Merav Socolovsky

Fetal Anemia and Apoptosis of Red Cell Progenitors in Stat5a−/−5b−/− Mice: A Direct Role for Stat5 in Bcl-XL Induction

The erythropoietin receptor (EpoR) is essential for production of red blood cells; a principal function of EpoR is to rescue committed erythroid progenitors from apoptosis. Stat5 is rapidly activated following EpoR stimulation, but its function in erythropoiesis has been unclear since adult Stat5a-/-5b-/- mice have normal steady-state hematocrit. Here we show that Stat5 is essential for the high erythropoietic rate during fetal development. Stat5a-/-5b-/- embryos are severely anemic; erythroid progenitors are present in low numbers, show higher levels of apoptosis, and are less responsive to Epo. These findings are explained by a crucial role for Stat5 in EpoRs antiapoptotic signaling: it mediates the immediate-early induction of Bcl-X(L) in erythroid cells through direct binding to the Bcl-X promoter.

Genetic analysis of BRCA1 function in a defined tumor cell line.

Retrovirally expressed, wild-type BRCA1 decreased the gamma radiation (IR) sensitivity and increased the efficiency of double-strand DNA break repair (DSBR) of the BRCA1-/- human breast cancer line, HCC1937. It also reduced its susceptibility to DSB generation by IR. By contrast, multiple, clinically validated, missense mutant BRCA1 products were nonfunctional in these assays. These data constitute the basis for a BRCA1 functional assay and suggest that efficient repair of double-strand DNA breaks is linked to BRCA1 tumor suppression function.

Ligand-independent oligomerization of cell-surface erythropoietin receptor is mediated by the transmembrane domain

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) is crucial for production of mature red cells. Although it is well established that the Epo-bound EpoR is a dimer, it is not clear whether, in the absence of ligand, the intact EpoR is a monomer or oligomer. Using antibody-mediated immunofluorescence copatching (oligomerizing) of epitope-tagged receptors at the surface of live cells, we show herein that a major fraction of the full-length murine EpoR exists as preformed dimers/oligomers in BOSC cells, which are human embryo kidney 293T-derived cells. This observed oligomerization is specific because, under the same conditions, epitope-tagged EpoR did not oligomerize with several other tagged receptors (thrombopoietin receptor, transforming growth factor β receptor type II, or prolactin receptor). Strikingly, the EpoR transmembrane (TM) domain but not the extracellular or intracellular domains enabled the prolactin receptor to copatch with EpoR. Preformed EpoR oligomers are not constitutively active and Epo binding was required to induce signaling. In contrast to tyrosine kinase receptors (e.g., insulin receptor), which cannot signal when their TM domain is replaced by the strongly dimerizing TM domain of glycophorin A, the EpoR could tolerate the replacement of its TM domain with that of glycophorin A and retained signaling. We propose a model in which TM domain-induced dimerization maintains unliganded EpoR in an inactive state that can readily be switched to an active state by physiologic levels of Epo.

Molecular insights into stress erythropoiesis.

Purpose of reviewIn addition to its essential role in baseline erythropoiesis, the hormone erythropoietin drives the erythropoietic response to hypoxic stress. A mechanistic understanding of stress erythropoiesis would benefit multiple clinical settings, and may aid in understanding leukemogenesis. Recent findingsThe spectrum of progenitors targeted by the erythropoietin receptor is broader during stress than during baseline erythropoiesis. Further, the requirement for erythropoietin receptor signaling is more stringent during stress. However, erythropoietin receptor signaling has been mostly studied in vitro, where it is difficult to relate signaling events to stress-dependent changes in erythroid homeostasis. Here we review advances in flow cytometry that allow the identification and study of murine erythroid precursors in hematopoietic tissue as they are responding to stress in vivo. The death receptor Fas and its ligand, FasL, are coexpressed by early splenic erythroblasts, suppressing erythroblast survival and erythropoietic rate. During stress, erythropoietin receptor signaling downregulates erythroblast Fas and FasL, consequently increasing erythropoietic rate. SummaryErythropoietic rate is regulated at least in part through the erythropoietin receptor-mediated survival of splenic early erythroblasts. Future research will delineate how multiple antiapoptotic pathways, potentially activated by the erythropoietin receptor, interact to produce the remarkable dynamic range of erythropoiesis.

Global DNA Demethylation During Mouse Erythropoiesis in Vivo

Erythroblasts undergoing differentiation into red cells lose one-third of DNA methylation marks at nearly all genomic loci. In the mammalian genome, 5′-CpG-3′ dinucleotides are frequently methylated, correlating with transcriptional silencing. Genome-wide demethylation is thought to occur only twice during development, in primordial germ cells and in the pre-implantation embryo. These demethylation events are followed by de novo methylation, setting up a pattern inherited throughout development and modified only at tissue-specific loci. We studied DNA methylation in differentiating mouse erythroblasts in vivo by using genomic-scale reduced representation bisulfite sequencing (RRBS). Demethylation at the erythroid-specific β-globin locus was coincident with global DNA demethylation at most genomic elements. Global demethylation was continuous throughout differentiation and required rapid DNA replication. Hence, DNA demethylation can occur globally during somatic cell differentiation, providing an experimental model for its study in development and disease.

The Prolactin Receptor and Severely Truncated Erythropoietin Receptors Support Differentiation of Erythroid Progenitors

Activation of the erythropoietin receptor is essential for the survival, proliferation, and differentiation of erythroid progenitors. To understand the role of erythropoietin receptor (EpoR) activation in erythroid differentiation, we infected primary erythroid progenitors with high-titer retrovirus encoding the non-hematopoietic prolactin receptor. The infected progenitors responded to prolactin in the absence of Epo by generating fully differentiated erythroid colonies. Therefore, differentiation of erythroid progenitors does not require an intracellular signal generated uniquely by the EpoR; the EpoR does not have an instructive role in erythroid differentiation. We also infected primary erythroid progenitors with retrovirus encoding chimeric receptors containing the extracellular domain of PrlR and the intracellular domain of either the wild-type or truncated EpoRs. A chimeric receptor containing only the membrane-proximal 136 amino acids of the EpoR cytoplasmic domain efficiently supported prolactin-dependent differentiation of erythroid progenitors. Substitution of the single cytoplasmic domain tyrosine in this receptor with phenylalanine (Y343F) eliminated its ability to support differentiation. The minimal EpoR cytoplasmic domain required for erythroid differentiation is therefore the same as that previously reported to be sufficient to support cell proliferation (D’Andrea, A. D., Yoshimura, A., Youssoufian, H., Zon, L. I., Koo, J. W., and Lodish, H. F. (1991) Mol. Cell. Biol. 11, 1980–1987; Miura, O., D’Andrea, A. D., Kabat, D., and Ihle, J. N. (1991) Mol. Cell. Biol. 11, 4895–4902; He, T.-C., Jiang, N., Zhuang, H., Quelle, D. E., and Wojchowski, D. M. (1994) J. Biol. Chem. 269, 18291–18294).

A Key Commitment Step in Erythropoiesis Is Synchronized with the Cell Cycle Clock through Mutual Inhibition between PU.1 and S-Phase Progression

During red blood cell development, differentiation and cell cycle progression are intimately and uniquely linked through interdependent mechanisms involving the erythroid transcriptional suppressor PU.1 and the cyclin-dependent kinase inhibitor p57KIP2.

Rb and N-ras Function Together To Control Differentiation in the Mouse

ABSTRACT The product of the retinoblastoma tumor suppressor gene (Rb) can control cell proliferation and promote dif-ferentiation. Murine embryos nullizygous for Rb die midgestation with defects in cell cycle regulation, control of apoptosis, and terminal differentiation of several tissues, including skeletal muscle, nervous system, and lens. Previous cell culture-based experiments have suggested that the retinoblastoma protein (pRb) and Ras operate in a common pathway to control cellular differentiation. Here we have tested the hypothesis that the proto-oncogene N-ras participates in Rb-dependent regulation of differentiation by generating and characterizing murine embryos deficient in both N-ras and Rb. We show that deletion of N-ras rescues a unique subset of the developmental defects associated with nullizygosity of Rb, resulting in a significant extension of life span. Rb−/−; N-ras −/− skeletal muscle has normal fiber density, myotube length and thickness, in contrast to Rb-deficient embryos. Additionally, Rb −/−; N-ras −/− muscle shows a restoration in the expression of the late muscle-specific gene MCK, and this correlates with a significant potentiation of MyoD transcriptional activity in Rb −/−; N-ras −/−, compared to Rb −/− myoblasts in culture. The improved differentiation of skeletal muscle in Rb −/−; N-ras −/− embryos occurs despite evidence of deregulated proliferation and apoptosis, as seen in Rb-deficient animals. Our findings suggest that the control of differentiation and proliferation by Rb are genetically separable.

Transgenic Analysis of the Stem Cell Leukemia +19 Stem Cell Enhancer in Adult and Embryonic Hematopoietic and Endothelial Cells

Appropriate transcriptional regulation is critical for the biological functions of many key regulatory genes, including the stem cell leukemia (SCL) gene. As part of a systematic dissection of SCL transcriptional regulation, we have previously identified a 5,245‐bp SCL +18/19 enhancer that targeted embryonic endothelium together with embryonic and adult hematopoietic progenitors and stem cells (HSCs). This enhancer is proving to be a powerful tool for manipulating hematopoietic progenitors and stem cells, but the design and interpretation of such transgenic studies require a detailed understanding of enhancer activity in vivo. In this study, we demonstrate that the +18/19 enhancer is active in mast cells, megakaryocytes, and adult endothelium. A 644‐bp +19 core enhancer exhibited similar temporal and spatial activity to the 5,245‐bp +18/19 fragment both during development and in adult mice. Unlike the +18/19 enhancer, the +19 core enhancer was only active in adult mice when linked to the eukaryotic reporter gene human placental alkaline phosphatase. Activity of a single core enhancer in HSCs, endothelium, mast cells, and megakaryocytes suggests possible overlaps in their respective transcriptional programs. Moreover, activity in a proportion of thymocytes and other SCL‐negative cell types suggests the existence of a silencer elsewhere in the SCL locus.

Tyrosine Residues within the Intracellular Domain of the Erythropoietin Receptor Mediate Activation of AP-1 Transcription Factors

Binding of erythropoietin (Epo) to the Epo receptor (EpoR) initiates a signaling cascade resulting in tyrosine phosphorylation of several proteins and induction of AP-1 transcription factor(s). While Epo is known to activate c-fos gene expression, the mechanism of AP-1 activation is unknown. Here we show that AP-1 activation by Epo requires tyrosine kinase activity and alsode novo protein synthesis. Using a mutant EpoR containing no cytosolic tyrosine residues, and a set of eight mutants containing a single cytosolic tyrosine residue, we show that multiple EpoR tyrosines, thought to activate multiple intracellular signal transduction proteins, can mediate AP-1 activation. An EpoR containing only tyrosine 343 or tyrosine 464 supports a maximal level of AP-1 activation. We also show that AP-1 activation does not require maximal STAT5 activation and may occur via a STAT5-independent signaling pathway.