Heme regulates gene expression by triggering Crm1-dependent nuclear export of Bach1

Abstract

Bach1 is a transcriptional repressor of heme oxygenase-1 and beta-globin genes, both of which are known to be transcriptionally induced by heme. To test the hypothesis that heme regulates the activity of Bach1, we expressed wild type and mutated versions of Bach1 together with or without its heterodimer partner MafK in human 293T and GM02063 cells and examined their subcellular localization. Inhibition of heme synthesis enhanced the nuclear accumulation of Bach1, whereas treating cells with hemin resulted in nuclear exclusion of Bach1. While the cadmium-inducible nuclear export signal (NES) of Bach1 was dispensable for the heme response, a region containing two of the heme-binding motifs was found to be critical for the heme-induced nuclear exclusion. This region functioned as a heme-regulated NES dependent on the exporter Crm1. These results extend the regulatory roles for heme in protein sorting, and suggest that Bach1 transduces metabolic activity into gene expression.

Figure 1

Cytoplasmic accumulation of Bach1 in response to heme. (A) 293T cells were cotransfected with FLAG-Bach1 and MafK expression plasmids. At the end of culturing, cells were treated with 10 μM CdCl₂ (second row) or hemin (third row) for 4 h. Bach1 and MafK were detected using anti-FLAG and anti-MafK antibodies, respectively. Images for Bach1, MafK, and DNA are shown (left to right). (B) Subcellular localization of Bach1 was classified into three categories: C>N, cytoplasmic-dominant accumulation (gray bar); C=N, roughly equal distribution in cytoplasmic and nuclear compartments (white bar); and C<N, nuclear-dominant accumulation (black bar). Results of counting 200 cells are shown. (C) Domains of Bach1. BTB and CLS facilitate cytoplasmic accumulation, whereas the basic region facilitates nuclear accumulation (Suzuki et al, 2003). CP motifs are shown with circled numbers. Bach1 derivatives tagged with FLAG at the N-termini are shown below, with the positions of amino-acid residues marked. (D, E) 293T cells were transfected with Bach1ΔBTB or Bach1ΔBTBΔC1 expression plasmids and treated with cadmium (second row) or hemin (third row). Images are DNA (right) and Bach1 (left). (F) Subcellular distributions of Bach1ΔBTB or Bach1ΔBTBΔC1 in 293T cells were counted as in (B). Results of counting 200 cells are shown.

Figure 2

Effects of inhibitors of heme synthesis on Bach1 localization. 293T cells were transfected with FLAG-Bach1 expression plasmid. Cells were maintained in medium lacking serum in the absence or presence of 5 mM SA or 100 μM DFO.

Figure 3

Involvement of heme-binding motifs in the heme response. (A, B) Schematic representation of Bach1 and Bach1 derivatives containing Cys-to-Ala mutations in the CP motifs 1–6 in various combinations. (C) 293T cells were transfected with FLAG-Bach1 or various FLAG-Bach1mCP expression plasmids. Extracts were analyzed by immunoblotting with anti-FLAG antibody. (D) 293T cells were transfected with FLAG-Bach1 or -Bach1mCP1–6, and treated with cadmium (third row) or hemin (fourth row).

Figure 4

Delineation of CP motifs involved in the heme-induced nuclear exclusion of Bach1. (A) Human fibroblast GM02063 cells were transfected with FLAG-tagged Bach1 or Bach1mCP derivative expression plasmids, together with MafK expression plasmid. Cells were treated with or without 10 μM hemin at the end of culturing. The subcellular localization of Bach1 was classified into three categories as in Figure 1. Results of counting 400 cells are shown. (B) Profile plots of signal intensity along the arrows are shown in green (Bach1, mCP34, or mCP1256) or blue (DNA). Without hemin treatment, the majority of cells showed a significant nuclear signal of Bach1 in confocal optical sections.

Figure 5

Delineation of heme-responsive CP motifs within Bach1. (A) Schematic representation of reporter proteins containing indicated regions of Bach1 fused with EGFP and GST. (B–D) GFP–B1(417–587) and its derivatives carrying indicated mutations were expressed in 293T cells. Hemin was added for the last 4 h before observation.

Figure 6

Involvement of Crm1 in the hemin-induced nuclear exclusion of Bach1. (A, B) Localization of GFP-GST-B1(417–587) in 293T cells (A) or Bach1 in GM02063 cells (B) in the presence of Crm1 or Crm1K1 was examined. Cells were treated with indicated reagents. (C) Glutathione beads (lane 4) or glutathione beads with GST–Bach1(417–645) (lane 5) were incubated with liver cell extracts and bound proteins were analyzed by immunoblotting analysis using anti-Crm1 antibodies. Fractions of input extracts were also examined (5.7, 1.9, or 0.6 μg in lanes 1–3, respectively).

Figure 7

Demarcation of heme-regulated NES. (A) Amino-acid sequence (residues 417–503) containing CP3–5 motifs (bold) is shown. Mutations in hydrophobic residues are shown above the sequence (mut 1, 2, and 3). Subfragments examined as EGFP fusion proteins are shown below the sequence. (B, C) Indicated EGFP fusions were expressed in 293T cells and their distribution was observed in the absence or presence of hemin.

Figure 8

Heme binding to the GST–Bach1 fragment fusion protein. Absorption spectra of hemin in the absence (dotted lines) and presence (solid lines) of the GST–Bach1(417–480) fragments at [Bach1]/[heme] ratio of 3 are shown on the left. Titration curves of hemin with increasing amounts of the fusion protein represented as absorbance at 371 nm as a function of the molar concentration ratios of the protein to heme are shown on the right. Lines are drawn as a visual aid for stoichiometic heme binding and do not carry any analytical meanings. From top to bottom panels, the wild-type, mut 1, and mCP3 GST–Bach1 fragments. A minor spectral change observed for mCP3 (the bottom panels) is probably due to nonspecific heme interaction with the fusion protein.

Figure 9

Effect of heme upon Bach1 in transcription reporter assays. 293T cells were transfected with the reporter pHO15luc together with or without FLAG-tagged Bach1 or a battery of Bach1mCP derivative expression plasmids. Cells were treated with 10 μM hemin for 4 h at the end of culturing. Relative reporter gene activities are shown from three experiments.

Figure 10

Regulation of DNA binding by heme in vivo. (A) GM02063 cells were transfected with the ho-1 reporter plasmid and indicated Bach1 and MafK expression plasmids. Cells were treated with or without hemin, and binding of Bach1 to the ho-1 E2 enhancer was examined by ChIP. (B) Binding of MafK or Nrf2 to the ho-1 E2 enhancer was examined as in (A). (C) Model describing the regulation of ho-1 or other target genes by Bach1 and heme. Besides MafK, other Maf-related factors may also serve as partners for Bach1. Bach1 occupies MARE enhancers to repress transcription under normal conditions. An increase in heme levels alleviates Bach1-mediated repression through inhibition of its DNA-binding activity and subsequent Crm1-dependent nuclear export, making MAREs available for activating Maf complexes including Nrf2 or p45 NF-E2.