Mice over-expressing human erythropoietin indicate that erythropoietin enhances expression of its receptor via up-regulated Gata1 and Tal1 | Haematologica

摘要

The development of medullary hematopoiesis is characterized by a specific expression profile of hematopoietic transcription factors, including GATA transcription factors. At mid-gestation, when hematopoiesis is newly established in the bone marrow of human fetuses, initially high GATA2 expression becomes subsequently down-regulated, while GATA1 expression increases in parallel.1 Both transcription factors bind to overlapping sets of hematopoietic downstream target genes, often at distinct sites, to regulate the balance between proliferation and differentiation. Chromatin occupancy by GATA1 and GATA2 can change in the course of hematopoietic differentiation, leading to the so-called GATA switch.2 Thus, a spatio-temporal regulation of GATA1 or GATA2 activities is required within lineage-specific differentiation. During erythroid differentiation GATA1 expression peaks at the level of colony-forming units (CFU-E),3 where erythropoietin (Epo) exerts most specifically its effects, but blocks terminal maturation if constitutively overexpressed.4 In CFU-E progenitors, the Epo receptor (EpoR) gene is a prerequisite downstream target of GATA1 that activates EpoR expression in concert with several co-factors.5 Notably, EpoR-mediated signals in turn strongly enhance GATA1 gene expression in erythroid progenitor cells in vitro.5,6 There is also in vitro evidence that Epo induces EpoR expression by activating the GATA1-mediated EpoR transcription.5An alternate link between Epo and excessive erythropoiesis, which includes GATA1 activity, is given by the basic helix-loop-helix protein TAL1 (T-cell Acute Leukemia-1 transcription factor).7 In vitro, Epo stimulates expression of TAL1 and phosphorylation of its protein products.8 TAL1 directly up-regulates EpoR transcription and increases by nucleosome shifting the association of a transcription factor complex that includes GATA1, TAL1, LMO2 (Lim-Only Protein-2) and LDB2 (Lim-Domain Binding Protein 2), to a regulatory domain in the 5′ untranslated region of the EpoR gene.7 In this way, TAL1 causes hypersensitivity to Epo and promotes excessive erythrocytosis.Transgenic mice, constitutively over-expressing the human EPO (hEPO) gene (tg6 mice), represent a valuable model to further elucidate the in vivo implication of Epo in fine-tuning transcriptional activities that may modulate EpoR expression and explain the gradual increase in erythropoiesis from normal hematocrit levels at birth to maximum hematocrit levels of up to 90% after several weeks.9Our analysis indicates that the two erythroid master regulators Gata1 and Tal1 co-operatively act as developmental-stage specific enhancers of EpoR expression in response to constitutive EPO overexpression.While Gata1 mRNA expression in the newly established bone marrow of wild-type mice declined with increasing postnatal age (Figure 1A), its expression remained on a constant and significantly higher level in hEPO over-expressing tg6 mice. However, Gata2 mRNA expression remained similar in hEPO over-expressing tg6 mice compared to controls throughout all ages (Figure 1B). In hEpo over-expressing tg6 mice, EpoR mRNA expression significantly increased with age, but declined in control animals (Figure 1C).Download figureOpen in new tabDownload powerpointFigure 1. Longitudinal expression of Gata1, Gata2 and EpoR mRNA transcripts in the bone marrow of tg6 and wild-type mice (at postnatal Day 7 (d7), d21, d49). The transgenic mouse line tg6, in which the hEPO transgene is transmitted in autosomal dominant manner, was bred at the Institute of Veterinary Physiology, University of Zurich. The protocol was approved by the local Institutional Review Board and all procedures were performed according to the Swiss Animal Protection Law. To obtain hematopoietic cells from the bone marrow, femurs and tibias of 7, 21 and 49 day-old mice were prepared and flushed with ice-cold PBS. Bone marrow specimens of each single animal were pooled. Total mRNA was purified following t