Developmental silencing of human zeta-globin gene expression is mediated by the transcriptional repressor RREB1

Abstract

The mammalian embryonic zeta-globin genes, including that of humans, are expressed at the early embryonic stage and then switched off during erythroid development. This autonomous silencing of the zeta-globin gene transcription is probably regulated by the cooperative work of various protein-DNA and protein-protein complexes formed at the zeta-globin promoter and its upstream enhancer (HS-40). We present data here indicating that a protein-binding motif, ZF2, contributes to the repression of the HS-40-regulated human zeta-promoter activity in erythroid cell lines and in transgenic mice. Combined site-directed mutagenesis and EMSA suggest that repression of the human zeta-globin promoter is mediated through binding of the zinc finger factor RREB1 to ZF2. This model is further supported by the observation that human zeta-globin gene transcription is elevated in the human erythroid K562 cell line or the primary erythroid culture upon RNA interference (RNAi)(2) knockdown of RREB1 expression. These data together suggest that RREB1 is a putative repressor for the silencing of the mammalian zeta-globin genes during erythroid development. Because zeta-globin is a powerful inhibitor of HbS polymerization, our experiments have provided a foundation for therapeutic up-regulation of zeta-globin gene expression in patients with severe hemoglobinopathies.

FIGURE 1.

Assessment of ζ-globin promoter activity in erythroid cell cultures. A, schematic illustrations of the α-like globin locus and the ζ-globin promoter region. The physical maps of the α-like globin gene cluster and the protein binding sites in the ζ-globin promoter are shown. Shown below the ζ-globin promoter is the ZF2 motif. The striped bar indicates the position of the ZF2 motif as mapped previously by footprinting analysis (24). B, the promoter-reporter construct is represented in the upper diagram. The reporter is hGH as driven by the human ζ-globin promoter (ζ) cis-linked with the HS-40 enhancer. The lowercase letters represent the mutated nucleotides in ZF2. The consensus GATA-1 and RREB1 sequences are boxed. Also listed is the consensus sequence of RREB1 binding sites (31). At 48 or 96 h after transfection, the culture media were collected, and the hGH levels were determined by radioimmunoassay. *, p < 0.05. The data were derived from three independent experiments.

FIGURE 2.

Analysis of transgenic mice. A, generation of the transgenic mice. Top, the diagram of the XhoI-NotI DNA fragment used for generation of the transgenic mice. The probes (PA1 and PA3) used for genotyping by Southern blotting are indicated by underlines under the reporter map. The copy numbers of the transgene were determined by Southern blotting as exemplified in the lower panels. The genomic DNAs from the tails were digested with BamHI and then hybridized with the probes. The DNA sizes of the markers are indicated on the right sides of the blots. The position of the head-to-tail tandem repeats of the transgene are marked on the side of the left blot. The endogenous DNA methyltransferase gene (MT) serves as the loading control on the blots. For copy number determination of the transgene, known copies of BamHI-digested XhoI-NotI fragment were loaded on gel and probed with PA3 (right). B, tissue-specific expression patterns of the wild type (wt) and mCC (mt) ζ-globin promoters in transgenic mice. The phenylhydrazine-treated, anemic mice were sacrificed, and the total RNAs were purified from several different adult tissues. The levels of the hGH RNAs were determined by semiquantitative RT-PCR using mouse G3PDH as the internal control. B, blood; S, spleen; L, liver; K, kidney; Br, brain. C, quantitative RT-PCR analysis of hGH mRNA in total RNAs isolated from E9.5 embryos and E14.5 fetal livers. Note the higher hGH mRNA levels in samples with mutant human ζ-globin promoter transgenes. wt, wild type.

FIGURE 3.

EMSA of factor-binding on the ZF2 motif. The identification of factors binding to the ZF2 motif in nuclear extract was analyzed by EMSA. A, nucleotide sequences of the oligonucleotides used for EMSA. Only one strand of each oligonucleotide is shown. The GATA-1 and RREB1-binding sequences on the wild type ZF2 are indicated. B, formation of DNA-protein complexes in nuclear extracts prepared from K562 (K), uninduced MEL (UM), HeLa (H), and 293T (T) cells. The four slowly migrating DNA-protein complexes formed in the K562 (K) extracts (a, b, G, and ?) are indicated. Note the presence of complex G only in the K and UM lanes. Also, the complex a is absent when the ZF2 (mCC) or ZF2 (3nt) oligonucleotide was used as the probe. C, competition among different ZF2 oligonucleotides. The factor-binding specificities on the ZF2 motif in K562 nuclear extract were determined with or without the presence of a 100-fold molar excess of unlabeled oligonucleotides, as indicated in the figure as the competitors. D, competition between ZF2 and GATA-1 oligonucleotides in EMSA. Both the K562 and uninduced MEL extracts were used. Note that complex G but not complex a disappeared (arrowhead) in the presence of a 100-fold molar excess of cold GATA-1 oligonucleotide. E, supershift assay using anti-GATA1. Nuclear extracts from three different cell types were prepared as described above, preincubated with the anti-GATA1 antibody, and then used in EMSA. Note the disappearance (arrowhead) of band G but not band a or band b upon preincubation with anti-GATA1 (lanes 5–7). F, competition between ZF2 and RREB1 oligonucleotides in EMSA. Note that complex a but not complex G or b formed on the ZF2 (wt) oligonucleotide disappeared upon use of a 100–200-fold molar excess of cold RREB1 oligonucleotide (lanes 5 and 6). All three complexes disappeared in the presence of cold ZF2 (wt) oligonucleotide (lanes 3 and 4). G, competition among ZF2, RREB1, X1, and X2 oligonucleotides. Note that complex a formed on the ZF2 oligonucleotide was competed out by an excess of cold ZF2 (lane 2) or RREB1 oligonucleotide (lane 3) but not by a 100-fold molar excess of X1 (lane 4) or X2 (lane 5). H, supershift assay using anti-Myc. Left, EMSA patterns using the ZF2 (wt) oligonucleotide (lanes 1–3) or ZF2 (mCC) oligonucleotide (lane 4) and nuclear extracts prepared from K562 cells transfected with pEF-Myc vector (lane 2) and pEF-Myc-RREB1 (lanes 3 and 4), respectively. Note the increase of the complex band a in lane 3. Right, patterns of EMSA using the ZF2 oligonucleotide and Myc-RREB1-overexpressing K562 nuclear extract without (lane 5) or with preincubation with increasing amounts of the anti-Myc antibody (lanes 6–8). wt, wild type.

FIGURE 4.

The expressional levels of α-like globin genes in RREB1-depleted cells. A, siRNA oligonucleotides were transiently transfected into K562 cells by electroporation. The cells were collected 48 h later to purify the RNA for analysis. The remnants were re-electroporated with the same siRNA oligonucleotides again. The upper histogram represents the data after 48 h of transfection. The bottom panel consists of data at 96 h post-transfection after the two sequential transfections of the RNAi oligonucleotides. A luciferase siRNA was used as the nonspecific control. Two independent siRNA oligonucleotides targeted to the RREB1 mRNA were used. B, lentivirus-mediated knockdown of RREB1 mRNA in K562 cells. Cells were infected with the indicated lentiviruses and then collected on the 10th day after viral infection for RNA analysis by quantitative RT-PCR. The panel shows the level of the RREB1 protein, as analyzed by Western blot (WB). C, lentivirus-mediated knockdown of RREB1 mRNA in primary human erythroid cells. Primary cultures of human erythroid cells were infected with lentivirus carrying shRNA2 targeting the RREB1 mRNA as described under “Experimental Procedures.” The total RNAs were isolated at the 10th day postinfection and subjected to quantitative RT-PCR analysis.