiation of Erythroid Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 568-568 ◽  
Author(s):  
Stephen Sawyer ◽  
Jingchun Chen
Keyword(s):  
Dna Binding ◽  
Transcriptional Control ◽  
Biological Significance ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Necrosis Factor Alpha ◽  
Erythroid Progenitor ◽  
Tnf Α

Abstract We recently reported that mouse and human primary erythroid progenitor cells and erythroid cell lines synthesize and respond to Tumor Necrosis Factor-alpha (TNF-α). The nuclear transcriptional control complex, NFκlB is central in signaling downstream from TNF-α; so we began to study the function of NFκlB in erythroid cells. We made three very interesting initial findings: 1) first we found that NFκlB binding to DNA increased very slowly in HCD57 erythroid cells treated with erythropoietin (EPO, the hormone required for red blood cell development). An inhibitory effect of adding a neutralizing antibody to TNF-α on EPO-stimulated NFκlB DNA suggested this increase in NFκlB was due to TNF-α rather than direct EPO signaling. 2) We also found that NFκlB binding to DNA increased 10-fold or greater during erythroid differentiation. We found greatly increased NFκlB DNA binding in HCD57 cells that differentiated due to over-expression of JunB, F-MEL induced by DMSO, or human UT7-EPO or murine HCD57 cells induced to differentiate with hemin. 3) Surprisingly, we found that the NFκlB DNA binding complex in mouse primary erythroid cells and the erythroid cells lines tested was almost exclusively composed of the atypical p50/p50/Bcl3 NFκlB rather than the canonical p65/p50 or the non-canonical p65/p52 NFκlB. When we begin to study the biological significance of this atypical NFκlB in EPO-mediated erythroid differentiation in vivo using genetic tools, we found marked deficiencies in the development of erythroid cells in either the nfkb1−/− mice (p50−/−) or the bcl3 −/− mice. The nfkb1−/− mice were mildly anemic. The number of red blood cells in the circulation of these mice was statistically lower than in control mice. The number of CFU-e was also reduced in nfkb1−/− mice. Using the Ter-119 and CD71 staining method, we noted that proerythroblasts and immature erythroid cells increased and mature erythroblasts decreased in either non-anemic bone marrow or anemic spleens of nfkb1−/− mice. Forward scatter of Ter-119+ cells also showed an increased size of the average immature erythroid cell in the bone a marrow of nfkb1 −/− mice, suggesting a block in differentiation and continued cell cycling of the immature erythroblasts. Similar erythroid defects were observed in the spleens of anemic bcl-3−/− mice. nfkb1−/− mice and bcl-3−/− mice are also apparently unable to produce new reticulocytes as effectively as wild type mice after induction of anemia. Our working hypothesis is without expression of either p50 or Bcl-3 NFκlB proteins, immature erythroid cells continue to proliferate and ineffectively differentiate. In summary, the atypical p50/p50/Bcl-3 NFκlB complex appears necessary for maximal differentiation of immature erythroid cells.

LSD1 Plays an Important Role in GATA Switch during Erythroid Differentiation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4846-4846
Author(s):  
Yue Jin ◽  
Yidi Guo ◽  
Dongxue Liang ◽  
Yue Li ◽  
Zhe Li ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Regulatory Element ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Hematopoietic Stem ◽  
Mouse Es Cells ◽  
Murine Erythroleukemia

Abstract GATA factors play important role in hematopoiesis. In particular, GATA2 is critical for maintenance of hematopoietic stem and progenitor cells (HS/PCs) and GATA1 is required for erythropoiesis. GATA1 and GATA2 are expressed in reciprocal patterns during erythroid differentiation. It was shown that GATA1 occupied the -2.8Kb regulatory element and mediated repression of the GATA2 promoter in terminally differentiating erythroid cells. However, the detailed molecular mechanisms that control the enhancer/promoter activities of the GATA2 gene remain to be elucidated. In this report, we found that LSD1 and TAL1 co-localize at GATA2 1S promoter through ChIP and double-ChIP assays in murine erythroleukemia (MEL) cells. To further test whether LSD1 and its mediated H3K4 demethylation is important for repression of the GATA2 gene during erythroid differentiation, we silenced LSD1 expression in both MEL cells and mouse ES cells using retrovirus mediated shRNA knockdown and induced them to differentiate into erythroid cells with DMSO and EPO, respectively. GATA2 expression was elevated while the level of GATA1 was repressed by RT-qPCR. Furthermore, consistent with the GATA witch hypothesis, ChIP analysis revealed that the levels of H3K4me2 were increased at the GATA2 1S promoter.  In addition, knock-down of LSD1 in MEL cells results in inhibition of erythroid cell differenciation and attenuation of MEL cell proliferation and survival. Thus, our data reveal that LSD1 involved in control of terminal erythroid differentiation by regulating GATA switch. The LSD1 histone demethylase complex may be recruited to the GATA2 1S promoter by interacting with TAL1. The H3K4 demethylation activity of LSD1 leads to downregulation of the active H3K4m2 mark at the GATA2 promoter that alters chromatin structure and represses transcription of the GATA2 genes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Distinct miRNA Signatures and Networks Discern Fetal from Adult Erythroid Differentiation and Primary from Immortalized Erythroid Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3626
Author(s):  
Panayiota L. Papasavva ◽  
Nikoletta Y. Papaioannou ◽  
Petros Patsali ◽  
Ryo Kurita ◽  
Yukio Nakamura ◽  
...  
Keyword(s):  
Mirna Expression ◽  
Regulatory Networks ◽  
Target Genes ◽  
Human Peripheral Blood ◽  
Expression Patterns ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Small Rna Sequencing ◽  
Erythroid Cells ◽  
Mirna Regulation

MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.

Several distinct types of sequence elements are required for efficient mRNA 3' end formation in a pea rbcS gene

1992 ◽  
Vol 12 (12) ◽  
pp. 5406-5414
Author(s):  
B D Mogen ◽  
M H MacDonald ◽  
G Leggewie ◽  
A G Hunt
Keyword(s):  
Cauliflower Mosaic Virus ◽  
Polyadenylation Signal ◽  
Cis Element ◽  
Rbcs Gene ◽  
Sequence Elements ◽  
Plant Genes ◽  
Cleavage And Polyadenylation ◽  
Polyadenylation Sites ◽  
Upstream Element ◽  
Polyadenylation Signals

We have conducted an extensive linker substitution analysis of the polyadenylation signal from a pea rbcS gene. From these studies, we can identify at least two, and perhaps three, distinct classes of cis element involved in mRNA 3' end formation in this gene. One of these, termed the far-upstream element, is located between 60 and 120 nt upstream from its associated polyadenylation sites and appears to be largely composed of a series of UG motifs. A second, termed the near-upstream element, is more proximate to poly(A) sites and may be functionally analogous to the mammalian polyadenylation signal AAUAAA, even though the actual sequences involved may not be AAUAAA. The third possible class is the putative cleavage and polyadenylation site itself. We find that the rbcS-E9 far-upstream element can replace the analogous element in another plant polyadenylation signal, that from cauliflower mosaic virus, and that one near-upstream element can function with either of two poly(A) sites. Thus, these different cis elements are largely interchangeable. Our studies indicate that a cellular plant gene possesses upstream elements distinct from AAUAAA that are involved in mRNA 3' end formation and that plant genes probably have modular, multicomponent polyadenylation signals.

Suppression of Erythroid-Specific 5-Aminolevulinate Synthase Using Short-Interfering RNA Alters Iron Metabolism and Inhibits Terminal Differentiation of Human Erythroleukemia Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1632-1632
Author(s):  
Kazumichi Furuyama ◽  
Kiriko Kaneko ◽  
Zhang Yongzhao ◽  
Patrick D. Vargas V. ◽  
Shigeru Sassa ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Mrna Level ◽  
Critical Role ◽  
Erythroid Differentiation ◽  
Iron Accumulation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Aminolevulinate Synthase ◽  
Heme Synthesis ◽  
Tgf Β1

Abstract Erythroid-specific 5-aminolevulinate synthase (ALAS2) is the first and the rate limiting enzyme for heme biosynthesis in erythroid cells. ALAS2 plays a critical role in hemoglobin synthesis and erythrocyte maturation, since targeting the ALAS2 gene results in embryonic death in mice because of severe anemia. In humans, heritable mutations of the ALAS2 gene are responsible for X-linked sideroblastic anemia (XLSA). However, the effect of suppressed expression of ALAS2 on erythroid cell differentiation has not been examined in human cells. We therefore addressed this question, by stably suppressing ALAS2 mRNA with short-interfering RNA (siRNA) in a human erythroleukemia cell line, YN-1. After cloning of cells expressing low ALAS2 (ALAS2low cells), cells were induced to undergo erythroid differentiation by treatment with transforming growth factor beta1 (TGF-β1). Gene expression profiles of induced and uninduced cells were examined, including genes involved in globin synthesis and iron metabolism. Hemoglobin production, as judged by o-dianisidine staining, was significantly lower in ALAS2low cells than in control cells both before and after erythroid differentiation. Both alpha and gamma globin mRNA levels were also reduced in ALAS2low cells, compared with control cells. Decreased heme synthesis as well as reduced globin production in ALAS2low erythroid cells are consistent with our previous findings in murine erythroleukemia cells studied by antisense technology (Meguro K, et al. Blood86:940–948, 1995), and extends our previous conclusion on the critical role of ALAS2 in heme and globin formation to human erythroid cells. Transferrin receptor (TFR) mRNA level was decreased in ALAS2low cells, and remained low following TGF-β1 treatment, whereas its level was increased in control cells during erythroid differentiation, which reflects enhanced iron uptake by differentiated control cells. Decreased TFR mRNA level in ALAS2low cells may suggest iron accumulation, since TFR mRNA is known to be unstable when intracellular iron level is increased. Notably, mitochondrial ferritin (MtF) mRNA level was decreased in control cells after differentiation, reflecting utilization of mitochondrial iron for heme synthesis, but it did not change in ALAS2low cells following TGF-β1 treatment. As accumulation of MtF protein is known to occur in iron-overloaded erythroid cells of patients with XLSA, our finding also suggests that there may be intramitochondrial iron accumulation in ALAS2low cells even after differentiation. In contrast to MtF mRNA, the level of cytosolic ferritin heavy chain mRNA was similar both in ALAS2low cells and control cells. These findings suggest that MtF levels, rather than cytosolic ferritin levels, may be a sensitive and specific indicator for iron accumulation in mitochondria. This study shows the critical role of ALAS2 not only in heme synthesis and hemoglobin formation, but also in iron metabolism in erythroid cells during their cell differentiation. An ALAS2low erythroid cell line, such as ALAS2-suppressed YN-1, will provide a good model for the study of relationship between heme biosynthesis and iron metabolism during terminal differentiation of human erythroid cells.

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