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

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.65 There is also in vitro evidence that Epo induces EpoR expression by activating the GATA1-mediated EpoR transcription.5

An 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.9

Our 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).

Figure 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 the TRIzol protocol (Invitrogen, Carlsbad, CA, USA), and cDNA was generated by a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). 5′-FAM-labeled probes were used for detection of murine Gata1 (Assay ID: Mm00484678_m1), Gata2 (Mm00492301_m1) or EpoR mRNA (Mm00438760_m1) transcripts and β-actin (ACTB 4352664-0602004; all from Applied Biosystems) by real-time PCR technology with TaqMan 2× Universal PCR Mastermix Mix as recommended in the manufacturer’s protocol (Applied Biosystems). Data (mean and standard deviation) are presented as ratio between Gata1, Gata2 or EpoR mRNA transcript and β-actin mRNA transcript levels (n=4–6 sets). All reactions were performed in duplicates, and all four transcripts were analyzed in parallel. Statistical significance was examined by one-way ANOVA post hoc comparisons. P<0.05 was considered significant. *P<0.05; ***P<0.001.

The significant upregulation of Gata1 and EpoR mRNA expression in hEPO over-expressing tg6 mice was confirmed by analyzing the spleen as a major source of hematopoiesis (Figure 2A and B). To further dissect the complexity of changes in the transcriptional network, the analysis of Myb mRNA expression served as marker for adult definitive erythroblasts,10 showing significantly reduced expression in hEPO over-expressing tg6 mice (Figure 2B). In contrast, expression levels of both Tal1 and Lmo2 were significantly up-regulated in hEPO over-expressing tg6 mice (Figure 2A).

Figure 2.(A and B) Expression analyses of Gata1, Lmo2, Tal1 (A), and Myb and EpoR mRNA transcripts (B) in the spleen of 6- to 8-week-old tg6 and wild-type mice (n=4 sets). (C) Relation between Gata1 and Tal1 mRNA expression in the bone marrow of tg6 and wild-type mice at different ages (postnatal Day 7 (d7), d21, d49). Q-PCR analysis was performed as described above using TaqMan Gene Expression Assays for Gata1 (Mm00484678_m1), Lmo2 (Mm01281680_m1), Tal1 (Mm01187033_m1) as well as Myb (Mm00501741_m1). All mRNA row data were normalized to β-actin transcript levels. Statistical significances were determined by using the unpaired t-test. (n=2 pairs; *P<0.05; **P<0.01: ***P<0.001).

The longitudinal analysis in the developing bone marrow also indicated increasing Tal1 mRNA levels, which were tightly correlated with Gata1 mRNA expression (Figure 2C).

The combined data confirm that Epo recruits the erythroid transcriptional network to enhance its erythropoietic effect by mechanisms that directly induce EpoR upregulation and hypersensitivity to Epo in vivo. While such effects, predominately mediated by Gata1 and Tal1, are attributed to the stage of BFU-E (burst-forming unit erythoid) and CFU-E, the reduced expression of Myb in the hEPO over-expressing tg6 mice may reflect its prerequisite role for Epo-induced differentiation commitment.11 However, evidence is given that Gata1 protein induces EpoR expression by activating the 5′ EpoR promoter.5 In response to acute anemic stress, the Epo-induced increase in the CFU-E population is concomitant with upregulation of EpoR, Gata1 and Bcl-XL expression in the murine bone marrow.12 Thus, Epo stimulates erythropoiesis not only by activation of ‘classical’ downstream signaling, but also further enhances its effect by the Gata1-induced upregulation of EpoR expression. As recently reported, Epo regulates GATA1 through protein kinase D activation, promoting histone deacetylase 5 dissociation from GATA1, and subsequent GATA1 acetylation.13 This post-translational modification resulted in increased DNA-binding activity of GATA1,14 which may contribute to enhanced EpoR expression. Such GATA1 function is highly specific for erythroid precursor cells, because on activated CD4-positive lymphocytes EpoR expression depends only on the transcription factor Sp1, but not on GATA1.15

The lack of any differences in Gata2 expression indicates that Epo does not directly interfere with the spatiotemporal regulation of Gata1 or Gata2 activities within the erythroid differentiation. The observation that Gata1 expression in the developing bone marrow of control mice declined with increasing age, while its expression in hEPO over-expressing tg6 mice remained constantly high, raises the question as to whether a critical threshold of the Gata1 induction has been reached during the observation period, and whether additional regulators are involved in the process of developing excessive erythropoiesis under constitutive hEPO overexpression. Indeed, an approximately 50-fold increase of Tal1 mRNA expression in adult hEPO over-expressing tg6 mice as well the tight correlation between gradually increasing Gata1 and Tal1 mRNA expression levels in their developing bone marrow direct to another mechanism of EpoR regulation. This mechanism has been previously explored in erythroid progenitor cells from a patient with excessive erythrocytosis.7 While Tal1 can directly induce EpoR expression by binding to E-box motifs in the 5′-untranslated EpoR locus, Tal1 may synergize with Gata1 activity by increasing the association of the GATA1-TAL1-LMO2-LDB2 transcription factor complex to 5′-GATA and 3′-E-box motifs flanking the EpoR transcription start site.7

In summary, longitudinal analysis of hEPO over-expressing tg6 mice provides novel in vivo information that Epo directly tunes activation of the erythroid transcriptional master regulators Gata1 and Tal1 to enhance EpoR gene expression. When a selective GATA1 inhibitor eventually becomes available, these data may be important for developing novel therapeutic concepts for chronic polycythemia.