An embryonic/fetal beta-type globin gene repressor contains a nuclear receptor TR2/TR4 heterodimer
2002; Springer Nature; Volume: 21; Issue: 13 Linguagem: Inglês
10.1093/emboj/cdf340
ISSN1460-2075
AutoresOsamu Tanabe, Fumiki Katsuoka, Andrew D. Campbell, Weimin Song, Masayuki Yamamoto, Keiji Tanimoto, J Engel,
Tópico(s)RNA modifications and cancer
ResumoArticle1 July 2002free access An embryonic/fetal β-type globin gene repressor contains a nuclear receptor TR2/TR4 heterodimer Osamu Tanabe Osamu Tanabe Department of Biochemistry, Molecular Biology and Cell Biology, and Robert H.Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208-3500 USA Search for more papers by this author Fumiki Katsuoka Fumiki Katsuoka Department of Biochemistry, Molecular Biology and Cell Biology, and Robert H.Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208-3500 USA Search for more papers by this author Andrew D. Campbell Andrew D. Campbell Department of Biochemistry, Molecular Biology and Cell Biology, and Robert H.Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208-3500 USA Search for more papers by this author Weimin Song Weimin Song Department of Biochemistry, Molecular Biology and Cell Biology, and Robert H.Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208-3500 USA Search for more papers by this author Masayuki Yamamoto Masayuki Yamamoto Centre for TARA, University of Tsukuba, Tsukuba, Ibaraki, 305-8577 Japan Search for more papers by this author Keiji Tanimoto Keiji Tanimoto Centre for TARA, University of Tsukuba, Tsukuba, Ibaraki, 305-8577 Japan Search for more papers by this author James Douglas Engel Corresponding Author James Douglas Engel Department of Biochemistry, Molecular Biology and Cell Biology, and Robert H.Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208-3500 USA Search for more papers by this author Osamu Tanabe Osamu Tanabe Department of Biochemistry, Molecular Biology and Cell Biology, and Robert H.Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208-3500 USA Search for more papers by this author Fumiki Katsuoka Fumiki Katsuoka Department of Biochemistry, Molecular Biology and Cell Biology, and Robert H.Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208-3500 USA Search for more papers by this author Andrew D. Campbell Andrew D. Campbell Department of Biochemistry, Molecular Biology and Cell Biology, and Robert H.Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208-3500 USA Search for more papers by this author Weimin Song Weimin Song Department of Biochemistry, Molecular Biology and Cell Biology, and Robert H.Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208-3500 USA Search for more papers by this author Masayuki Yamamoto Masayuki Yamamoto Centre for TARA, University of Tsukuba, Tsukuba, Ibaraki, 305-8577 Japan Search for more papers by this author Keiji Tanimoto Keiji Tanimoto Centre for TARA, University of Tsukuba, Tsukuba, Ibaraki, 305-8577 Japan Search for more papers by this author James Douglas Engel Corresponding Author James Douglas Engel Department of Biochemistry, Molecular Biology and Cell Biology, and Robert H.Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208-3500 USA Search for more papers by this author Author Information Osamu Tanabe1, Fumiki Katsuoka1, Andrew D. Campbell1, Weimin Song1, Masayuki Yamamoto2, Keiji Tanimoto2 and James Douglas Engel 1 1Department of Biochemistry, Molecular Biology and Cell Biology, and Robert H.Lurie Comprehensive Cancer Center, Northwestern University, Evanston, IL, 60208-3500 USA 2Centre for TARA, University of Tsukuba, Tsukuba, Ibaraki, 305-8577 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3434-3442https://doi.org/10.1093/emboj/cdf340 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We recently described an erythroid ϵ-globin gene repressor activity, which we named DRED (direct repeat erythroid-definitive). We show that DRED binds with high affinity to DR1 sites in the human embryonic (ϵ-) and fetal (γ-) globin gene promoters, but the adult β-globin promoter has no DR1 element. DRED is a 540 kDa complex; sequence determination showed that it contains the nuclear orphan receptors TR2 and TR4. TR2 and TR4 form a heterodimer that binds to the ϵ and γ promoter DR1 sites. One mutation in a DR1 site causes elevated γ-globin transcription in human HPFH (hereditary persistence of fetal hemoglobin) syndrome, and we show that this mutation reduces TR2/TR4 binding in vitro. The two receptor mRNAs are expressed at all stages of murine and human erythropoiesis; their forced transgenic expression reduces endogenous embryonic ϵy-globin transcription. These data suggest that TR2/TR4 forms the core of a larger DRED complex that represses embryonic and fetal globin transcription in definitive erythroid cells, and therefore that inhibition of its activity might be an attractive intervention point for treating sickle cell anemia. Introduction Changes in the program of expression of each of the five human β-type globin genes during embryonic development are a consequence of the influences of both local and distant transcriptional control elements. Transcript abundance from the globin genes is determined by the locus control region (LCR), a dispersed group of DNase I hypersensitive sites lying 5′ to the five human genes (Forrester et al., 1987; Grosveld et al., 1987). In contrast, temporal control of individual globin genes during erythropoiesis appears to be largely a property of more local regulatory influences: even individual human β-type globin genes are generally expressed at their proper developmental times (albeit at quite low levels) in transgenic mice in the absence of the LCR (Magram et al., 1985; Townes et al., 1985; Raich et al., 1990; Dillon and Grosveld, 1991). In order to investigate one aspect of how globin gene temporal specificity might be established, we showed that clustered mutations in the ϵ-globin promoter, incorporated into a 150 kb human β-globin yeast artificial chromosome (YAC), induced this exclusively embryonic gene to continued high-level expression into adulthood (Tanimoto et al., 2000). This mutation was originally intended to generate a high affinity binding site for an erythroid transcriptional activator, EKLF, since EKLF had been shown to be mandatory for adult β-globin transcriptional activation (Perkins et al., 1996; Wijgerde et al., 1996). To our surprise, we found that this mutational effect acted independently of EKLF (by breeding the mutant β-globin YAC transgene into an EKLF-null mutant background). We therefore investigated the possibility that continued adult erythroid expression of ϵ-globin in the mutant YAC might have been due to inadvertent mutation of a previously unrecognized ϵ-globin repressor binding site. We discovered that definitive erythroid cells indeed harbored a novel DNA binding activity that could represent this purported repressor, which we named DRED (direct repeat erythroid-definitive), and we showed that DRED bound to the ϵ-globin DR1 (direct repeat) promoter elements. We further demonstrated that DRED was not COUP-TF2, a nuclear receptor expressed exclusively in embryonic erythroid cells (Filipe et al., 1999). Of numerous genetic lesions shown to affect human β-globin transcription, one specific class of mutations cause continued synthesis of γ-globin from the fetal stage into adulthood. These mutations are collectively termed HPFH (hereditary persistence of fetal hemoglobin) syndromes, and lead to elevated synthesis (up to 30%) of γ-globin in definitive erythroid cells which normally have only very low levels (usually <1%) of tetrameric hemoglobin F (HbF, α2γ2). HPFH mutations include both small and large deletions in the β-globin locus, as well as point mutations in the two γ-globin gene promoters (Stamatoyannopoulos and Neinhuis, 1994). Of particular interest here, one non-deletion HPFH mutant allele (referred to as the Greek −117 Aγ HPFH) was found to lie near the CCAAT box of the Aγ-globin gene; importantly, this mutation alters a DR1 sequence element in the Aγ-globin gene promoter (Gelinas et al., 1985). Here we report the purification of DRED binding activity, as well as its cloning and initial characterization. The DR1-specific binding activity of DRED is composed of two nuclear orphan receptors that are expressed in both embryonic and adult erythroid cells. These two proteins have been shown to repress transcription collaboratively in other physiological settings, and we show that they do so here as well, implying that these orphan receptors are likely candidates to be a part of the DRED ϵ- and γ-globin gene repressor complex. Results DRED binding site specificity and purification After testing several strategies for purification based on the recovery of DRED electrophoretic gel mobility shift assay (EMSA) activity, we finally adopted differential precipitation, gel exclusion and ion exchange chromatography steps followed by a final DNA sequence affinity column (see Materials and methods). Nuclear extracts from mouse erythroleukemia (MEL) cells were tested for their degree of purification and percentage of EMSA activity recovered after individual fractionation steps. We first selected DRED activity by ammonium sulfate precipitation followed by gel filtration. The peak of DRED binding activity eluted from the gel exclusion column (Figure 1A) as a species of ∼540 kDa (Figure 1B), suggesting either that DRED was huge or that it was composed of a relatively stable admixture of multiple components. Figure 1.DNA binding characteristics and specificity of DRED. (A) MEL cell nuclear extracts were separated by filtration on a Superdex 200 column (Materials and methods) relative to known molecular mass standards: a, thyroglobulin (669 kDa); b, ferritin (440 kDa); c, catalase (232 kDa); d, aldolase (158 kDa); e, albumin (67 kDa); f, ovalbumin (43 kDa); and g, chymotrypsinogen A (25 kDa). The peak of DRED EMSA activity (horizontal arrow) elutes coincident with a predicted molecular mass of 540 kDa (B). (C) Oligonucleotides used in EMSA and DRED purification. Unless otherwise stated, each of the oligonucleotides referred to in the text were blunt ended, double-stranded DNAs. The top line shows the position of two inverted DR1 consensus sequence elements aligned above the two DR1 elements in the ϵ-globin gene promoter (epsi), while the remaining oligonucleotides are described in the text. The Greek −117 Aγ HPFH point mutation [gamma(HPFH)/CAAT] is indicated in lower case bold, as are all of the oligonucleotides that differ from parental (wild type) sequences. Genuine or postulated (test) DR1 elements are underlined. (D) EMSA was performed as described in Materials and methods using radiolabeled 'epsi' bearing two DR1 elements (Tanimoto et al., 2000) as the probe. MEL cell nuclear extract (5 μg) was incubated with a 200-fold molar excess of indicated competing oligonucleotides before the addition of radiolabeled probe. Download figure Download PowerPoint When crude nuclear extracts from MEL cells were tested for DRED binding specificity using the relevant region where we originally detected DRED binding activity (the 'epsi' oligonucleotide; Figure 1C) as a radiolabeled probe, we found that both individual DR1 elements in the ϵ-globin promoter ('epsi/proxi' and 'epsi/CAAT'; Figure 1C) competed effectively for DRED complex formation (Figure 1D, lanes 4 and 5), as did the 'gamma/CAAT distal' and consensus retinoic acid response element (RARE) oligonucleotides (Figure 1D, lanes 6 and 12). In contrast, a comparable segment of the adult β-globin gene promoter that lacks a DR1 element (β/CAAT), or a segment of the γ-globin promoter that contains a duplicated CAAT element lying closer to the gene (Figure 1C, γ/CAAT proxi) were far less effective competitors for DRED binding. To examine more closely the details of sequence requirements for DRED binding, we found that an oligonucleotide containing a two-base mutation far away from the DR1 CAAT box of the Aγ gene (Figure 1C, AG-1) was an effective competitor (Figure 1D, lane 9), while mutation of an additional 3 bp within the DR1 element (AG-1M) abrogated competition (Figure 1D, lane 10). Since the mutation in AG-1M lies within the DR1 element, but does not affect the partially overlapping 'CCTTG' motif, DRED must be a different protein than the one proposed to bind and repress transcription from the multiple γ-globin CCTTG promoter elements (Lee et al., 2000). In summary, these data confirmed our earlier suspicion that the DNA binding component of DRED was probably a member of the extended family of nuclear receptors (Tanimoto et al., 2000). To examine DRED affinity for the DR1 element in greater detail, we performed competitive equilibrium EMSA experiments. To do so, a constant concentration of MEL cell nuclear extract was added to an aliquot of radiolabeled probe (Epsi) (Tanimoto et al., 2000) plus varying quantities of different competitor oligonucleotides (see Materials and methods). After electrophoresis, the amount of DRED complex in each reaction was quantified on a phosphorimager. The data depicted in Figure 2 show that the ϵ-globin CAAT probe containing one DR1 element competes most effectively, and that the same protein–DNA complex is strongly inhibited by a γ-globin DR1 oligonucleotide (γ/CAAT; Figure 1C), while the analogous segment of the adult β-globin gene promoter (β/CAAT), which contains no DR1 element, is an ineffective competitor. An oligonucleotide bearing the −117 Aγ HPFH point mutation [γ(HPFH)/CAAT; Figure 1C] is a less effective competitor (∼3-fold) than the wild-type γ/CAAT DR1 oligonucleotide. Thus, a single DR1 element in the ϵ-globin gene promoter binds to DRED with a higher affinity than does the γ-globin DR1 element, but the −117 HPFH point mutation in the DR1 sequence that leads to elevated γ-globin transcription in vivo binds to DRED with 3-fold lower affinity than the wild-type γ-globin DR1 sequence. Figure 2.Competitive EMSA analysis of DRED binding to CAAT boxes from the β-like globin gene promoters. (A) Equilibrium competition EMSA was performed as described in Materials and methods using radiolabeled 'epsi' as probe. A 32-, 63-, 125-, 250-, 500- or 1000-fold molar excess of competing oligonucleotide [β/CAAT, γ(HPFH)/CAAT, γ/CAAT or ϵ/CAAT] was incubated with MEL cell nuclear extract before the addition of the radiolabeled probe. (B) The DRED EMSA bands shown in (A) were quantified on a phosphorimager and the relative amount of complex remaining at each competitor concentration was plotted relative to the same sample with no added competitor (set at 100%). The values represent the average of two independent experiments. Download figure Download PowerPoint In executing the final phase of the overall purification strategy that we finally adopted, the peak of binding activity from a DEAE column was twice applied to, and eluted from, an ϵ-globin DR1 sequence-specific affinity column (Figure 3A). The 0.4 M eluate from the sequence-specific affinity column containing peak DRED activity revealed four major protein species (Figure 3B, p37 to p190) as well as a number of other bands that were either less abundant or did not correlate well with DRED EMSA activity eluted from the final column. Figure 3.DRED purification. The DRED complex was purified as described in Materials and methods. (A) EMSA was performed by incubating crude nuclear extract (NE; 2 μg of protein), the pooled DEAE Sepharose fraction (DEAE, 0.2 μg, 10 μl) or the peak fraction from the DNA sequence affinity column (DR1, 10 μl) with radiolabeled 'epsi' probe with (+) or without (−) preincubation with excess unlabeled probe. The arrow indicates the mobility of the DRED complex. (B) Proteins recovered in the peaks of the DEAE Sepharose and DR1 sequence affinity fractions (40 μl each) were separated by SDS–PAGE and silver-staining. Download figure Download PowerPoint Nanoelectrospray mass spectrophotometric sequence analysis of peptides generated from all four major bands recovered from the SDS–polyacrylamide gel (Figure 3B) revealed co-purification of several well characterized nucleic acid binding proteins: the CDP (CCAAT displacement protein) homeoprotein in the p190 band, the leader-binding protein-1a (LBP-1a) and orphan nuclear receptors TR2 and TR4 in p64, and the upstream stimulatory factor-1 (USF-1) in p43. In particular, detection of unique signature peptides for murine (m)TR2 (S203PLAATPT FVTDSETAR219; DDBJ/EMBL/GenBank accession No. U28265) and mTR4 (I35QIVTAVDASGSSK48; DDBJ/EMBL/GenBank accession No. U11688) nuclear orphan receptors were of particular interest, since we had already concluded that the DRED DNA binding activity might contain nuclear receptor family members. We therefore focused on whether TR2 and/or TR4 might comprise the DNA binding component of DRED. DRED is partially composed of a heterodimer of the nuclear orphan receptors TR2 and TR4 In order to determine whether or not the EMSA band that we identified as DRED contained TR2 and/or TR4, we performed further EMSA studies on MEL cell nuclear extracts, by including in the reactions antibodies that might be expected to compete with, or block the interactions with, possible constituents of the DRED repressor. The addition of anti-TR2 (αTR2a) or anti-TR4 antibodies (αTR4a and αTR4b) to the EMSA reactions ablated the entire DRED band (Figure 1D, lanes 13–15). Importantly, addition of any of these three antibodies affected the whole complex, allowing us to conclude that TR2 and TR4 both participate in DRED DNA binding. Inclusion of an antibody to the nuclear orphan receptor COUP-TF2, which is expressed exclusively in primitive erythroid lineage cells (Filipe et al., 1999), did not affect the DRED EMSA band (Figure 1D, lane 16; Tanimoto et al., 2000), nor did inclusion of antibodies recognizing the CAAT-box factor CDP (Barberis et al., 1987; Mantovani et al., 1988, 1989; Superti-Furga et al., 1988) or USF-1 (Gregor et al., 1990; data not shown), which co-purified with TR2 and TR4 (Figure 1D). The inclusion of antibodies against another CAAT factor (NF-Y) or antibodies that recognize proteins known to associate with transcription factors as part of chromatin co-repressor complexes (e.g. mSin3; Ayer et al., 1995) also failed to produce a reaction (data not shown). In contrast to the single EMSA product observed in nuclear extracts prepared from definitive erythroid cells, several complexes from primitive erythroid cells appeared to bind specifically to the ϵ-globin DR1 elements: the supershift experiments show that these factors include both DRED and COUP-TF2 (Figure 4, lanes 9–11). Thus, embryonic erythroid cells contain multiple protein species that can bind to the DR1 element (Filipe et al., 1999), including at least two different factors that are not present in definitive erythroid cells. Figure 4.Embryonic erythroid cells contain multiple DR1 binding proteins. MEL (lanes 2 and 3) or K562 (lanes 4–11) nuclear extracts (5 μg of protein each) were incubated with the indicated competing oligonucleotides (Figure 1C) or antibodies before the addition of radio labeled 'epsi proxi' probe. See the text and Figure 1D legend for details. Download figure Download PowerPoint We then tested TR2 and TR4, both individually and together, for binding specificity after transfection into non-erythroid cells. Upon transfection of the wild-type receptors, nuclear extracts were prepared and examined for 'epsi/proxi' (Figure 1C) binding activity. When TR2 alone is transfected into quail fibroblast (QT6) cells, no new EMSA bands appear (Figure 5A, lane 2), while transfection of TR4 alone yields a weak EMSA product (Figure 5A, lane 3) that co-migrates with authentic MEL cell-derived DRED binding activity (Figure 5A, lane 5, arrow). This complex is more intense (suggesting either higher affinity or more abundant binding) when both TR2 and TR4 are co-transfected into QT6 cells (Figure 5A, lane 4). Figure 5.Reconstitution of DRED binding by expression of TR2 and TR4 in tissue culture cells. (A) TR2 or TR4 expression plasmids were transiently transfected into QT6 quail fibroblasts. Two days after transfection, nuclear extracts were prepared and subjected to EMSA using the 'epsi proxi' probe. MEL cell nuclear extract was used as a control. (B) Amino/Flag-epitope-tagged or wild-type TR2 or TR4 expression plasmids were transiently transfected into 293T cells. Two days after transfection, nuclear extracts were prepared and subjected to EMSA with the 'epsi proxi' probe. Nuclear extracts were preincubated with anti-Flag monoclonal antibody (+) or not (−), before the addition of the radiolabeled 'epsi proxi' probe. The arrows indicate the migration position of authentic DRED. Download figure Download PowerPoint To confirm that TR2 and TR4 are able to bind to the ϵ-globin promoter DR1 elements as a heterodimer, we prepared N-terminal Flag epitope-tagged versions of both TR2 and TR4, and repeated the transfections into the human embryonic kidney (293T) cell line. As shown in this experiment, the only EMSA product formed with nuclear extracts from TR2 plus TR4-transfected cells is a heterodimer, regardless of which molecule contains the Flag epitope (Figure 5B). Since inclusion of the anti-Flag antibody supershifts the entire EMSA band (i.e. there is no remaining antibody-unreactive EMSA product) when either the TR2 or TR4 molecules are epitope tagged, we conclude that DRED is composed of a heterodimer of TR2 and TR4 orphan nuclear receptors. TR2 and TR4 are expressed in both primitive and definitive murine and human erythroid cells We next examined embryonic and adult blood cells for the presence and relative abundance of both TR2 and TR4 mRNAs. For RNA analysis, primers corresponding to unique sequences within either cDNA were generated (Materials and methods). RNA was isolated from murine embryos throughout gestation from yolk sacs and fetal livers as the source of primitive and definitive embryonic erythroid cells, respectively, or anemic adult spleens (AdS) as the source of definitive adult erythroid cells. The level of HPRT mRNA was used as the internal co-amplification control. Figure 6 shows that TR2 mRNA is present in yolk sac erythrocytes at 9.5 days post-coitum (d.p.c.), and at this stage of erythroid development the mouse embryo contains only primitive, nucleated erythrocytes. The abundance of TR2 mRNA in erythroid cells diminished modestly during embryonic development, and is relatively constant even during adult erythropoiesis (in anemic adult spleen cells; Figure 6, upper left panel). TR4 mRNA levels during murine gestation remained essentially constant (Figure 6, lower left panel). Thus, both mRNAs were expressed in both primitive and definitive murine erythroid lineage cells. We also tested human CD34+ and CD34– adult bone marrow cells for TR2 and TR4 mRNA accumulation (using an S14 co-amplification control; right panels), and found that both TR2 and TR4 were modestly more abundant (<50%) in the CD34– fraction, but both were significantly more abundant in K562 (primitive erythroid) cultured cells. We conclude that TR2 and TR4 are expressed in both primitive and definitive murine and human erythroid lineage cells, but clearly during murine development the abundance of TR2 and TR4 mRNA does not vary significantly between primitive and definitive erythropoiesis. Figure 6.TR2 and TR4 are both expressed throughout murine and human hematopoiesis. Total RNA was prepared from mouse tissues or from cell lines, and used for cDNA synthesis (see Materials and methods). TR2 (top panels) and TR4 (bottom panels) transcript abundances were normalized to the expression levels of co-amplified mouse HPRT mRNA (C, left panels) or human ribosomal protein S14 mRNA (C, right panels). RT–PCR was performed with primers corresponding to TR2 (top panels) or TR4 (bottom panels). cDNAs used for the reactions corresponded to the cell types shown above each lane. For the human erythroid cells, total RNA was prepared from fractionated human bone marrow cells (CD34+ or CD34−) and was used for cDNA synthesis. RNAs recovered from the murine definitive erythroid cell line MEL or the human primitive erythroid cell line K562 were used as the positive controls. Download figure Download PowerPoint Forced expression of TR2 and TR4 in myeloerythroid lineages reduces endogenous murine ϵy gene transcription Finally, we addressed the possible functional consequences of TR2/TR4-forced expression on globin gene transcription in vivo. To do so, we linked the Flag-tagged TR2 and TR4 cDNAs to an erythroid expression vector, placing them under GATA1-HRD (GATA-1 gene hematopoietic regulatory domain) transcriptional control. The 6.1 kb GATA1-HRD cassette consists of 3.9 kb of 5′ DNA flanking the GATA-1 gene erythroid first exon (exon IE), the first intron and the second exon, including the GATA-1 translational start site. This segment of the GATA-1 gene has been shown to be necessary and sufficient for generating high level expression of linked cDNAs in primitive and definitive erythroid cells (Onodera et al., 1997). We have previously employed this vector to investigate the specific requirements of erythroid cells for GATA-1 (as compared with GATA-2 or GATA-3; Takahashi et al., 2000), and the role of small Maf proteins in megakaryopoiesis and platelet generation (Motohashi et al., 2000). During the abbreviated developmental process of erythropoiesis in the mouse, the βH1 gene is expressed most abundantly at ∼10.5 d.p.c. in the yolk sac, where it comprises ∼40% of total β-type mRNA, while ϵy transcription comprises the remaining 60% (Whitelaw et al., 1990). ϵy contains two promoter DR1 sites, while βH1 has one, and thus we assumed that any effect of increased TR2/TR4 levels would be most sensitively reflected in altered ϵy transcription. Since the ratios of adult α-globin to GATA-1 transcript levels do not vary significantly in the yolk sac between 9.5 and 11.0 d.p.c. (Whitelaw et al., 1990), we normalized the expression of ϵy and βH1 at 10.5 d.p.c. to α-globin mRNA levels, and the abundance of transgenic TR2 and TR4 mRNAs separately to GATA-1 abundance. We then examined the consequences of expressing variously elevated levels of transgenic TR2 and TR4 mRNAs on the endogenous murine embryonic β-type globin genes. The GATA1-HRD.TR2 and GATA1-HRD.TR4 DNAs were co-injected into fertilized eggs; the injected embryos were implanted into pseudopregnant foster mothers and allowed to develop to 10.5 d.p.c. The yolk sacs from injected embryos were then analyzed for approximate TR2/TR4 transgene copy number using PCR, and then for TR2 and/or TR4 transgene-derived mRNA levels as well as endogenous murine GATA-1, adult α-, embryonic ϵy- and embryonic βH1-globin mRNA accumulation, by RT–PCR. Transgene-specific TR2 or TR4 transcripts were detected by PCR using a GATA1 exon IE sense primer, which was common to both transgenes, and individual antisense TR2 or TR4 cDNA primers. The abundance of transgene-derived TR2 and TR4 transcripts was normalized to endogenous GATA-1 RT cDNA co-amplified by duplex PCR, and expression of ϵy- or βH1-globins was normalized to co-amplified α-globin. The normalized data (Figure 7) show that ϵy abundance diminishes markedly with increasing TR2 or TR4 transgene-derived transcript accumulation, while βH1 abundance does not vary significantly, or might even increase slightly (the result anticipated if ϵY and βH1 are in 'competition' for LCR activity at this stage) (Choi and Engel, 1988; Wijgerde et al., 1995). The correlation between TR2 or TR4 expression and diminished ϵy abundance is statistically significant (P < 0.5 and < 0.1%, respectively, by Pearson's correlation coefficient test). These data demonstrate that TR2 and TR4, when forcibly expressed in myeloerythroid lineage cells, repress endogenous ϵy transcription, and thus strongly support the working hypothesis that DRED is an erythroid repressor of DR1-dependent β-type globin transcription, and also that the TR2/TR4 heterodimer is the DNA recognition component of DRED. Figure 7.TR2 and TR4 erythroid-restricted expression represses ϵy-globin transcription. Transgenic founder embryos co-injected with TR2 and TR4 cDNAs transcriptionally directed by GATA1-HRD (see text) were analyzed for accumulation of transgenic TR2 or TR4 mRNAs, as well as for ϵy- and βH1-globin mRNAs in E10.5 yolk sacs by semi-quantitative RT–PCR. TR2 and TR4 mRNA levels were normalized to GATA-1 mRNA abundance, and ϵy- and βH1-globin mRNA levels were normalized to endogenous α-globin mRNA abundance (see text). Symbols: crosses, embryos expressing no transgene (n = 25); open circles, embryos expressing both TR2 and TR4 (n = 9); triangles, embryos expressing only TR2 (n = 1); inverted triangles, embryos expressing only TR4 (n = 4). Each of the lines was drawn by the least squares method. Analyzed by Pearson's correlation coefficient test, the correlation between TR2 or TR4 expression and diminished ϵy abundance was statistically significant (A and B), while there was no significant correlation between expression of TR2 or TR4 and βH1 (C and D). Download figure Download PowerPoint Discussion We showed here that DRED is (initially) a high molecular weight complex of ∼540 kDa, and that this complex binds with high affinity to both the human ϵ- and γ-globin DR1 promoter elements. We also showed, through equilibrium EMSA competition experiments, that DRED binds to the Greek HPFH point mutation with 3-fold lower affinity than it does to the wild-type Aγ gene promoter DR1
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