Developmental Silencing of Human ζ-Globin Gene Expression Is Mediated by the Transcriptional Repressor RREB1^*

Plasmids

The construct pBS-HS40-ζ-hGH described previously was used in the current study but with replacement of the backbone with that of pBluescript II KS(−) (Stratagene) (21). This new pBS-HS40-ζ-hGH plasmid was then used as the parental plasmid to introduce different mutations into the ζ-globin promoter with the use of the QuikChange site-directed mutagenesis kit from Stratagene. pBS-HS40-α-hGH was generated by replacement of the ζ-globin promoter in pBS-HS40-ζ-hGH with a 1.5-kb PstI fragment of the α-globin promoter. pEF-Myc-RREB1 was constructed by cloning of the RREB1 cDNA (GenBank^TM accession number NM_001003699) amplified by PCR using the PfuUltra^TM II Fusion HS DNA polymerase (Stratagene), in the SalI/XhoI sites of the pEF/myc/cyto vector (Invitrogen).

Cell Cultures and DNA Transfection

K562 cells were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin, in a 37 °C chamber under a 5% CO₂ humidified atmosphere. MEL, HeLa, and 293T cells were cultured in the same conditions but in Dulbecco's modified Eagle's medium (Invitrogen). For DNA transfections, the cells were harvested at a density of 5–8 × 10⁵ (K562) or 8–10 × 10⁵ (MEL, 293T, and HeLa) per ml. The transfection was carried out using the Neon^TM transfection system (Invitrogen). 2 × 10⁶ cells were transfected with 5 μg of the test plasmids and 1 μg of pCMV-β-gal. Following microporation, the K562 cells were seeded in 6-well plates with 5 ml of antibiotic-free RPMI for 48 h before the human growth hormone (hGH) assay. The MEL cells were seeded with 5 ml of antibiotic-free Dulbecco's modified Eagle's medium for 24 h and then induced with 2% DMSO for 96 h before the hGH assay. The 293T and HeLa cells were seeded in 6-well plates with 5 ml of antibiotic-free Dulbecco's modified Eagle's medium for 48 h before the hGH assay.

Generation and Genotype Analysis of Transgenic Mice

The transgenic mice were generated in the transgenic core facility at the Institute of Molecular Biology using the standard pronuclei microinjection method. The XhoI-NotI DNA fragments isolated from the pBS-HS40-ζ-GH plasmid series (see Fig. 1B) were used for microinjection. For genotyping of the mouse tail DNAs, the transgene was detected with use of the primers (5′-TGCTTGTCAGGGGACAGATCC-3′ and 5′-ATTGGTCAGGTGAGGGGAGG-3′), which amplified a 464-bp product. For further structural and copy number analysis, the tail DNAs were digested with BamHI and analyzed by Southern blotting with several probes. The PA1 probe (1,026 bp) hybridized to the 5′-end of the transgene, whereas the PA3 probe (1,051 bp) hybridized to a 2.2-kb BamHI fragment. The 960-bp methyltransferase (MT) probe from the DNA methyltransferase I gene was used as the loading control. The XhoI-NotI fragment of the transgene was also used as the copy number standard in the Southern blotting analysis (Fig. 2A). After PA1 hybridization, the integrity and the single copy nature of the transgene could be determined. Use of the PA3 probe and comparison of the hybridization intensities with those of the copy number standard blot provided the copy numbers of the transgene in different lines (Table 1). Quantitation of the band intensities on the blots was carried out in a Fuji FLA-5000 phosphor imager.

Semiquantitative RT-PCR Analysis

For induction of anemia, 8-month-old mice were injected with phenylhydrazine (40 μg/g of body weight), twice separated by 8 h. The treated mice were sacrificed on day 6, and the tissue RNAs were isolated by TRIzol reagent (Invitrogen). Each RT reaction was performed with the use of SuperScript II reverse transcriptase (Invitrogen) and 1 μg of RNA. One-tenth of the RT products was used as the template in PCR (Fermentas). The amplifications were carried out at the thermal cycle of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. The products of hGH and mouse G3PDH are 313 and 525 bp, respectively. The sequences of the primers are as follows: 5′-hGH, 5′-AGGAAGGCATCCAAACGCTG-3′; 3′-hGH, 5′- ATTAGGACAAGGCTGGTGGG-3′; 5′-mouse G3PDH, 5′- GGTCATCCATGACAACTTTGG-3′; 3′-mouse G3PDH, 5′-TCTTACTCCTTGGAGGCCATG-3′.

Electrophoretic Mobility Shift Assays (EMSA)

Nuclear extracts were prepared from K562, MEL, HeLa, and 293T cells as described previously (26), in the presence of protease inhibitors (Roche Applied Science protease inhibitor mixture tablets). The oligonucleotides used are listed in Fig. 3A. All of the DNA binding reactions were performed as described by Wen et al. (27) with minor modifications. The double-stranded probes were 5′-end-labeled with ³²P by T4 polynucleotide kinase (New England Biolabs) and then purified over a Sephadex G-25 column (Roche Applied Science). 5 or 10 μg of the nuclear extracts were incubated with the probe (100,000 cpm) at room temperature for 15 min in 20 μl of 20 mm HEPES (pH 7.9), 50 mm KC1, 1 mm MgCl₂, 0.5 mm dithiothreitol, 4% glycerol, and 1 μg of poly(dI-dC). For competition EMSA, excess cold oligonucleotides were used (see legend to Fig. 3 for more details).

For further identification of the complexes by a supershift assay, the antibody anti-GATA1 (sc-265, Santa Cruz Biotechnology, Inc. (Santa Cruz, CA)) or anti-Myc (LTK BioLaboratories) was preincubated with the nuclear extract on ice for 30 min before use in EMSA. Normal IgG was used as the control.

siRNA Interference

The target sequences for siRNA interference of RREB1 mRNA (siRNA 1, 5′-GGGCAGACCUUUCAUACAGUU-3′; siRNA 2, 5′-GAAGAAAGCUGAUGAAGUCUU-3′) were identified using the manufacturer's design (ON-TARGETplus, Dharmacon). One strand of the control duplex RNA targeting the firefly luciferase mRNA is 5′-CUUACGCUGAGUACUUCGAUU-3′. 2 × 10⁶ K562 cells were transfected with the duplex RNA oligonucleotides at a concentration of 100 nm. Using the Neon^TM transfection system (Invitrogen), cells were microporated at 1,300 V with a 30-ms width and one pulse and then reseeded in 2 ml of antibiotic-free RPMI. 2 ml more of the medium were added to the cells 6 h later. After incubation for 48 h, half of the cells were harvested for assay of the gene expression. The remnants were remicroporated with siRNA oligonucleotides using the same conditions and incubated for another 48 h before the assay.

Lentivirus-mediated Knockdown Experiments

Lentiviral plasmids (pLKO.1-shRNA) expressing short hairpin RNAs (shRNA1, 5′-CCGGCCAGGAAACGAAAGAGGAGAACUCGAGUUCUCCUCUUUCGUUUCCUGGUUUUU-3′; shRNA2, 5′-CCGGCGACGAUGACAAGAAACCAAACUCGAGUUUGGUUUCUUGUCAUCGUCGUUUUU-3′) targeting the RREB1 mRNA were acquired from the TRC (The RNAi Consortium) lentiviral shRNA library (28). pLKO.1sh expressing a scrambled shRNA and the shRNA-null puromycin-resistant vector (pLKO.1) were used to produce the control lentiviruses. The viruses were prepared by co-transfecting 293T cells with the plasmid (pLKO.1-shRNA, pLKO.1sh, or pLKO.1), the packaging plasmid (pCMV-ΔR8.91), and the envelope plasmid (pMD.G). The culture medium containing lentiviruses was harvested at 64 h post-transfection for the estimation of the viral titer. For RNAi knockdown in K562 cells, spin infection (multiplicity of infection = 2) was carried out at 2,750 g in 6-well plates for 30 min at 25 °C, with a final concentration of 8 μg/ml polybrene in the culture medium. After 24 h of lentiviral infection, the cells were selected with 2.5 μg/ml puromycin for another 4 days. The total RNAs were harvested from cells at 5 days and 10 days postinfection, respectively, for further quantitative RT-PCR analysis.

For RNAi knockdown experiments of primary human erythroid culture, the culture was initiated and prepared following the standard protocol (29) except that StemSpan SFEM medium (StemCell Technologies) was used for culturing and maintenance of the cells (14). The cells were maintained in the differentiation medium at a density of 0.1–1 × 10⁶ cells/ml. The lentivirus transductions were carried out on day 2 of the phase II culture of erythroid differentiation. Puromycin (2 μg/ml) selection was started at 24 h post-transduction for 9 days, and the total RNAs were then isolated using the RNAqueous®-Micro Kit (Ambion) for analysis by quantitative RT-PCR.

Quantitative RT-PCR Analysis

RNAs from E9.5 mouse embryos with the yolk sac and the E14.5 mouse fetal liver were isolated with use of TRIzol reagent (Invitrogen). The RNAs of the RNAi knockdown cells were purified using the RNAspin mini kit (GE Healthcare). cDNA synthesis was carried out using SuperScript II reverse transcriptase (Invitrogen). Quantitative RT-PCR was performed by using the SYBR Green PCR Master Mix (Applied Biosystems) and ABI 7500 real-time system. All data were analyzed after normalization to the expression level of mouse glycophorin A or human β-actin gene. The sequences of the primers used for the quantitative RT-PCR are as follows: 5′-hGH, 5′-TAGAGGAAGGCATCCAAACG-3′; 3′-hGH, 5′-GTCTGCTTGAAGATCTGCCC-3′; 5′-mouse GPA, 5′-GCCGAATGACAAAGAAAAGTTCA-3′; 3′-mouse GPA, 5′-TCAATAGAACTCAAAGGCACACTGT-3′; 5′-human β-actin, 5′-CCTGAACCCCAAGGCCAACC-3′; 3′-human β-actin, 5′-CAGGGATAGCACAGCCTGGA-3′; 5′-RREB1, 5′-CGACTTAGGATTCACGGACTTC-3′; 3′-RREB1, 5′-CAGACAAAACGGTGTTGCTC-3′; 5′-human GATA1, 5′-TGGCCTACTACAGGGACGCT-3′; 3′-human GATA1, 5′-CATATGGTGAGCCCCCTGG-3′; 5′-human G3PDH, 5′-CAACTTTGGTATCGTGGAAGGACTC-3′; 3′-human G3PDH, 5′-AGGGATGATGTTCTGGAGAGCC-3′.

Functional Role of the ZF2 Motif in Erythroid Cell Cultures

The factor-binding motifs in the human ζ-globin promoter region from −250 to −70, as determining previously by footprinting analysis in K562 nuclear extract (24), are displayed in Fig. 1A. Among these factor-binding motifs, ZF2 (−169 to −148) (Fig. 1A) consists of a GATA-1 binding site followed by a sequence homologous to the consensus of the binding sites of the factor RREB1 (Fig. 1B). To examine the contributions of the putative GATA-1 and RREB1 binding sites within the ZF2 motif to the ζ-globin promoter activity, we introduced three different types of mutations (mT, mCC, and 3nt; Fig. 1B) into the ZF2 motif of the promoter. The activities of the wild type ζ-promoter and the three mutants were then compared by hGH reporter assay in transiently transfected K562 and MEL cells (Fig. 1B). As seen, the abolishment of the GATA-1 binding site on ZF2 (mutant mT) caused significant reduction of the ζ-promoter activity in K562 as well as in MEL cells (Fig. 1B, gray bars). On the other hand, mutation of the putative RREB1 binding site (mCC) resulted in ∼2-fold higher promoter activity in either K562 or MEL cells (Fig. 1B, black bars) but not in the non-erythroid 293T and HeLa cells in which the ζ-globin promoter activities were very low (Table 2). The elevated activity of the mCC mutant promoter in the erythroid cell lines was consistent with the previous study (25), further suggesting that a factor(s) binding to the predicted RREB1 site of ZF2, possibly RREB1, was a repressor of the human ζ-globin promoter.

Physiological Role of ZF2 Motif in Transgenic Mice

To further address the physiological role of ZF2 in the regulation of the ζ-globin promoter, we analyzed transgenic mice carrying the same ζ-GH reporter fragments as used in Fig. 1, with or without the mCC mutation (Table 1 and Fig. 2). The positive lines were first identified by the presence of a 464-bp PCR fragment amplified from the human ζ-promoter region (see “Experimental Procedures”) (data not shown). These lines were further analyzed by Southern blotting, as exemplified in Fig. 2A. The copy numbers of the transgene were determined and listed in Table 1. They varied from 1 to 60 for the wild type transgene and from 1 to 40 for the mutant transgene (Table 1). As measured by the hGH assay of the plasma samples from the adult transgenic mice, the activities of the wild type human ζ-globin promoter in most of the transgenic lines were very low, as also observed previously (23). In contrast to the wild type, the mutant lines, except for line 251, showed a significantly higher level of hGH per copy of the transgene. In particular, for the low copy lines (e.g. 221, 222, and 223), the levels of the plasma hGH per copy of the mutant transgene were 10–40 times higher than in mice carrying the wild type promoter (Table 1).

We have also analyzed the hGH expression in the mutant lines by RT-PCR analysis. First, to analyze the level in the adult mice, the mice were treated with phenylhydrazine to increase the erythropoiesis. RNAs were then isolated from the blood, spleen, liver, kidney, and brain and analyzed by semiquantitative RT-PCR. As shown in Fig. 2B, for both the wild type and the mutant transgenic lines, the RT-PCR signals were detected mainly in the blood samples (Fig. 2B, lanes B), although a minor signal could also be seen in the spleen samples (Fig. 2B, lanes S). Finally, quantitative RT-PCR analysis showed that the mutant ζ-globin promoter was also derepressed in the yolk sac of E9.5 embryos and fetal liver of E14.5 embryos (Fig. 2C). The data of Fig. 2 suggested that ZF2 also played a repressive role in the regulation of the human ζ-globin promoter activity in vivo during erythroid development.

Factor Binding at the ZF2 Motif

Following the above, we have used EMSA to examine the nature of the complex(es) formed on the ZF2 motif, in particular at the RREB1 sequence. For this, four oligonucleotides containing the wild type and the three different mutant ZF2 sequences listed in Fig. 1B were used as the EMSA probes (Fig. 3A). As shown in lanes 2–5 of Fig. 3B, four major complexes (a, b, G, and ?) were present on the EMSA gels when the wild type (wt) ZF2 oligonucleotide was used. Of the four, the band marked with a question mark was present only in K562 nuclear extract, and it formed with all four oligonucleotides (Fig. 3B). This complex band was not studied further. Of the other three, complex a formed in all four nuclear extracts tested, whereas complex G appeared only in the erythroid extracts (compare lanes 2 and 3 with lanes 4 and 5; Fig. 3B). Complex b was not present in the uninduced MEL extract (Fig. 3B, lane 3).

As described in Fig. 1 already, ZF2 consisted of two factor-binding sites, one for GATA-1 and the other homologous to the RREB1 consensus binding sequence. To correlate the three EMSA complexes (complexes a, b, and G) with these factors, oligonucleotides containing mutations at the GATA-1 site (mT), the RREB1 sequence (mCC), and both (3nt), respectively (Fig. 3A), were used in EMSA. As shown, when oligonucleotide ZF2 (mCC) was used, mainly band a disappeared (Fig. 3B, compare lanes 7 and 8 with lanes 2 and 3), suggesting that band a was a protein-DNA complex formed at the RREB1 sequence. On the other hand, band G disappeared when oligonucleotide ZF2 (mT) was used (Fig. 3B, compare lanes 10 and 11 with lanes 2 and 3), suggesting that it was a GATA-1-DNA complex. At the same time, the amounts of both complexes a and b increased in EMSA with the mT oligonucleotide (Fig. 3B, lanes 10 and 11). We interpreted the data of Fig. 3B as partly the result of the close proximity and possibly the overlapping nature of the RREB1 sequence and the GATA-1 binding site (see sequence of the oligonucleotide ZF2 (wt) in Fig. 3A). When GATA-1 binding site was mutated, more bindings of factors (e.g. RREB1) to the RREB1 sequence and other yet-to-be-determined factor-binding sites on the oligonucleotide became possible, thus forming more complexes a and b, respectively. In agreement with the EMSA data above, on the gel with use of the ZF2 (3nt) oligonucleotide, in which both the GATA-1 binding site and RREB1 sequence were mutated, complex a as well as complex G disappeared, whereas complex b increased significantly (Fig. 3B, lanes 13 and 14).

The above suggested factor-binding scenario at the ZF2 motif in the K562 nuclear extracts was further confirmed by EMSA with ³²P-labeled probe(s), a 50–200-fold molar excess of cold oligonucleotide competitors, and supershift assays (Fig. 3, C–H). For example, the formation of complex G with the ZF2 (wt) oligonucleotide as the probe was abolished when a 100-fold excess of cold, GATA-1-binding site-containing oligonucleotides (i.e. ZF2 (wt) (Fig. 3C, lane 2), ZF2 (mCC) (Fig. 3C, lane 5), and GATA-1 (Fig. 3D, lanes 3 and 5)) (30) were preincubated with the nuclear extracts. That complex G, a GATA-1-DNA complex, was also confirmed with the use of an anti-GATA-1 antibody in a supershift assay (Fig. 3E). Similarly, the formation of the complex a was competed away with the use of the ZF2 (wt) or ZF2 (mT) oligonucleotide as the competitor (Fig. 3C, lanes 2 and 3) but not by the ZF2 (3nt) or ZF2 (mCC) oligonucleotide (Fig. 3C, lanes 4 and 5). More significantly, the formation of complex a on the wild type ZF2 oligonucleotide in the K562 nuclear extract could be effectively competed by an excess of either cold ZF2 oligonucleotide itself or by cold RREB1-binding site-containing oligonucleotide (Fig. 3F, left), the latter of which was shown before to bind the RREB1 factor specifically (31). The reverse competitive EMSA experiment gave a similar result (Fig. 3F, right). Furthermore, the use of the oligonucleotide X1 or X2, both of which consisted of sequences unrelated to the RREB1-binding consensus, as the competitor could not abolish the formation of complex a, whereas the ZF2 and RREB1 oligonucleotides could (Fig. 3G, compare lanes 4 and 5 with lane 3).

That band a was a RREB1-DNA complex was further supported by a supershift assay. Because no anti-RREB1 of supershift quality was available, we overexpressed Myc-tagged RREB1 by transient transfection of K562 cells with the plasmid pEF-Myc-RREB1 (Fig. 3H, left, lane 3). As seen in the right panel of Fig. 3H, preincubation of the Myc-RREB1-containing nuclear extract with anti-Myc resulted in the decrease of the intensity of band a (Fig. 3H, compare lanes 7 and 8 with lanes 5 and 6). The data of Fig. 3 together strongly suggested that RREB1 was the factor binding to the RREB1 sequence in the ZF2 motif.

Elevated Expression of the Human ζ-Globin Gene in RREB1-depleted Cells

To further verify the negative regulatory effect of RREB1 on the ζ-globin promoter activity, as suggested above by data from Figs. 1–3, we first knocked down the RREB1 expression in K562 cells by RNAi. For this, two different siRNA oligonucleotides were used, both of which were targeted at two specific sequences on the human RREB1 mRNA. After the RNAi treatment of K562 cells, the RNA was isolated and analyzed by quantitative RT-PCR. Of the two siRNA oligonucleotides, siRNA 1 reduced the level of RREB1 mRNA by 67%, and siRNA 2 reduced the level by 35% after 48 h of treatment. A small increase of the ζ-globin mRNA (1.4-fold) was observed. To our surprise, reduction of RREB1 also caused an increase in the level of the α-globin mRNA by ∼1.7-fold, whereas those of the GATA-1 mRNA and G3PDH mRNA were not altered (Fig. 4A, top). At 96 h of the siRNA treatment, more increases of the two globin mRNAs were observed, with the ζ-globin mRNA elevated by 2-fold and the α-globin mRNA elevated by 2.7-fold in siRNA 1-treated samples (Fig. 4A, bottom).

We also analyzed the level of the ζ-globin mRNA in K562 cells after more persistent reduction of the RREB1 expression with the use of two different recombinant lentiviruses, each expressing an shRNA targeting the RREB1 mRNA. The empty lentiviral vector was used as the control. As seen, the RREB1-shRNAs reduced the RREB1 mRNA levels to 40% after 5 days of infection (data not shown), and the knockdown of RREB1 mRNA could last for 10 days (Fig. 4B). Western blot using an anti-RREB1 antibody from Rockland indicated that the RREB1 protein level was indeed lowered after knockdown of the RREB1 mRNA (Fig. 4B, inset). The ζ-globin mRNA also remained at a high level after 10 days of infection, ∼4–8-fold higher than the controls (Fig. 4B). Notably, the increases of the ζ- and α-globin gene expression, as shown in Fig. 4, were not by an indirect effect due to the induction of erythroid differentiation because the mRNA levels of neither erythroid-related genes (e.g. GATA-1 and NF-E2) nor non-erythroid genes (e.g. G3PDH) were significantly different between K562 cells with and without depletion of the RREB1 mRNA by RNAi knockdown (Fig. 4) (data not shown).

Finally, we have tested the effect of knockdown of RREB1 expression by lentivirus-based shRNA in primary culture of adult human erythroid cells. Although shRNA1 could not effectively knock down the level of RREB1 mRNA in the primary culture (data not shown), expression of the lentivirus-based shRNA2 consistently reduced the RREB1 mRNA level by 50% (Fig. 4C). Interestingly, this reduction greatly increased the level of the ζ-globin mRNA, by 70-fold, but not that of the α-globin mRNA (Fig. 4C). As in RNAi-knockdown K562 cells, the levels of the GATA1 and G3PDH mRNAs were not altered either (Fig. 4C). These data of Fig. 4 strongly suggested that the RREB1 played a repressive role in the human ζ-globin gene expression in vivo.