The Transactivation Domain AF-2 but Not the DNA-Binding Domain of the Estrogen Receptor Is Required to Inhibit Differentiation of Avian Erythroid Progenitors

Marieke von Lindern, Liesbeth Boer, Oliver Wessely1, Malcolm Parker2 and Hartmut Beug

Institute of Molecular Pathology Vienna Biocenter A 1030 Vienna, Austria


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Earlier work demonstrated that an activated estrogen receptor (ER) is required for long-term self-renewal of c-ErbB-expressing avian erythroid progenitors. Here, we demonstrate that activation of the ER does not only arrest or retard differentiation of early progenitors but that it affects erythroid differentiation at all stages of erythroid maturation. A search for genes whose expression is affected by the ER showed that the 17ß-estradiol-activated receptor suppressed the differentiation-associated up-regulation of Gata-1, SCL-1, and globin genes in partially mature cells. In the same cells, the expression of carbonic anhydrase II (CAII) and histone H5 was enhanced. This led to premature expression of CAII, a possible explanation for the toxic effects of overexpressed ER. Repression specifically required the transactivation domain AF-2, but neither an intact DNA-binding domain (DBD) nor the AF-1 domain. The transcriptional activation of CAII, however, required both an intact AF-2 and a functional DBD. The requirement for the AF-2, but not the DBD, suggested that the ER may compete with other nuclear hormone receptors for transcriptional coactivators that bind AF-2, a domain well conserved within this family of transcription factors. We show, however, that this model does not apply for the most likely candidate, the avian thyroid hormone receptor.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pluripotent stem cell is generally considered the only cell in bone marrow able to undergo self-renewal, i.e. to perform cell divisions that do not alter the differentiative potential of the daughter cells as compared with the mother cell. However, some exceptions to this rule have been described (1). A particularly well studied example are fully committed erythroid progenitors of avian origin. Typical erythroid progenitors of the BFU-E/CFU-E stage are normally unable to self-renew, but they can develop into cells that self-renew until the end of their in vitro life span (~40 generations; Ref.2). This development is triggered by the combined action of stem cell factor (SCF) and transforming growth factor-{alpha} (TGF{alpha}), ligands of the receptor tyrosine kinases c-Kit and c-ErbB, respectively, together with steroid hormones activating the estrogen and glucocorticoid receptors (ER, GR; Refs. 3–7). A low proportion of cells capable of self-renewal in the presence of TGF{alpha}, estradiol (E2), and dexamethasone is already present in bone marrow (6, 7).

Terminal differentiation of progenitor cells is induced when self-renewal factors are removed and replaced by differentiation factors [erythropoietin (Epo) and insulin]. During this differentiation, the cells undergo four to five cell divisions, reduce their size by a factor of 3–4, and accumulate hemoglobin (2, 8). Proper regulation of the balance between self-renewal and terminal differentiation is of extreme importance to generate adequate numbers of mature erythrocytes in the blood stream during the entire lifetime of the organism.

Recent experiments showed that activation of both the GR and the estrogen receptor (ER) is required to induce self-renewal of avian erythroid progenitors. The two receptors cannot replace each other’s function but have to cooperate (7). The GR is expressed in many tissues including hematopoietic cells, and activation of the GR does influence erythropoiesis (9, 10, 11). Although the major function of the ER appears to be in the female reproductive system, a regulatory function of estrogen has been demonstrated in bone homeostasis (reviewed in Ref.12) and in neuronal tissue (Refs. 13 and 14 and references therein). Estrogens also increase the number of erythroid cells in mice in vivo (15, 16) and are required for proliferation of erythroid chicken cells in vitro (4).

Accumulating evidence suggests that the ER can be activated in the absence of hormone through phosphorylation, e.g. by dopamine or insulin-like growth factor I (IGF-I) in neuronal cells (13, 14, 17) or by epidermal growth factor (EGF) or IGF-I in uterine tissue (18, 19, 20). Also in the presence of estradiol, signal transduction from activated receptor tyrosine kinases (IGF-I and EGF receptor) further enhances ER activity through phosphorylation of N-terminal residues (21, 22). Thus, the synergistic effect between TGF{alpha} and estradiol in avian erythroid progenitors may well be analogous to the synergistic effects of EGF and estradiol in mammary epithelial cells and uterine tissue (18, 19).

The cooperation of c-ErbB with the ER to cause self-renewal in normal erythroid progenitors closely resembles the cooperation between two retroviral oncoproteins, the avian erythroblastosis virus (AEV)-encoded proteins, v-ErbB and v-ErbA, in the induction of erythroleukemia (reviewed in Ref.2). V-ErbB is a mutated and constitutively active derivative of the avian EGF receptor, while V-ErbA is a mutated version of the thyroid hormone receptor. It was recently shown that v-ErbA has the ability to functionally replace both the ER and the GR in inducing sustained self-renewal of erythroid progenitors and to block terminal differentiation (23). Whereas investigations regarding the role of the GR in erythroid proliferation and differentiation are reported elsewhere (7), this paper concentrates on the role of the ER in erythropoiesis.

Earlier work indicated that the ER is able to arrest or retard erythroid differentiation, depending on the expression level (4). Activation of the ER inhibited the expression of several genes induced late in erythroid differentiation, such as the ion exchanger Band 3 and the globin genes. Blobel et al. (24) showed that the ER can form a protein complex with the erythroid-specific transcription factor Gata-1 in cells overexpressing the ER. This interaction was dependent on hormone and suppressed the transactivation of reporter genes by Gata-1. Gata-1 plays an essential role in erythroid development (25). Thus, this inactivation of Gata-1 by the E2-activated ER seemed difficult to reconcile with the essential role of the ER in erythroid self-renewal. Recently, however, it was shown that self-renewing avian erythroid progenitors express Gata-1 mainly as a cytoplasmic protein, essentially unable to bind DNA. Only upon induction of terminal differentiation in the presence of Epo, is Gata-1 translocated to the nucleus and acquires the capacity to bind DNA (26). It is possible that partial inactivation of Gata-1 by the ER can contribute to the differentiation arrest occurring during self-renewal.

In this paper, two complementary cell systems are used to demonstrate that the ER can affect erythroid differentiation at any stage and that the ER exhibits a dual role with respect to transcriptional control of genes up-regulated during erythroid differentiation. Gata-1, SCL-1, and the globin genes were repressed dependent on estradiol, while the expression of carbonic anhydrase II (CAII) and histone H5 was enhanced by estradiol. Repression and activation involved distinct mechanisms. Since the ER inhibits and the thyroid hormone receptor enhances erythroid differentiation, these receptors could interfere with each other’s function. However, activation of the ER had no effect on differentiation induction and target gene induction by the avian thyroid hormone receptor.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of the ER Can Inhibit Erythroid Differentiation at Any Stage of Maturation
Previously, we demonstrated that activation of an overexpressed human ER in the erythroid cell line HD3 inhibits or retards erythroid differentiation and suppresses the expression of several genes activated late in erythropoiesis (4). During the course of these studies the effect of E2 or the ER-antagonist ICI164,384 (ICI) was compared with steroid-stripped media. In stripped media, as well as upon addition of ICI, erythroid differentiation takes place with almost similar kinetics, whereas differentiation is inhibited by addition of E2. In most of the experiments described, however, anemic serum (AS) was required as a source for avian Epo. Since we were unable to strip AS from steroids without losing Epo activity, we had to use the antagonist ICI. Very recently, we successfully stripped AS from steroids without loss of activity. In respective control experiments, we again could not detect significant differences between stripped media and ICI (data not shown). Thus, antiestrogen treatment was used in all subsequent experiments to achieve estrogen-free cultures.

Next, we sought to determine whether the activated receptor blocks entry into a terminal differentiation program, or whether the active ER is able to arrest ongoing differentiation at any stage. Therefore, we wanted to activate the ER in erythroid cells at various stages of differentiation and monitor its effect on the continuation of the differentiation program. Since normal progenitors down-regulate expression of the endogenous ER during terminal differentiation, we employed a clone of the erythroid cell line HD3 that lacks the endogenous chicken ER and constitutively expresses the human ER from a retroviral vector (clone 4; Ref.4). In this way, transcriptional regulation of the ER could be circumvented.

HuER-HD3 clone 4 cells were induced to differentiate for 64 h in steroid-free medium containing anemic chicken serum and insulin. Since synchrony of differentiation was limited, homogenous cell populations at different stages of maturity were obtained by purification on Percoll gradients (Fig. 1AGo; see Materials and Methods). Two aliquots of these fractions were reseeded in the same differentiation medium either containing estradiol (E2) or the estrogen antagonist ICI 164,384. During the next 4 days, cells were assayed for various differentiation parameters, i.e. proliferation, hemoglobin accumulation, and cell morphology.



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Figure 1. Effect of E2 on Erythroid Cells of Increasing Maturity

A, Schematic flow diagram of the purification and stimulation with hormones of HD3 cells constitutively expressing HEO (clone 4). Cells were first differentiated in steroid-free medium for 64 h. This yields a somewhat heterogeneous population, containing cells at various stages of maturation, which will eventually all terminally differentiate (top horizontal arrows). To purify cells of the same differentiation stage, the above partially mature cell population was fractionated on a Percoll step gradient. These purified fractions are reseeded in differentiation medium with either E2 or the E2 antagonist ICI 164,384 (ICI) and characterized for their state of differentiation. Cells mature to erythrocytes in ICI, but are arrested in the initial, partially mature state in E2. B, C, and D, results obtained from a cell fraction with a density of 1.075–1.080 g/cm3, purified and treated with hormones for the times indicated as shown in panel A. [3H]Thymidine incorporation (B) was measured of the fraction isolated 64 h past the onset of differentiation (t = 64) and 24 h after the fraction was reseeded in E2 or ICI 164,384 (t = 64+24). Similarly, hemoglobin content normalized to cell number (C) was determined before differentiation (t = 0), of the isolated fraction (t = 64) and 72 h after the fraction was reseeded in E2 or ICI 164,384 (t = 64 + 72). Finally, cells from the isolated fraction (D, left), as well as cells from that fraction 72 h after the fraction was reseeded in E2 (D, middle) or ICI 164,384 (D, right), were cytocentrifuged and stained with histological dyes plus neutral benzidine. Images were taken using a CCD camera (Photometrics) and a blue filter (480 nm) to detect hemoglobin. Mature cells appear dark under these conditions. Images were processed with Adobe Photoshop.

 
During normal differentiation, the cell proliferation rate first increases as the cells go through ~four cell divisions with a drastically shortened G1 period and then declines upon G1 arrest of the terminally differentiated cells (8). This can easily be measured by [3H]thymidine incorporation. Simultaneously, cell size is reduced to that of a mature erythrocyte. During all stages of maturation, there is a progressive accumulation of hemoglobin. Mature avian erythrocytes are highly hemoglobin-positive, which can be shown by neutral benzidine staining, and they have an oval shape and a small condensed nucleus (27).

E2 clearly affected the progress of maturation in partially differentiated cells. Upon E2 treatment, cells with a density between 1.075 and 1.080 g/cm3 almost retained their high thymidine incorporation level, while this decreased by a factor of 4 in the presence of the E2 antagonist (Fig. 1BGo). Concurrently, hemoglobin levels increased only moderately in the presence of E2, but strongly (5.5-fold) in the presence of ICI 164,384 (Fig. 1CGo). Together, this indicated that ongoing maturation was arrested or heavily retarded in the presence of E2, but that the cells continued to mature in the presence of ICI 164,384. Morphological and histochemical analyses of cytospins confirmed these results (Fig. 1DGo). Before splitting, the cells consisted of late reticulocytes, as shown by their small size, and light hemoglobin staining (Fig. 1DGo, left). Upon addition of ICI 164,384, the cells progressed in differentiation and formed oval, well hemoglobinized, mature erythrocytes (Fig. 1DGo, right). Upon addition of E2, however, the cells were mostly arrested at the late reticulocyte state, with only a few being able to mature into erythrocyte-like cells (Fig. 1DGo, middle).

A result similar to that obtained for this cell fraction (density 1.075–1.080 g/cm3) was obtained with all other fractions. Regardless of their initial stage of maturation, the cells were arrested at their particular stage of maturity when treated with E2, but continued to mature into erythrocytes in ICI. This indicated that addition of estrogen can block differentiation at any stage and that it does not merely affect the onset of the differentiation program. However, E2 was unable to cause retrodifferentiation of the partially mature cells and did not cause them to turn on a self-renewal program again.

The E2-Activated ER Suppresses Transcription of Erythrocyte-Specific Genes
Activation of the erythroid differentiation program induces the transcription of a number of genes whose products are required for maturation of erythrocytes. A transient up-regulation of the transcription factors Gata-1 and SCL-1 is followed by expression of the erythrocyte-specific genes, histone H5, band 3, band 4.1, CAII, Ala-S, and the globin genes (8). Previously, it was shown that cells arrested in differentiation by the ER showed decreased mRNA levels of band 3, band 4.1, {alpha}-globin, and ß-globin (4), both in HD3 cells that overexpress the human ER and in primary erythroid cells that express the endogenous receptor. Since activation of the ER appeared to block differentiation at any stage, it was of interest to determine whether the ER could repress the above genes after they had been up-regulated by differentiation induction.

Several of the late erythroid genes encode relatively stable mRNAs. Therefore, down-modulation of expression could not be studied using Northern blots. Rather, simplified run-on assays using the coding regions of the genes to be studied were performed. The initial studies were performed in HD3 cells that constitutively express the human ER and do not down-regulate the ER during differentiation. To rule out artefacts by the overexpressed ER, or by a possible residual activity of the v-ErbA oncoprotein in the HD3 cells, the experiments were repeated in primary cells that lack the v-ErbA oncoprotein and express the endogenous chicken ER.

HuER-HD3 clone 4 cells were induced to differentiate and were density purified as described above. The partially mature cells obtained were then treated with E2 or ICI 164,384 and processed for run-on analysis. To determine suitable times for predifferentiation and hormone treatment, cells were induced to differentiate for either 24 or 40 h and then subjected to estrogen/ICI treatment for 8 and 24 h. Figure 2AGo shows that the times required for optimal differences between E2 and ICI varied with the gene tested. After 24 h of predifferentiation, Gata-1 had not yet reached maximal transcription levels. In the presence of E2, the transcription rate remained constant, while it increased 2-fold in a time-dependent fashion upon treatment with ICI 164,384. After 40 h of predifferentiation, Gata-1 transcription was nearly maximal and thus only moderately reduced by subsequent hormone treatment. In contrast, transcription of ß-globin was submaximal both after 24 and 40 h of predifferentiation, allowing the detection of a time-dependent, 5-fold increase upon ICI 164,384 treatment in both cases. Interestingly, E2 did not only block the further increase in ß-globin transcription, but even caused a moderate decrease in transcriptional rate after hormone treatment, both after 24 and 40 h of predifferentiation.



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Figure 2. Transcription of Differentiation-Specific Genes in the Presence of E2 or Its Antagonist ICI 164,384 in Partially Mature Erythroid Cells

A, To determine suitable times for predifferentiation and hormone treatment (see legend to Fig. 1Go), HD3 HEO clone 4 cells were predifferentiated for either 24 or 40 h and then subjected to E2/ICI treatment for 8 and 24 h. Radiolabeled RNA was synthesized and hybridized against Gata-1 (left panel) or ß-globin (right panel). Values obtained were corrected for transcription of the constitutively expressed ER. B, In a similar experiment, radiolabeled mRNA obtained from HD3 HEO clone 4 cells predifferentiated 24 h and incubated with E2 or ICI 164,384 for again 24 h each was hybridized against cDNAs of Gata-1, Gata-2, {alpha}-globin, ß-globin, ALA-S, CAII, and histone H5 (H5), and signals were corrected for the constitutive transcription of the ER as in panel A. The results from a representative experiment are expressed as the percent difference of the rate of transcription (r.t.) in the presence of E2 compared with that obtained with ICI 164,384 [(r.t. E2 corr. - r.t. ICI corr.)/r.t. ICI corr. x 100%]. This calculation yields neagtive values if transcription is inhibited by E2. Similar results were obtained in three further, independent experiments using slightly different incubation times. N.D., Not determined. C, Primary erythroid progenitors were predifferentiated 24 h and subsequently treated with E2 or ICI 164,384 for 24 h each, and radioactive mRNA was hybridized against Gata-1, Gata-2, SCL-1, {alpha}-globin, ß-globin, ALA-S, CAII, and histone H5 (H5) as in panel A. The average of three independent experiments is shown. Signals were corrected for transcription of ribosomal RNA. Results are expressed as described in the legend to panel B. The E2-induced increase in transcription of CA II and H5 was calculated by the same formula as in panel B, yielding positive values. N.D., Not determined.

 
The effects of E2 on the transcription of other erythroid-specific genes was determined similarly. For Gata-2, {alpha}-globin, and Ala-S the results obtained after 24 h of predifferentiation and 24 h of hormone treatment were plotted as percent reduction by E2, as compared with ICI. A representative experiment is shown in Fig. 2BGo. Similar results were obtained in several additional, independent experiments. All three genes were found to be repressed by the ER, in addition to ß-globin and Gata-1. Trials to determine transcriptional rates of c-myb, c-KIT, and band 3 failed due to insufficient sensitivity of the assay used.

Next, we sought to confirm these data in primary, erythroid progenitors. Although the endogenous ER is down-regulated during progenitor differentiation, it is possible to delay this down-regulation, if TGF{alpha} plus ICI 164,384 is added together with the differentiation factors (4). Under these conditions, differentiation is induced but the ER is down-regulated only after 72 h. Primary SCF/TGF{alpha} progenitors (5) were predifferentiated under these conditions for 24 h, washed, split into two aliquots, incubated with E2 or ICI 164,384 for another 24 h in the absence of TGF{alpha}, and processed for run-on analysis.

The combined results from three independent experiments are shown in Fig. 2CGo. They were highly reproducible with respect to which genes were activated or repressed as a consequence of ER activation, but the extent of the repression or activation was somewhat variable, perhaps due to variations in the expression of the endogenous ER. Transcription of the nuclear proteins, Gata-1 and SCL-1, was repressed ~2-fold, as was transcription of the erythrocyte genes {alpha}- and ß-globin. In contrast to the HD3 cells, Gata-2 transcription was not affected by the ER. In these run-on assays, the transcription rate of CAII was not affected by E2 in HD3 cells, while transcription was clearly enhanced in primary cells. Steady state levels of CAII are increased by E2 in HD3 cells as judged on Northern blots (Fig. 4Go). The inability to see an up-regulation of CAII in these run-on assays may be due to the choice of 24-h predifferentiation.



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Figure 4. Expression of Differentiation-Specific Genes in HD3 Cells Expressing Mutant ERs

HD3 cells expressing either {Delta}1–121 MOR (panel A; identical results obtained with the wt MOR), MOR C241A,C244 (B) or {Delta}1–121 MOR L543A,L544A (C) were induced to differentiate under the appropriate conditions (see Materials and Methods), in the presence of either E2 (E) or ICI164,384 (I). RNA was prepared from cells harvested at the onset of the experiment (day 0), and 1, 2, 4, and 6 days after differentiation induction. In the case of the MOR C241A,C244 construct, the experiment was terminated at day 4 because the cells were disintegrating in the presence of E2. Northern blots were hybridized with probes for Gata-1, SCL-1, ECH, and CA II. Ethidium staining of rRNA demonstrated equal loading (data not shown).

 
In summary, activation of the ER inhibited differentiation at any stage. It decreased expression of distinct genes during induction of terminal differentiation, but enhanced expression of CAII in primary cells.

Requirement of Functional Domains of the ER for Its Effects on Erythroid Differentiation
To investigate by what mechanisms the ER may regulate its target genes, we first determined which domains of the ER are required for the observed differentiation arrest and for the repression or activation of late erythroid genes. The ER has two transactivation domains, the N-terminal AF-1 and the C-terminal AF-2. These domains were shown to act independently and subject to both cell type and promoter context (28, 29). In addition to regulating gene transcription via specific binding to estrogen response elements (EREs), the ER also interferes with the function of other transcription factors through protein-protein interactions. Notably, the E2-activated ER can interact with Gata-1 (24) or nuclear factor (NF)-{kappa}B and AP-1 family members, which involves both N-terminal and C-terminal domains of the ER that are different from the transactivation domains (30, 31, 32, 33).

ER constructs mutated in one or both of the transcription activation domains, or mutated in the DNA-binding domain (DBD) (Refs. 34 and 35; Fig. 3AGo) were cloned into a retroviral expression vector (pCRN, Ref.36) and transfected into HD3 cells. Single-cell colonies were isolated, grown into clonal cultures, and tested 1) for expression of the ER construct by Western blot (data not shown) and 2) for its ability to terminally differentiate in the presence of ICI under standard conditions as described above. For every ER construct, a representative clone positive for ER expression by Western blot was selected and induced to differentiate in the presence of either E2 or ICI 164,384. During 4 days of differentiation, hemoglobin levels, cell numbers, and cell volumes were determined. As a representative parameter, the ability of the different clones to differentiate in the presence of E2 was expressed as percent reduction of hemoglobin per cell volume by E2, relative to the ICI control at day 3 (Fig. 3BGo). The wt mouse ER (MOR) and mutant constructs carrying either a deletion in the AF-1 domain (amino acids 1–121; Ref.34) or a mutation of the second zinc finger of the DBD that disrupts DNA binding (C241A, C244A; Ref.35) caused a distinct, E2 dependent arrest of differentiation (Fig. 3BGo). In contrast, mutant receptors carrying a mutated, nonfunctional AF-2 domain (L543A, L544A; Ref.34) failed to affect differentiation, either in the presence or absence of a functional AF-1 domain. These findings could be corroborated by evaluation of stained cytospins from the same cell populations (data not shown).



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Figure 3. Effect of Mutations in the ER on Its Ability to Inhibit Erythroid Differentiation

A, Schematic diagrams of the mutant ER constructs expressed in either HD3 cells or primary erythroid progenitors. These included the wt mouse estrogen receptor (MOR), a mutation in the first Zinc finger (MOR C241A,C244A), a mutation in the AF-2 domain (MOR L543A,L544A), deletion of the AF-1 (MOR {Delta}1–121), and deletion of the AF-1 plus mutation of the AF-2 (MOR {Delta}1–121, L543A,L544A). B and C, HD3 cells (B) or primary erythroid progenitors (C) constitutively expressing the constructs shown in panel A were induced to differentiate and subjected to determination of differentiation parameters as described in Materials and Methods. As a representative parameter, the percent difference in hemoglobin level per cell volume in the presence of E2 vs. ICI 164,384 [(HbE2-HbICI)/HbICI x 100%] is shown. N.D., Not determined.

 
We then sought again to confirm these findings in primary erythroid progenitors. Some of the ER constructs (particularly the one with a mutant DBD) were toxic to primary cells, since SCF/TGF{alpha} progenitors require E2 for sustained self-renewal (4, 5) and could not be induced and expanded in the presence of ICI. Thus, clonal cultures of normal progenitors expressing the mutant ERs were not accessible. We therefore chose mass cultures of SCF progenitors which 1) can be effectively infected by retroviruses within 2–3 days (37), 2) proliferate for 8–10 days even in the presence of ICI (5) and 3) tolerated the expression of mutant ER constructs under these conditions. Only the ER construct with a DBD mutation (C241A,C244A) could not be expressed in these cells.

Avian bone marrow cells were cocultivated with an excess of fibroblasts, which produced retroviruses expressing the various ER mutants (Fig. 3AGo). Five days after infection of the bone marrow cells, the resulting mass cultures were tested for expression of the mutant ER proteins, using the monoclonal antibody H222, which recognizes the mammalian, but not the avian, ER. All ER constructs were expressed at comparable levels except for the DBD mutant (data not shown). We repeatedly failed to obtain proliferating erythroid progenitors from bone marrow infected with this mutant. The DBD mutant was already shown to be toxic in HD3 cells upon E2 addition. However, HD3 cells can be grown in the presence of ICI, and activation of this protein can be inhibited.

At the same time expression of the ER constructs was examined, the mass cultures were induced to differentiate in the presence of Epo/Insulin plus either E2 or the antagonist ICI 164,384. During the next 4 days, proliferation, hemoglobin accumulation, and cell volume were monitored. Data obtained at day 3 were expressed as the E2-induced percent decrease in hemoglobin per cell volume (Fig. 3CGo; see above).

The primary cells infected with empty vector already showed an E2-induced decrease in hemoglobin per cell volume, due to the endogenous avian ER that is down-regulated during terminal differentiation only after 24–48 h (4). However, constitutive expression of the wt ER resulted in a significantly stronger inhibition of differentiation. In agreement with what was found in HD3 cells, constitutive expression of a mutant construct lacking AF-1 resulted in an arrest of differentiation comparable with the activity of the wt ER (Fig. 3CGo). The AF-2/AF-1 mutant was inactive, causing an E2 effect comparable to that of the control cells. Expression of the AF-2 mutant, however, seemed to even stimulate erythroid differentiation, which may be due to a dominant negative effect of the mutant protein on the endogenous ER (Fig. 3CGo). In line with this, the inductive effect of AF-1 was not detected in the HD3 cell line lacking the endogenous ER.

In conclusion, the experiments in the HD3 cells combined with those in the primary cells show that an intact AF-2 domain, but neither the AF-1 domain nor the DBD, is required for the ER to arrest or delay erythroid differentiation.

Functional ER Domains Required for Regulation of Expression of Erythrocyte-Specific Genes
Next, we wanted to determine which ER domains are required for regulation of erythrocyte-specific genes. The results of the run-on assays suggested that this regulation was complex, causing repression of some erythroid target genes by the E2-activated ER, but induction of others, e.g. CAII. It was therefore of interest to determine whether genes found to be repressed by the ER (Fig. 2Go) in both HD3 cells and primary cells required an intact AF-2, but not the AF-1 or the DBD. Furthermore, such studies should reveal whether or not the domains required for up-regulation of CAII were similar. Previous experiments had established that the results on repression of Gata-1 and the globin genes obtained with HD3 clones closely matched those obtained with primary cells (Figs. 2Go and 3Go). It therefore seemed safe to use the HD3 clones expressing the various mutant ERs (see above) for these experiments, since they do not show the variability seen in infected mass cultures of primary cells (Fig. 2Go). These HD3 clones also have the advantage that they lack the endogenous ER.

In the previous run-on experiments we sought to identify target genes whose transcription could be reduced by the activated ER. Having established Gata-1 and globin genes as target genes for the ER, we employed Northern blots to test all mutant constructs for their effect on the accumulation of these and other erythrocyte genes, turned on at different times after differentiation induction. In these experiments, gene repression by the ER would be indicated as lower levels of accumulated mRNA induced by E2.

HD3 cells expressing the various constructs were induced to differentiate in the presence of E2 or its antagonist ICI 164,384. RNA was isolated at the onset of differentiation and 1, 2, 4, and 6 days later. Northern blots containing these RNAs were then probed for the expression of a set of erythroid-specific genes (Fig. 4Go). In HD3 cells, as well as in primary cells, terminal differentiation involves a transient up-regulation of Gata-1 and SCL1, which is followed by expression of CAII, Band3, and the globin genes (8). In HD3 cells expressing the wt MOR or expressing the MOR with a deletion of AF-1, Gata-1 and SCL1 were transiently up-regulated upon differentiation induction in the presence of ICI. This transient up-regulation was largely inhibited in the presence of E2, as can be seen when expression levels are compared between E2 and ICI at days 1, 2, and 4. The reduction of gene expression by E2 was similar in clones expressing wt MOR or the AF-1 deletion mutant (Fig. 4AGo and data not shown; the data for the {Delta} AF-1 ER are shown rather than the wt ER data, since the former are better comparable with the AF-1/AF-2 double mutant in Fig. 4CGo). In contrast, transient up-regulation of another erythroid transcription factor, ECH, the chicken homolog of NF-E2 (38), was similar in the presence of E2 and ICI, thus hardly affected by the E2-activated ER. As expected from previous results (8, 39), CAII was up-regulated only at late stages of differentiation in the presence of ICI 164,384. In contrast, E2 caused an unexpected, premature activation of CAII.

In line with the biological data, mutation of the DBD did not interfere at all with the repression of Gata-1 and SCL-1 by E2. Since this mutant receptor interfered so severely with differentiation, inhibition of mRNA accumulation by E2 was much stronger than with the wt ER or the {Delta} AF-1 ER, even affecting the expression of ECH. Interestingly, however, the DBD mutation abrogated the up-regulation of CAII by E2 (Fig. 4BGo).

In cells expressing the AF-2 mutant protein, either alone or in addition to a deletion of AF-1, gene expression was not affected by E2 as compared with ICI, suggesting that these mutant receptors were inactive. Both the repression of Gata-1 and SCL-1 by E2 and the E2-induced up-regulation of CAII were abrogated (Fig. 4CGo and data not shown). A small repression by E2 is visible at day 1, but ethidium bromide staining and additional experiments strongly suggest that this is due to small RNA loading differences.

Thus, the AF-2, but not AF-1 or the DBD, is required for E2-dependent repression of the transcription factors Gata-1 and SCL-1. The same was true for the {alpha}- and ß-globin genes (data not shown). However, the premature up-regulation of CAII requires an intact AF-2 and an intact DBD.

The ER Does Not Inhibit Acceleration of Erythroid Differentiation by the Thyroid Hormone Receptor TR-{alpha}
Phenotypically, the arrest of differentiation and the repression of erythrocyte-specific genes resemble respective functions of the oncoprotein v-ErbA. However, v-ErbA lacks a functional AF-2 domain and seems to require specific DNA binding for its function (40) while the ER requires an intact AF-2 domain but no intact DBD for differentiation arrest and gene repression (see above). As a part of its multiple functions (23) v-ErbA suppresses the acceleration of erythroid differentiation by the endogenous, T3-activated thyroid hormone receptor (TR-{alpha}/c-ErbA, (41). It was therefore of interest to study whether the ER would also interfere with TR-{alpha}/c-ErbA function. This appeared possible since the AF-2 domain is well conserved among all nuclear hormone receptors (31, 42, 43), and specific putative coactivators interacting with the AF-2 domain have recently been described (44, 45, 46, 47, 48, 49, 50, 51, 52).

To test whether retardation of erythroid differentiation by E2 was dominant over the acceleration of maturation by thyroid hormone (T3), erythroid progenitors (grown in TGF{alpha} and E2) were induced to differentiate in the presence of either E2, T3, or combinations thereof. Two days later, hemoglobin content, cell numbers, and cell volumes were determined. Figure 5AGo shows that T3-enhanced hemoglobin levels normalized to cell volume, whereas addition of E2 resulted in a retardation of hemoglobin accumulation. In the presence of E2, T3 caused a similar or even stronger acceleration of differentiation, suggesting that the ER is completely unable to interfere with TR-{alpha}/c-ErbA action. This result was obtained both with normal progenitors expressing the endogenous ER and with cells constitutively expressing the human ER from a retroviral vector. Thus, the apparent inability of the ER to interfere with T3 action cannot be due to down-regulation of the ER during differentiation.



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Figure 5. The E2-Activated ER Does Not Impair TRa/c-ErbA Function

A, Primary erythroid progenitors, expressing the endogenous avian ER (control cells) or constitutively expressing the human estrogen receptor (h-ER), were induced to differentiate in medium depleted from steroids and thyroid hormone (T3), in the presence of differentiation factors and either E2, T3, or E2 plus T3. Differentiation parameters were measured as in Fig. 3Go, and the percent difference in hemoglobin per cell volume in media minus or plus hormones was determined after 3 days of differentiation [(HbE2,T3,E2/T3-Hbno add)/Hbno add x 100%]. B, Primary erythroid progenitors were cultivated in media either sustaining proliferation in the presence of TGF{alpha} (left panel) or inducing differentiation in the presence of AS, insulin, and SCF (right panel). To these media, T3 plus E2, E2 alone, T3 alone, or no hormones were added. After 30 h, cells were harvested, and total RNA was isolated and probed for the expression of histone H5 (H5), CAII, and c-myb mRNAs by Northern blotting.

 
To analyze whether activation of the ER was also unable to interfere with the up-regulation of erythroid-specific genes by TR-{alpha}/c-ErbA, the expression of erythrocyte-specific genes was determined. Erythroid progenitors grown in TGF{alpha} and E2 were kept under proliferation conditions (TGF{alpha}) or induced to differentiate by addition of differentiation factors (Epo, Insulin, SCF), both in the presence of either thyroid hormone (T3), E2, or T3 plus E2 for 30 h. Total RNA was extracted and used to assay mRNA levels of CAII, histone H5, and c-myb by Northern blot analysis. CAII was previously shown to be a direct target gene for the thyroid hormone receptor (TR) (53). Both T3 and E2 enhanced the expression of CAII and histone H5 mRNA, both under proliferation and differentiation conditions (Fig. 5BGo). Interestingly, the combination of E2 and T3 resulted in considerably higher levels of CAII and histone H5 mRNA than induced by each hormone alone. At least in the regulation of these two genes, the ER and the TR were synergistic rather than competitive, arguing against competition of the two receptors for joint coactivators. As expected, c-myb expression was clearly higher in proliferating than in differentiating cells (8). In line with the fact that T3 accelerated differentiation even in the presence of an E2-activated ER, the combination of T3 and E2 reduced c-myb mRNA levels almost to the extent seen with T3 alone (Fig. 5BGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Previously, we showed that overexpression and activation of the human ER in the avian erythroid cell line HD3 can block differentiation (4). We now establish that a E2-activated ER is able to block differentiation at any stage of the differentiation process and that the expression of target genes is regulated via distinct mechanisms. Multiple erythroid-specific genes are repressed upon E2 activation of the ER in a fashion independent of DNA binding, but requiring a functional AF-2. Other erythrocyte genes, such as carbonic anhydrase II (CAII), were up-regulated, requiring both an intact AF-2 and an intact DBD.

Repression of Gene Expression by Nuclear Hormone Receptors
Several nuclear hormone receptors are known to repress transcription of specific genes. The thyroid hormone receptor (TR) is present in the nucleus and binds to thyroid hormone response elements both in the absence and presence of hormone. In the absence of ligand, however, the TR represses transcription (54, 55, 56), which is mediated by a transferable repression domain (57). Repression occurs independently of the conserved AF-2 domain. Instead, the repression domain is located in the hinge region and binds a corepressor, N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator for retinoid and thyroid receptors) (47, 58, 59).

Repression of erythroid differentiation by the ER specifically required an intact AF-2 domain. Therefore, it is unlikely that a corepressor, as described to bind the TR{alpha} and v-ErbA, is involved in repression of differentiation by the ER. Moreover, repression by v-ErbA or the TR{alpha} requires DNA binding to allow interaction of the corepressor with the target genes. In contrast, repression of erythroid differentiation and erythroid gene transcription by the ER is independent of DNA binding.

Repression of gene transcription by steroid hormone receptors is commonly due to protein-protein interactions between the steroid hormone receptors and other transcription factors among which AP-1 or NF-{kappa}B (reviewed in Ref.60). Since the result of AP-1/receptor interactions is largely cell type and promoter dependent and since several domains of the hormone receptors were reported to be involved, distinct mechanisms may underly repression of gene transcription by the ER and the GR (31, 32, 33). Mutation alanalysis and the use of steroid analogs such as Tamoxifen or RU38486, which block transactivation via the AF-2 domain of the ER and the GR, respectively, showed that repression is mainly independent of homodimerization, DNA recognition, and the transactivating AF-2 domain (31, 61, 62). Similarly, both the ER and the GR were reported to interfere with transcriptional activation by NF-{kappa}B (30, 63), but again independently of an active AF-2 domain. Since repression of differentiation in avian erythroblasts required an intact AF-2, the underlying mechanism of repression is most likely different from the ones described above. In addition, transcriptional activation of a downstream repressor by the ER is excluded, since no intact DBD is required.

Instead, the specific requirement for an intact AF-2 suggests that this transactivation domain may bind ("squelch") coactivators that mediate contacts between specific transcription factors and the basic transcription machinery (for reviews see Refs. 64 and 65). As a result, these coactivators would be less available for other transcription factors that enhance erythroid differentiation. The AF-2 domains of the ER and the thyroid/retinoid receptors are highly homologous, and squelching of factors bound to the AF-2 domain have been described between the ER and the TR (43). The E2-bound ER inhibits differentiation markedly, while both retinoids and thyroid hormone enhance erythroid differentiation (41, 66).

Several proteins were recently isolated that interact with the AF-2 domain of E2-activated nuclear hormone receptors, namely RIP (receptor-interacting protein)-140 and RIP-160 (45, 67), ERAP (estrogen receptor-associated protein)-140 and ERAP-160 (44), TIF (transcriptional intermediary factor)-1 (46), a number of isoforms of SRC-1 (steroid receptor coactivator-1) (49, 50), TRIP1 (thyroid hormone receptor interacting protein 1)/SUG-1 (48, 51), TIF2 (52), AIB1 (amplified in breast cancer) (68), and ACTR (69). SRC-1, TIF2, ALB1, and ACTR have been shown to stimulate the transcriptional activity of nuclear receptors and warrant the name coactivators. The roles of the remaining receptor-interacting proteins are still unclear.

We showed that the ER was not effectively competing with T3-activated TR in the erythroid progenitors. In contrast, the activated ER and TR cooperated in the induction of CAII and histone H5 expression in primary cells. This does not necessarily exclude the possibility that common coactivators are used by the TR and the ER. On promoters harboring binding sites for both receptors, the joint binding of certain coactivators may result in synergism rather than competition. Yet, an antagonistic effect may be seen on promoters that harbor a thyroid hormone response element, but not an estrogen response element (ERE).

Does the ER Directly Interact with Gata-1?
Recently it was shown by Blobel et al. (24) that the ER and Gata-1 form a complex both in vitro and upon overexpression of the ER in MEL cells. Upon activation of Gata-1, Gata-1 expression is increased via an autoregulatory loop. Since most erythroid-specific genes contain Gata-1 response elements in their promoter, partial inactivation of Gata-1 could be responsible for the observed repression of differentiation. Both in primary cells and in the cell line HD3 we observed lower Gata-1 levels in the presence of E2. Our data showing that the AF-2- but not the DBD domains of the ER are essential for Gata-1 repression is in line with Blobel’s observation that tamoxifen acts as a full antagonist of the ER in transient transactivation assays using a part of the EKL-F promoter that has a Gata site but no ERE (24).

Gata-1 has an essential function in erythropoiesis, and total inactivation of the gene in mice is lethal due to the absence of mature erythrocytes in the embryo (25). However, regulation of Gata-1 function during differentiation is unexpectedly complex. In self-renewing, primary avian erythroid cells, Gata-1 was shown to be located mainly in the cytoplasm. Only induction of terminal differentiation by Epo resulted in translocation to the nucleus, DNA binding, and up-regulation of expression (26). Gata-1 controls maturation of erythrocytes, but also survival of erythroid progenitors (70). Blobel and Orkin (71) showed that MEL cells overexpressing the ER become apoptotic in the presence of estrogen. However, we found that activation of the ER is essential to induce self-renewal of avian erythroid progenitors. At the expression levels seen in our cells, the ER does not completely suppress Gata-1 expression, but rather reduces its up-regulation during terminal differentiation. Therefore, the ER may inhibit the up-regulation of late erythroid differentiation genes that are dependent on high levels of Gata-1 in the nucleus, while essential functions of Gata-1 in proliferating progenitors may not be impaired. The apoptotic effects seen by Blobel and Orkin (71) may well be the result of overexpression, since we also observed growth arrest and cell death in addition to a differentiation arrest in clones expressing very high levels of the ER (Ref. 4 and M. von Lindern, and H. Beug, unpublished observations). Another reported effect of E2 during erythroid differentiation, i.e. an E2-induced reduction of erythroid colony number determined in a BFU-E assay (24), is also not necessarily the result of an adverse effect on erythropoiesis. Rather, it could be due to repression of terminal differentiation, which would result in reduced or absent hemoglobinization of the growing colonies and thus in a highly reduced score of red colonies. The reverse, a higher score of colonies because of increased hemoglobinization, has been reported in the presence of retinoic acid, which enhances hemoglobin expression (72). In line with this, we never observed an adverse effect of E2 on cell proliferation in liquid cultures of human erythroid progenitors (M. von Lindern, submitted).

Clearly, not all genes harboring Gata-1 response elements in their promoter are repressed in the presence of E2. The E2-induced genes CAII and histone H5 are also transcriptionally regulated by Gata-1 (73). The affinity of Gata-1 for multiple interaction partners, possibly including the ER, is likely to determine what processes may be affected by activation of the ER.

Both the ER and v-ErbA Repress Erythroid Differentiation
The cooperation between v-ErbB and v-ErbA in avian erythroleukemia resembles the cooperation between endogenous receptor kinases and steroids in self-renewal of primary erythroid cells (7). The ER and the GR were shown to have distinct functions in stimulating proliferation and inhibiting differentiation, while v-ErbA abrogated the requirement for both steroid ligands (23). Like the ligand-activated GR, v-ErbA may enhance the expression of genes involved in proliferation. The repression of erythroid differentiation genes, however, seems to parallel the function of the E2-activated ER.

However, the mechanisms employed by the two nuclear hormone receptors to repress differentiation are clearly distinct. In contrast to the ER, V-ErbA requires its DBD, while its AF-2 domain is deleted (see review in Ref.74). Binding of the co-repressor N-CoR or SMRT is essential for the function of v-ErbA (59). Instead, the requirement for an intact AF-2 domain in the ER suggests "squelching" of co-activators. It is possible that these two mechanisms could lead to the same biological effect and that the two receptors even interact with the same set of target genes. V-ErbA needs DNA binding in order to target the corepressor to a certain promoter. In contrast, the ER may bind coactivators required by other transcription factors that could activate that gene. To deplete these other transcription factors from their coactivators, the ER does not have to bind DNA, but its AF-2 domain must be available.

v-ErbA expressing HD3 cells may constitute a less suitable system, since v-ErbA and ER action may interfere. The observation that the effect of E2 on the expression of Gata-2 and histone H5 is different in HD3 cells compared with primary cells (Fig. 2BGo) necessitated further caution in the use of HD3 cells. However, it was previously shown that in the presence of H7 and upon inactivation of ts c-ErbB at 42 C, v-ErbA is inactive (75). Recent experiments, in which v-ErbA is expressed in primary erythroid cells, convincingly showed that v-ErbA effects are completely dependent on the presence of activated c-Kit or c-ErbB (23). In the absence of c-Kit or c-ErbB signaling, v-ErbA-expressing cells were unable to inhibit the differentiation-associated up-regulation of CAII and histone H5. As experiments using HD3 cells expressing the ER constructs were performed under conditions rendering v-ErbA inactive, we are confident that ER effects can be accurately measured in HD3 cells. The discrepancies in expression of Gata-2, CAII, and histone H5 in primary vs. HD3 cells may be due to differences in the stage of erythroid development, represented by these different cells. When the effect of E2 on Gata-1 is measured at subsequent stages of differentiation (Fig. 2BGo), the inhibiting effect of the ER is obvious when Gata-1 expression is increasing 24 h after differentiation induction. However, once Gata-1 expression is at its maximum 48 h after differentiation induction, E2 has no significant effect anymore. HD3 cells express clearly detectable levels of Gata-2, which can be repressed by E2. In primary SCF cells, however, Gata-2 expression is hardly detectable in a run-on experiment, which explains why an inhibitory effect is not detected either. Similarly, histone H5 is expressed in HD3 cells and can be repressed by E2, while it is hardly expressed in self-renewing primary cells, where E2 enhances its expression. The function of the ER in the development of mature erythroid cells may not simply be to support self-renewal of immature cells through inhibition of differentiation, but the ER may have a role in the timing of the proliferation and differentiation program. Dependent on the stage of the cells, dependent on other transcription factors present, expression of some genes is inhibited by a mechanism independent of DNA binding, while others can be activated by a mechanism requiring binding of the ER to EREs.

In conclusion, the E2-activated ER has an important, though possibly subtle, role in regulating the balance between erythroid progenitor proliferation and differentiation. Since the ER is down-regulated at the onset of terminal differentiation, it may contribute to a proper timing of terminal differentiation in primary erythroid cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Cell Culture
The v-erbA/ts-v-erbB transformed cell line HD3 was described by Beug et al. (27). SCF and SCF/TGF{alpha} progenitors were generated from freshly prepared bone marrow cells and grown in modified CFU-E medium as described by Beug and co-workers (4, 5). Avian recombinant SCF (100 ng/ml) (76), 5 ng/ml TGF{alpha} (Promega, Madison, WI), 5 x 10-7 M estradiol, or 5 x 10-7 M ICI 164,384 was added as indicated. For differentiation assays, cells were seeded in CFU-E medium without chicken serum, with a double concentration of iron-saturated chicken transferrin (Sigma, St. Louis, MO) and in the presence of 2% high titer AS and 1.5 nM insulin (4). HD3 cells were differentiated at 42 C in the additional presence of 25 µM H7 (75). Chicken embryo fibroblasts (CEFs) were isolated and grown as described (4).

Viruses, Transfections, and Infections
DNA fragments containing the open reading frame of the wt mouse estrogen receptor (MOR) (77); MOR 121–599 (AF-1 deleted); MOR L543A/L544A (mutated AF-2); MOR 121–599,L543A/L544A [AF-1 deleted and AF-2 mutated (34)]; and MOR C241A,C244A [no DNA binding (35)] were excised from the expression vector pJ3 and inserted behind the CMV promoter of the avian retroviral expression vector pCRNCM (36). The retroviral constructs were transfected together with RCAN helper virus DNA in HD3 cells using diethylaminoethyl-dextran, and single cell derived colonies were selected in G418-containing methocell (36). The same retroviral constructs plus RCAN DNA were also transfected into CEFs using calcium phosphate precipitates, and G418-resistent cultures were selected (36). To generate mass cultures of SCF progenitors expressing the various MOR constructs, 40 x 106 Ficoll purified bone marrow cells were cocultivated with 1–2 x 106 virus producing CEFs for 1 day in 10 ml modified CFU-E medium supplemented with 100 ng/ml avian recombinant SCF. Next day nonadherent bone marrow cells were transferred to a fresh Petri dish and grown for 4 days in the same medium. The cells were kept at a density of 2–4 x 106/ml, daily transferred to a fresh dish to remove adherent cells, and daily provided with fresh medium and factors.

Measurement of Differentiation Parameters
Cell numbers and cell volume were measured with an electronic cell counter (CASY-1, Schärfe-System, Reutlingen, Germany). Aliquots of 1–2 x 105 cells (50 µl of an average culture) were transferred to 96-well plates to assay for [3H]thymidine incorporation, hemoglobin determination, or cytocentrifugation (see below).

Analysis of Differentiation by Cell Morphology and Staining
Cells were cytocentrifuged and subsequently stained with histological dyes and neutral benzidine for hemoglobin as described by Beug et al. (27). Images were taken using a CCD camera (Photometrics, Tucson, AZ) and a blue filter (480 nm), so that mature cells (stained yellow to brownish) appear darkly stained. Images were processed with Adobe Photoshop (Adobe Systems, Inc., Mountain View, CA).

[3H]Thymidine Incorporation
Aliquots (3 x 50 ml) of the cultures were dispensed into the wells of a 96-well plate and labeled for 2 h with 0.8 mCi [3H]thymidine per well ([3H]-TdR, Amersham, Arlington heights, IL; ~30 Ci/mmol). Labeled cells were harvested onto glass fiber filters using an automated cell harvester (Tomtec, Orange, CT), and the cell-bound radioactivity was determined in an 96-well scintillation counter (Microbeta 1450; Wallac, Gaithersburg, MD) essentially as described earlier (37, 78).

Photometric Hemoglobin Assay
Aliquots (3 x 50 ml) of the cultures were removed and processed for photometric determination of hemoglobin as described (79).

Northern Blot
RNA was isolated from HD3 cells or erythroid progenitor cells using the method of Chomczynski and Sacchi (80) with minor modifications. Cells were lysed in guanidinium isothiocyanate buffer and NaAc, pH 4.0, was added to 25 mM. The solution was extracted with H2O-saturated phenol, chloroform, and isoamylalcohol. RNA was precipitated from the aqueous phase with isopropanol, all as described. Subsequently, the pellet was dissolved in 10 mM Tris, pH 7–1 mM EDTA-0.2% SDS, and proteinase K was added to 200 µg/ml. After 30 min incubation at 37 C, the solution was extracted with phenol-chloroform-isoamylalcohol (25:24:1), pH 7, and precipitated with ethanol. RNA (10–20 µg) was run on a formaldehyde-containing agarose gel and transferred to nylon filters (Gene Screen, Life Science Products, Boston, MA) using conventional procedures (81). The cDNA probes used have been described previously described (8, 82)

Run-on Analysis
HD3 cells or primary erythroid progenitors were differentiated in medium with freon-stripped serum to reduce serum levels of estradiol, 2% AS, and insulin as described above. HD3 cells were differentiated in the presence of 25 µM H7 at 42 C. To the primary cells 2 ng/ml TGF{alpha} were added. After differentiation for the times indicated in the text, the cells were split in two aliquots, and either estradiol or ICI 164,384 was added to 106 M. At times indicated, the cells were harvested and nuclei were isolated and processed for run-on analysis as described (40). Radioactively labeled RNA was hybridized to filters on which total plasmid DNA containing the probes mentioned above (8) was blotted. Radioactive signals were measured on a PhosphoImager (Molecular Dynamics, Sunnyvale, CA) using the Imagequant program (Molecular Dynamics). Signals were corrected for small differences in the amount of nuclei used using the transcriptional signals for the constitutively expressed HEO (HD3 clone 4) or for rRNA.


    ACKNOWLEDGMENTS
 
The authors wish to thank Dr. S. Green (Chicago) for his kind gift of monoclonal antibody to the ER (H222), M. Busslinger (IMP, Vienna, Austria), and Henk Stunnenberg (Heidelberg, Germany) for helpful discussions and suggestions, and Evi Deiner for expert technical assistance.


    FOOTNOTES
 
Address requests for reprints to: Marieke von Lindern, Institute of Hematology, Erasmus University, Box 1738, NL-3000 DR Rotterdam, The Netherlands.

M. von Lindern and H. Beug were recipients of funding from the Austrian Research Promotion Fund.

1 Present adress: Department of Biological Chemistry, University of California, Los Angelos, CA 90095-1737. Back

2 Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A-3PX U.K. Back

Received for publication April 18, 1997. Revision received November 17, 1997. Accepted for publication November 19, 1997.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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