Heme mediates derepression of Maf recognition element through direct binding to transcription repressor Bach1

Abstract

Heme controls expression of genes involved in the synthesis of globins and heme. The mammalian transcription factor Bach1 functions as a repressor of the Maf recognition element (MARE) by forming antagonizing hetero-oligomers with the small Maf family proteins. We show here that heme binds specifically to Bach1 and regulates its DNA-binding activity. Deletion studies demonstrated that a heme-binding region of Bach1 is confined within its C-terminal region that possesses four dipeptide cysteine-proline (CP) motifs. Mutations in all of the CP motifs of Bach1 abolished its interaction with heme. The DNA-binding activity of Bach1 as a MafK hetero-oligomer was markedly inhibited by heme in gel mobility shift assays. The repressor activity of Bach1 was lost upon addition of hemin in transfected cells. These results suggest that increased levels of heme inactivate the repressor Bach1, resulting in induction of a host of genes with MARES:

Fig. 1. Recombinant Bach1 derivatives. (A) Schematic representation of mouse Bach1, Bach1ΔBTB and fragments fused with MBP or GST. CP motifs are numbered above the first line. (B) Purified fusion proteins were analyzed by Coomassie Blue staining after SDS–PAGE. Lanes 1–3: BA1G174–739, BA1G174–415 and BA1G417–739, respectively. M, size markers. (C) Purified fusion proteins were analyzed as above. Samples were BA1G417–739 (lane 1), BA1G417–645 (lane 2), BA1G558–739 (lane 3), BA1G417–564 (lane 4), BA1G417–739CP3AP (lane 5), BA1G417–739CP4AP (lane 6), BA1G417–739CP5AP (lane 7), BA1G417–739CP6AP (lane 8), BA1G417–739CP3-5AP (lane 9) and BA1G417–739CP3-6AP (lane 10).

Fig. 2. Binding of hemin to Bach1. (A) Hemin (2.7 µM) was incubated with 2.7 µM BA1G174–739 and the absorbtion spectrum was measured. The dotted line represents the absorption spectrum of hemin alone. (B) Scatchard plot analysis of heme binding to BA1G174–739 was carried out using data from several heme-binding assays. The K_d value was 140 nM using a curve-fitting program (DeltaGraph).

Fig. 3. Binding of hemin to Bach1 subfragments. GST–Bach1 fusions (1 nmol) were immobilized onto Sepharose beads and incubated with 1 µM hemin. The amounts of bound hemin were determined using spectrofluorometry. Each data bar represents the average and SEM of several independent experiments.

Fig. 4. Effects of cysteine to alanine substitutions of CP motifs of Bach1 on heme binding. (A) Heme binding assays were carried out as in Figure 3 using GST fusion proteins with single or multiple mutation(s) within the CP motifs in the context of BA1G417–739. Results are the mean ± SEM of several independent experiments. (B and C) Scatchard plot analyses of BA1G417–739 (B) and BA1G417–739CP3–6AP (C). The K_d values were 170 nM (BA1G417–739) and 1220 nM (BA1G417–739CP3-6AP).

Fig. 5. Inhibition of DNA binding of Bach1 by heme. (A) EMSA was carried out using 20 ng of each recombinant Bach1 and MafK and a 240 bp enhancer fragment from the β-globin LCR containing two tandem MAREs as described previously (Igarashi et al., 1998). Complexes were separated on a 1.0% agarose gel. Heme concentrations were 0.03 µM (lane 3), 0.1 µM (lane 4), 0.3 µM (lane 5) and 1 µM (lane 6). (B) EMSA was carried out using a CβE oligonucleotide probe as described previously (Igarashi et al., 1998) using 40 ng of Bach1 or Bach1ΔBTB and 10 ng of MafK in the combinations indicated above the panels. Heme concentrations were 0.1 µM (lanes 4 and 9), 0.3 µM (lanes 5 and 10), 0.6 µM (lanes 6 and 11) and 1 µM (lanes 7 and 12). Complexes were separated through 4% polyacrylamide gels. Arrow heads indicate specific heterodimer binding complexes. The arrow indicates binding complexes that may be due to degradation products. (C) EMSA was carried out as in (B) using 40 ng of Bach1ΔBTB (lanes 2–6) or Nrf2 (lanes 7–11). Heme concentrations were 0.1 µM (lanes 3 and 8), 0.3 µM (lanes 4 and 9), 0.6 µM (lanes 5 and 10) and 1 µM (lanes 6 and 11).

Fig. 6. Involvement of the CP motifs of Bach1 in regulation of its DNA-binding activity by heme. BA1G417–739 or BA1G417–739CP3-6AP and MafK were incubated in the absence or presence of various concentrations of hemin. Binding to the CβE probe was examined by EMSA. Heme concentrations were 0.1 µM (lanes 4 and 9), 0.3 µM (lanes 5 and 10), 0.5 µM (lanes 6 and 11) and 1.0 µM (lanes 7 and 12).

Fig. 7. Effects of heme on formation of Bach1–MafK heterodimers. FLAG-tagged MafK and GST–Bach1 fusion proteins (BA1G417–739, BA1G417–739CP3–6AP or BA1G417–564) were incubated in the presence or absence of 1 µM heme. The resulting complexes were resolved in native polyacrylamide gels, transferred onto membranes and detected with anti-FLAG (upper panel) and anti-GST (lower panel) antibodies. Bach1–MafK complexes are indicated with small dots along the lanes. Asterisks indicate the slower mobility complex of MafK.

Fig. 8. Effects of heme on the transcriptional activity of Bach1. Reporter gene assays were carried out using 293 cells to compare repressor activity of the wild-type Bach1 (lanes 2, 3, 7 and 8) or Bach1 with mutations in the CP motifs (Bach1 mCP1–6, lanes 4, 5, 9 and 10) in the absence (lanes 1–5) or presence (lanes 6–10) of 10 µM hemin. Either 300 or 900 ng of the Bach1 expression plasmids were used. Reporter gene expression in the absence of any effector plasmid was set to 100% for each experimental group.

Fig. 9. Transcription regulation by heme through Bach1. (A) Sequence alignment of human, mouse and Xenopus Bach1. Amino acid sequences surrounding the three clustered CP motifs (CP3–CP5, underlined) of human, mouse and Xenopus (deduced from AW147501) Bach1 are compared. Asterisks indicate residues that are conserved among the three species. (B) A model based on our observations. In the presence of lower concentrations of heme within a cell, Bach1 represses genes with MAREs together with small Maf proteins. In the presence of higher concentrations of heme, Bach1 is inactivated by binding to heme, leaving MAREs available for the activator class of factors such as Nrf2 or other MARE-binding proteins. In this way, heme directly regulates the balance of repression and activation of gene expression.