scholarly journals Similarities and differences in the structure and function of 4.1G and 4.1R135, two protein 4.1 paralogues expressed in erythroid cells

2010 ◽  
Vol 432 (2) ◽  
pp. 407-416 ◽  
Author(s):  
Wataru Nunomura ◽  
Kengo Kinoshita ◽  
Marilyn Parra ◽  
Philippe Gascard ◽  
Xiuli An ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Erythroid Differentiation ◽  
Band 3 ◽  
Erythroid Cells ◽  
Protein 4.1 ◽  
Calcium Dependent ◽  
Calmodulin Binding ◽  
And Function ◽  
Glycophorin C ◽  
In Erythroid Cells

Membrane skeletal protein 4.1R is the prototypical member of a family of four highly paralogous proteins that include 4.1G, 4.1N and 4.1B. Two isoforms of 4.1R (4.1R135 and 4.1R80), as well as 4.1G, are expressed in erythroblasts during terminal differentiation, but only 4.1R80 is present in mature erythrocytes. Although the function of 4.1R isoforms in erythroid cells has been well characterized, there is little or no information on the function of 4.1G in these cells. In the present study, we performed detailed characterization of the interaction of 4.1G with various erythroid membrane proteins and the regulation of these interactions by calcium-saturated calmodulin. Like both isoforms of 4.1R, 4.1G bound to band 3, glycophorin C, CD44, p55 and calmodulin. While both 4.1G and 4.1R135 interact with similar affinity with CD44 and p55, there are significant differences in the affinity of their interaction with band 3 and glycophorin C. This difference in affinity is related to the non-conserved N-terminal headpiece region of the two proteins that is upstream of the 30 kDa membrane-binding domain that harbours the binding sites for the various membrane proteins. The headpiece region of 4.1G also contains a high-affinity calcium-dependent calmodulin-binding site that plays a key role in modulating its interaction with various membrane proteins. We suggest that expression of the two paralogues of protein 4.1 with different affinities for band 3 and glycophorin C is likely to play a role in assembly of these two membrane proteins during terminal erythroid differentiation.

GATA-1 Forms Distinct Activating and Repressive Complexes in Erythroid Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 356-356
Author(s):  
John Strouboulis ◽  
Patrick Rodriguez ◽  
Edgar Bonte ◽  
Jeroen Krijgsveld ◽  
Katarzyna Kolodziej ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Chromatin Remodeling ◽  
Gene Activation ◽  
Erythroid Differentiation ◽  
Specific Gene ◽  
Erythroid Cells ◽  
In Erythroid Cells

Abstract GATA-1 is a key transcription factor essential for the differentiation of the erythroid, megakaryocytic and eosinophilic lineages. GATA-1 functions in erythropoiesis involve lineage-specific gene activation and repression of early hematopoietic transcription programs. GATA-1 is known to interact with other transcription factors, such as FOG-1, TAL-1 and Sp1 and also with CBP/p300 and the SWI/SNF chromatin remodeling complex in vitro. Despite this information the molecular basis of its essential functions in erythropoiesis remains unclear. We show here that GATA-1 is mostly present in a high (> 670kDa) molecular weight complex that appears to be dynamic during erythroid differentiation. In order to characterize the GATA-1 complex(es) from erythroid cells, we employed an in vivo biotinylation tagging approach in mouse erythroleukemic (MEL) cells1. Briefly, this involved the fusion of a small (23aa) peptide tag to GATA-1 and its specific, efficient biotinylation by the bacterial BirA biotin ligase which is co-expressed with tagged GATA-1 in MEL cells. Nuclear extracts expressing biotinylated tagged GATA-1 were bound directly to streptavidin beads and co-purifying proteins were identified by mass spectrometry. In addition to the known GATA-1-interacting transcription factors FOG-1, TAL-1 and Ldb-1, we describe novel interactions with the essential hematopoietic transcription factor Gfi-1b and the chromatin remodeling complexes MeCP1 and ACF/WCRF. Significantly, GATA-1 interaction with the repressive MeCP1 complex requires FOG-1. We also show in erythroid cells that GATA-1, FOG-1 and MeCP1 are stably bound to repressed genes representing early hematopoietic (e.g. GATA-2) or alternative lineage-specific (e.g. eosinophilic) transcription programs, whereas the GATA-1/Gfi1b complex is bound to repressed genes involved in cell proliferation. In contrast, GATA-1 and TAL-1 are bound to the active erythroid-specific EKLF gene. Our findings on GATA-1 complexes provide novel insight as to the critical roles that GATA-1 plays in many aspects of erythropoiesis by revealing the GATA-1 partners in the execution of specific functions.

GATA-1-Dependent and -Independent Modes of Establishing the Erythroid Cell Genetic Network.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2083-2083
Author(s):  
Nathaniel James Pope ◽  
Emery H Bresnick
Keyword(s):  
Target Genes ◽  
Genetic Network ◽  
Cell Maturation ◽  
Mediator Complex ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Cell Type ◽  
Cell Type Specific ◽  
And Function ◽  
In Erythroid Cells

Abstract Abstract 2083 The constant physiological demand to generate large numbers of red blood cells requires a complex genetic network established by the master regulatory transcription factor GATA-1, which orchestrates erythroblast survival, proliferation, and differentiation. Many questions remain regarding how GATA-1 instigates genetic networks and to what extent GATA-1-independent mechanisms regulate erythropoiesis. Med1, a component of the broadly expressed Mediator complex (Mediator), facilitates GATA-1-dependent transcriptional activation at select target genes, although its contribution to GATA-1 function in cell-based assays is considerably less than that of the cell type-specific coregulator Friend of GATA-1. Med1-nullizygous mice have hematopoietic, cardiac, and vascular defects, though the underlying mechanisms are not defined. Furthermore, whether Med1 coactivator activity is dedicated to GATA-1 in erythroid cells and whether it controls numerous or a restricted cohort of genes is also unclear. Using a genetic complementation assay in GATA-1-null erythroid cells and a functional genomics approach, we demonstrate that Med1 regulates a restricted gene ensemble in erythroid cells, consisting predominantly of genes not controlled by GATA-1. Of the 265 Med1-regulated genes and 1054 GATA-1-regulated genes, only 35 genes were regulated by Med1 and GATA-1. Given the preponderance of GATA-1-independent Med1 targets, it is attractive to propose that Med1 has important GATA-1-independent functions required to exert its crucial hematopoietic activities. Since Med1 is a Mediator subunit, it is presumed to function through Mediator to regulate target genes. However, Med1 interacts with various trans-acting factors, and therefore its gene regulatory activity may not invariably rely on Mediator or a Mediator subcomplex. As Mediator is largely unstudied in erythroid cells, we asked whether Mediator subunit expression is regulated upon primary human erythroid cell maturation ex vivo. Mining the Human Erythroblast Maturation Database revealed that Med25 is strongly up-regulated during maturation. Knockdown of Med25 significantly dysregulated all ten of the highest responding Med1 target genes. Simultaneous knockdowns of Med1 and Med25 altered expression of 9 of the 10 top Med1 target genes, resembling the individual factor knockdowns. These results support the hypothesis that Med1 and Med25 function in the erythroid Mediator complex to regulate these genes. Med1 regulated these genes in a cell type-specific manner, as 8 of the 10 top Med1 targets in G1E-ER-GATA-1 proerythroblast-like cells and Mouse Erythroleukemia Cells were not dysregulated upon Med1 knockdown in Mouse Embryonic Fibroblasts. As Med1 modulated, but was not essential for, GATA-1-dependent transcription, we reasoned that certain Med1 target genes may exert GATA-1-independent activities to control erythroid cell development and/or function. The Med1 target gene Rrad encodes a small GTPase induced during primary human erythroid cell maturation, but its regulation/function has not been described in the hematopoietic system. Loss-of-function analysis in G1E-ER-GATA-1 cells indicated that Rrad confers survival. Knocking-down Rrad increased early apoptosis 2.5 fold (p < 0.05). The Rrad requirement for survival was more pronounced when cells were deprived of Erythropoietin (Epo) and Stem Cell Factor (SCF). In cells cultured without Epo, early apoptosis increased 7.0 fold upon Rrad knockdown [from 1.0% ± 0.1% to 7.2% ± 0.5% (p < 0.05)]. Removing SCF from the media significantly increased apoptotic cells, and Rrad knockdown elevated this further from 28% ± 2.4% to 46% ± 2.8% (p < 0.01), while the number of live cells decreased 4.7 fold (p < 0.01). These studies established a dual role for Mediator in erythroid cell regulation as a context-dependent GATA-1 coregulator and a GATA-1-independent regulator of cell type-specific genes, including potentially critical regulators of erythroid cell development, survival, and function. Mechanistically, given the greater than twenty components of the canonical Mediator, it will be particularly instructive to compare our findings to that of other key Mediator components, which shall yield a comprehensive understanding of their regulation and function during the progressive transitions from erythroid precursors to the erythrocyte. Disclosures: No relevant conflicts of interest to declare.

Elucidation of the Role of LMO2 (LIM-only protein 2) in Erythroid Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3443-3443
Author(s):  
AI Inoue ◽  
Tohru Fujiwara ◽  
Yoko Okitsu ◽  
Noriko Fukuhara ◽  
Yasushi Onishi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cord Blood ◽  
Western Blotting ◽  
Target Genes ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Dependent Manner ◽  
Erythroid Cells ◽  
In Erythroid Cells

Abstract Abstract 3443 Background: Developmental control mechanisms often utilize multimeric complexes containing transcription factors, coregulators, and additional non-DNA binding components. It is challenging to ascertain how such components contribute to complex function at endogenous loci. LMO2 (LIM-only protein 2) is a non-DNA binding transcriptional coregulator, and is an important regulator of hematopoietic stem cell development and erythropoiesis, as mice lacking this gene show defects in blood formation as well as fetal erythropoiesis (Warren et al. Cell. 1994). In the context of erythropoiesis, LMO2 has been demonstrated to be a part of multimetric complex, including master regulators of hematopoiesis (GATA-1 and SCL/TAL1), chromatin looping factor LDB1 and hematopoietic corepressor ETO2 (referred as GATA-SCL/TAL1 complex). As LMO2 controls hematopoiesis, its dysregulation is leukemogenic, and its influence on GATA factor function is still not evident, we investigated here the transcriptional regulatory mechanism via LMO2 in erythroid cells. Methods: For LMO2 knockdown, anti-LMO2 siRNA (Thermo Scientific Dharmacon) and pGIPZ lentiviral shRNAmir system (Open Biosystems) were used. Western blotting and Quantitative ChIP analysis were performed using antibodies for GATA-1, LMO2 (abcam), GATA-2, TAL1 and LDB1 (Santa Cruz). To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in liquid suspension culture. For transcription profiling, human whole expression array was used (Agilent), and the data was analyzed with GeneSpring GX software. To induce erythroid differentiation of K562 cells, hemin was treated at a concentration of 30 uM for 24h. Results: siRNA-mediated LMO2 knockdown in hemin-treated K562 cells results in significantly decreased ratio of benzidine-staining positive cells, suggesting that LMO2 has an important role in the erythroid differentiation of K562 cells. Next, we conducted microarray analysis to characterize LMO2 target gene ensemble in K562 cells. In contrast to the predominantly repressive role of LMO2 in murine G1E-ER-GATA-1 cells (Fujiwara et al. PNAS. 2010), the analyses (n = 2) demonstrated that 177 and 78 genes were upregulated and downregulated (>1.5-fold), respectively, in the LMO2-knockdowned K562 cells. Downregulated gene ensemble contained prototypical erythroid genes such as HBB and SLC4A1 (encodes erythrocyte membrane protein band 3). To test what percentages of LMO2-regulated genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n= 5,749, Fujiwara et al. Mol Cell. 2009), and demonstrated that 26.4% and 23.1% of upregulated and downregulated genes, respectively, contained significant GATA-1 peaks in their loci. Furthermore, whereas LMO2 knockdown in K562 cells did not affect the expression of GATA-1, GATA-2 and SCL/TAL1 based on quantitative RT-PCR as well as Western blotting, the knockdown resulted in the significantly decreased chromatin occupancy of GATA-1, GATA-2, SCL/TAL1 and LDB1 at beta-globin locus control region and SLC4A1 locus. We subsequently analyzed the consequences of LMO2 knockdown in primary erythroblasts. Endogeneous LMO2 expression was upregulated along with the differentiation of cord blood cell-derived primary erythroblasts. shRNA-mediated knockdown of LMO2 in primary erythroblasts resulted in significant downregulation of HBB, HBA and SLC4A1. Conclusion: Our results suggest that LMO2 contributes to the expression of GATA-1 target genes in a context-dependent manner, through modulating the assembly of the components of GATA-SCL/TAL1 complex at endogeneous loci. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Runx1 promotes murine erythroid progenitor proliferation and inhibits differentiation by preventing Pu.1 downregulation

2019 ◽  
Vol 116 (36) ◽  
pp. 17841-17847 ◽  
Author(s):  
Michael A. Willcockson ◽  
Samuel J. Taylor ◽  
Srikanta Ghosh ◽  
Sean E. Healton ◽  
Justin C. Wheat ◽  
...  
Keyword(s):  
Ectopic Expression ◽  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Chromatin Accessibility ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Down Regulation ◽  
Erythroid Progenitor ◽  
Terminal Erythroid Differentiation ◽  
In Erythroid Cells

Pu.1 is an ETS family transcription factor (TF) that plays critical roles in erythroid progenitors by promoting proliferation and blocking terminal differentiation. However, the mechanisms controlling expression and down-regulation of Pu.1 during early erythropoiesis have not been defined. In this study, we identify the actions of Runx1 and Pu.1 itself at the Pu.1 gene Upstream Regulatory Element (URE) as major regulators of Pu.1 expression in Burst-Forming Unit erythrocytes (BFUe). During early erythropoiesis, Runx1 and Pu.1 levels decline, and chromatin accessibility at the URE is lost. Ectopic expression of Runx1 or Pu.1, both of which bind the URE, prevents Pu.1 down-regulation and blocks terminal erythroid differentiation, resulting in extensive ex vivo proliferation and immortalization of erythroid progenitors. Ectopic expression of Runx1 in BFUe lacking a URE fails to block terminal erythroid differentiation. Thus, Runx1, acting at the URE, and Pu.1 itself directly regulate Pu.1 levels in erythroid cells, and loss of both factors is critical for Pu.1 down-regulation during terminal differentiation. The molecular mechanism of URE inactivation in erythroid cells through loss of TF binding represents a distinct pattern of Pu.1 regulation from those described in other hematopoietic cell types such as T cells which down-regulate Pu.1 through active repression. The importance of down-regulation of Runx1 and Pu.1 in erythropoiesis is further supported by genome-wide analyses showing that their DNA-binding motifs are highly overrepresented in regions that lose chromatin accessibility during early erythroid development.

scholarly journals Unequal synthesis and differential degradation of alpha and beta spectrin during murine erythroid differentiation.

1988 ◽  
Vol 107 (2) ◽  
pp. 413-426 ◽  
Author(s):  
M E Lehnert ◽  
H F Lodish
Keyword(s):  
Single Injection ◽  
Erythroid Differentiation ◽  
Band 3 ◽  
Cytoskeletal Proteins ◽  
Protein 4.1 ◽  
Stage Of Development ◽  
Alpha Spectrin ◽  
Actin Mrna ◽  
Murine Erythroleukemia ◽  
Differentiation Period

Murine erythroleukemia (MEL) cells represent a valuable system to study the biogenesis of the cytoskeleton during erythroid differentiation. When attached to fibronectin-coated dishes MEL cells induce, upon addition of DMSO, a 7-d differentiation process during which they enucleate and reach the reticulocyte stage (Patel, V. P., and H. F. Lodish. 1987. J. Cell Biol. 105:3105-3118); they accumulate band 3, spectrin, and ankyrin in amounts equivalent to those found in mature red blood cells. To follow the biosynthesis of spectrin during differentiation, membranes and cytoskeletal proteins of cells metabolically labeled with [35S]methionine were solubilized by SDS and alpha and beta spectrins were recovered by specific immunoadsorption. In both uninduced and 3-d induced cells, the relative synthesis of alpha/beta spectrin is approximately 1:3. In uninduced MEL cells newly synthesized alpha and beta spectrins are degraded with a similar half-life of approximately 10 h. In contrast, in 3-d differentiated MEL cells newly made beta spectrin is much more unstable than alpha spectrin; the half-lives of alpha and beta spectrin chains are approximately 22 and 8 h, respectively. Thus, accumulation of equal amounts of alpha and beta spectrin is caused by unequal synthesis and unequal degradation. As judged by Northern blot analyses, the level of actin mRNA is relatively constant throughout the 7-d differentiation period. alpha and beta spectrin mRNAs are barely detectable in uninduced cells, increase during the first 4 d of induction, and remain constant thereafter. In contrast, band 3 mRNA is first detectable on day 4 of differentiation. Thus, most of the spectrin that accumulates in enucleating reticulocytes is synthesized during the last few days of erythropoiesis, concomitant with the onset of band 3 synthesis. To determine whether this was occurring in normal mouse erythropoiesis, we analyzed the rate of appearance of labeled membrane proteins in mature erythrocytes after a single injection of [35S]methionine. Our results show that most of the spectrin and band 3 in mature erythrocytes is synthesized during the last days of bone marrow erythropoiesis, and that, in the marrow, band 3 and protein 4.1 are synthesized at a somewhat later stage of development than are alpha and beta spectrin, ankyrin, and actin.

Hydroxyurea Induced Expression of gamma Globin and Erythrocyte Membrane Proteins in Cultured Erythroid Progenitor Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3797-3797
Author(s):  
Gloria A. Green ◽  
Beau R. Braden ◽  
Obianuju Mba ◽  
Stacy A. Chivira ◽  
Laleh Ramezani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sickle Cell ◽  
Band 3 ◽  
Erythroid Cells ◽  
Band 3 Protein ◽  
Erythroid Progenitors ◽  
Gamma Globin ◽  
Mouse Antibody ◽  
Reactive Mechanism ◽  
In Erythroid Cells

Abstract BACKGROUND: Hydroxyurea (HU) an S-phase specific cytotoxic agent has been used for the treatment of patients with sickle cell anemia and beta-thalassemia. The clinical efficacy of HU is due primarily to increases in fetal hemoglobin (HbF) levels. HU increases the %HbF and the %F cells. The HU reactive mechanism(s) in erythroid cells, however, have not been clearly defined. Patients receiving HU therapy develop subpopulations of macrocytic erythrocytes. Our previous studies demonstrate that sickle cell patients treated with low dose HU develop subpopulations of RBCs that express greater relative levels of the erythrocyte anion exchange protein (AE1) per cell as compared with untreated individuals. The frequency of cells expressing greater levels of (AE1) per cell was increased in each serial blood sample. We propose that part of the HU reactive mechanism will include the upmodulation of non-gamma globin erythroid proteins that contribute to the macrocytic structures. As part of our investigation of the development of RBCs expressing increased band 3 protein per cell, we examined the possibility that HU induced AE1 synthesis can be detected in vitro using cultured erythroid progenitors. METHODS: HU induced protein synthesis was investigated as a function of HU concentration. Erythroid progenitors were cultured in serum free semisolid media containing different concentrations of HU [0–40 micromolar]. BFU-E were scored and harvested at day15 in culture, then assayed. BFU-E derived cells were first labeled with monoclonal anti-band 3 antibody. The change in the frequency of cells positive for band 3 protein was determined by flow cytometry. In separate studies BFU-E derived cells were fixed, permeabilized, labeled with anti-spectrin. These cells were labeled with monoclonal anti-band 3 and PE-labeled anti-mouse antibody. The labeled cells were assayed by confocal microscopy. For the next set of experiments, BFU-E cultured in increasing concentrations of HU were assayed for the presence of band 3 protein and gamma globin. BFU-E cells were fixed, permeabilized, and then labeled with tricolor-conjugated-anti-gamma globin. These cells were labeled with monoclonal anti-band 3 and PE-labeled anti-mouse antibody; then assayed by flow cytometry. RESULTS: Results show that the frequency of cells positive for band 3 protein [AE1] was increased at increasing concentrations of HU as compared to controls. The band-3 upmodulation appears to start to plateau at 12.5 micromolar HU. Band 3 protein derived from BFUE was confirmed by Western blot analysis. The frequency of cells positive for both band 3 and spectrin was similarly increased in cells cultured in the presence of increasing concentrations of HU. In separate studies, cells were assayed for the expression of both gamma globin and AE1. Results show a 2–3-fold increase in the % band 3 [AE1] plus gamma globin positive cells in HU [5–40 micromolar] treated cells as compared to untreated controls. CONCLUSIONS: These results demonstrate that HU induces the expression of band 3 and gamma globin in cultured erythroid progenitors. These findings suggest that part of the mechanism of HU action in erythroid cells involves the induction of erythroid structural proteins concordant with the induction of gamma globin.

scholarly journals Erythroid anion transporter assembly is mediated by a developmentally regulated recruitment onto a preassembled membrane cytoskeleton.

1987 ◽  
Vol 105 (3) ◽  
pp. 1405-1416 ◽  
Author(s):  
J V Cox ◽  
J H Stack ◽  
E Lazarides
Keyword(s):  
Steady State ◽  
Embryonic Development ◽  
Binding Sites ◽  
Band 3 ◽  
Erythroid Cells ◽  
Developmentally Regulated ◽  
Protein 4.1 ◽  
Membrane Cytoskeleton ◽  
Anion Transporter ◽  
Time Band

Analysis of the expression and assembly of the anion transporter by metabolic pulse-chase and steady-state protein and RNA measurements reveals that the extent of association of band 3 with the membrane cytoskeleton varies during chicken embryonic development. Pulse-chase studies have indicated that band 3 polypeptides do not associate with the membrane cytoskeleton until they have been transported to the plasma membrane. At this time, band 3 polypeptides are slowly recruited, over a period of hours, onto a preassembled membrane cytoskeletal network and the extent of this cytoskeletal assembly is developmentally regulated. Only 3% of the band 3 polypeptides are cytoskeletal-associated in 4-d erythroid cells vs. 93% in 10-d erythroid cells and 36% in 15-d erythroid cells. This observed variation appears to be regulated primarily at the level of recruitment onto the membrane cytoskeleton rather than by different transport kinetics to the membrane or differential turnover of the soluble and insoluble polypeptides and is not dependent upon the lineage or stage of differentiation of the erythroid cells. Steady-state protein and RNA analyses indicate that the low levels of cytoskeletal band 3 very early in development most likely result from limiting amounts of ankyrin and protein 4.1, the membrane cytoskeletal binding sites for band 3. As embryonic development proceeds, ankyrin and protein 4.1 levels increase with a concurrent rise in the level of cytoskeletal band 3 until, on day 10 of development, virtually all of the band 3 polypeptides are cytoskeletal bound. After day 10, the levels of total and cytoskeletal band 3 decline, whereas ankyrin and protein 4.1 continue to accumulate until day 18, indicating that the cytoskeletal association of band 3 is not regulated solely by the availability of membrane cytoskeletal binding sites at later stages of development. Thus, multiple mechanisms appear to regulate the recruitment of band 3 onto the erythroid membrane cytoskeleton during chicken embryonic development.

cAMP and in vivo hypoxia inducetob,ifr1, andfosexpression in erythroid cells of the chick embryo

2002 ◽  
Vol 282 (4) ◽  
pp. R1219-R1226 ◽  
Author(s):  
Stefanie Dragon ◽  
Nina Offenhäuser ◽  
Rosemarie Baumann
Keyword(s):  
Adaptive Modulation ◽  
Erythroid Differentiation ◽  
Cell Types ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Erythroid Cells ◽  
Proliferation And Differentiation ◽  
Key Events ◽  
In Erythroid Cells

During avian embryonic development, terminal erythroid differentiation occurs in the circulation. Some of the key events, such as the induction of erythroid 2,3-bisphosphoglycerate (2,3-BPG), carbonic anhydrase (CAII), and pyrimidine 5′-nucleotidase (P5N) synthesis are oxygen dependent (Baumann R, Haller EA, Schöning U, and Weber M, Dev Biol 116: 548–551, 1986; Dragon S and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 280: R870–R878, 2001; Dragon S, Carey C, Martin K, and Baumann R, J Exp Biol202: 2787–2795, 1999; Dragon S, Glombitza S, Götz R, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R982–R989, 1996; Dragon S, Hille R, Götz R, and Baumann R, Blood 91: 3052–3058, 1998; Million D, Zillner P, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 261: R1188–R1196, 1991) in an indirect way: hypoxia stimulates the release of norepinephrine (NE)/adenosine into the circulation (Dragon et al., J Exp Biol 202: 2787–2795, 1999; Dragon et al., Am J Physiol Regulatory Integrative Comp Physiol 271: R982–R989, 1996). This leads via erythroid β-adrenergic/adenosine A2receptor activation to a cAMP signal inducing several proteins in a transcription-dependent manner (Dragon et al., Am J Physiol Regulatory Integrative Comp Physiol 271: R982–R989, 1996; Dragon et al., Blood 91: 3052–3058, 1998; Glombitza S, Dragon S, Berghammer M, Pannermayr M, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R973–R981, 1996). To understand how the cAMP-dependent processes are initiated, we screened an erythroid cDNA library for cAMP-regulated genes. We detected three genes that were strongly upregulated (>5-fold) by cAMP in definitive and primitive red blood cells. They are homologous to the mammalian Tob, Ifr1, and Fos proteins. In addition, the genes are induced in the intact embryo during short-term hypoxia. Because the genes are regulators of proliferation and differentiation in other cell types, we suggest that cAMP might promote general differentiating processes in erythroid cells, thereby allowing adaptive modulation of the latest steps of erythroid differentiation during developmental hypoxia.

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