Loss of Tup1p relieves heme-deficient repression of FET3. (A) A strain was generated that contained an AFT1-1up plasmid, FET3-lacZ reporter plasmid, and a genomic FET3-GFP. The strain was UV mutagenized, and a β-galactosidase plate assay was performed. Blue colonies (positives) were collected, as they indicated loss of FET3-lacZ repression. To rule out mutations in the FET3-lacZ reporter construct, positives were then viewed by epifluorescence microscopy. The parent strain (repressed) and one representative positive clone, 3-13A, are shown. (B) A Δhem1 strain or a Δhem1/Δtup1 strain was transformed with a FET3-lacZ reporter construct. Strains were induced in the presence of 50 μM FeSO₄ or 80 μM BPS in the indicated media. Cells were grown for 6 h, harvested, and a β-galactosidase assay was performed. (C) The Δhem1 strain or the Δhem1/Δtup1 strain was grown for 6 h in the media as indicated. Cells were harvested and total RNA was isolated. S1 nuclease protection assay was performed on FTR1 and FET3, with CMD1 as a positive control. (D) The Δhem1 or the Δhem1/Δtup1 strain was transformed with the indicated reporter constructs. Cells were grown in metal-replete (50 μM) or metal-depleted media for the respective construct in the indicated media. Cells were grown for 6 h, harvested, and a β-galactosidase assay was performed.

Tup1p is recruited to iron-responsive promoters in response to changes in media iron. The Δhem1 strain was transformed with an epitope-tagged allele of TUP1 (pTUP1-HA). Cells were grown in the indicated media for 6 h and Tup1p-HA was crosslinked to DNA using formaldehyde treatment. Cells were harvested, disrupted, and chromatin was isolated. Chromatin was sheared using sonication to achieve an average fragment size of approximately 500 bp, with a range between 100 and 1500 bp. Chromatin was immunoprecipitated using the HA epitope of Tup1p. After crosslinking was reversed, PCR was performed using amplicons, which flank the Aft1p binding site of the indicated promoters, with CMD1 used as a positive control.

Mapping the differential response to heme deficiency. (A) Site-directed base-pair changes were made in the full-length wild-type ARN1 and FET3 constructs as shown. The Δhem1 strain was transformed with the indicated reporter constructs. Strains were induced in the presence of 50 μM FeSO₄ or 80 μM BPS in the indicated media. Cells were grown for 6 h, harvested, and a β-galactosidase assay was performed. (B) Δhem1 cells were transformed with the indicated reporter constructs. Cells were induced in the presence of 50 μM CuSO₄ or 40 μM BCS. Cells were grown for 6 h, harvested, and a β-galactosidase assay was performed.

Model for heme regulation of FET3 and ARN1 expression. Under normal heme conditions, Aft1p binds to the promoters of both FET3 and ARN1, leading to the recruitment of Tup1p and Cti6p. Under low-iron conditions in the absence of heme, Tup1p is able to mediate repression of FET3 through the chromatin remodeling protein Hda1p. The sequence immediately downstream of the Aft1p binding site of ARN1 mediates retention of Cti6p under heme-depleted conditions, resulting in loss of Tup1p and expression of ARN1.

The Aft1p binding site in FET3 is necessary and sufficient for repression. (A) The Δhem1 strain was transformed with the indicated reporter constructs. Strains were induced in 80 μM BPS in the indicated media. Cells were grown for 6 h, harvested, and a β-galactosidase assay was performed. (B) The Δhem1 strain was transformed with the indicated reporter constructs. Strains with FET3 constructs were induced in 80 μM BPS, and strains with CTR1 constructs were induced in the presence of 50 μM CuSO₄ or 40 μM bathocuproine sulfonate (BCS). Cells were grown for 6 h, harvested, and a β-galactosidase assay was performed.

Loss of Hda1p relieves heme-deficient repression of FET3. (A) The Δhem1, Δhem1/Δhda1, and Δhem1/Δhda1/pRS413-HDA1 strains were transformed with a FET3-lacZ reporter construct. Strains were induced in the presence of 50 μM FeSO₄ or 80 μM BPS in the indicated media. Cells were grown for 6 h, harvested, and a β-galactosidase assay was performed. (B) The Δhem1, Δhem1/Δhda1, and Δhem1/Δhda1/pRS413-HDA1 strains were grown for 6 h under low-iron conditions in the indicated media. Cells were harvested and total RNA was isolated. S1 nuclease protection assay was performed on FTR1 and FET3, with CMD1 as a positive control.

Aft1p-regulated genes respond differently to heme deficiency. (A) The Δhem1 strain was grown for 6 h in the media as indicated. Cells were harvested and total RNA was isolated. S1 nuclease protection assay was performed on ARN1 and FIT1 (not shown), with CMD1 as a positive control. (B) Δhem1 cells were transformed with the indicated reporter constructs. Strains were induced in the presence of 50 μM FeSO₄ or 80 μM BPS in the indicated media. Cells were grown for 6 h, harvested, and a β-galactosidase assay was performed. (C) Chromatin samples were prepared as in Figure 4. PCR was performed using amplicons, which flank the Aft1p binding site of the indicated promoters, with CMD1 used as a positive control.

Cti6p mediates induction of ARN1 when heme biosynthesis is disrupted. (A) The Δhem1/Δcti6 strain was transformed with the indicated reporter constructs. Strains were induced in the presence of 50 μM FeSO₄ or 80 μM BPS in the indicated media. Cells were grown for 6 h, harvested, and a β-galactosidase assay was performed. (B) The Δhem1/Δcti6 strain was transformed with a myc epitope-tagged Cti6p. Chromatin was prepared as in Figure 4. PCR was performed using amplicons, which flank the Aft1p binding site of FET3 and ARN1, with CMD1 used as a positive control. (C) Strains were induced in the indicated media for 6 h. Cells were harvested and total RNA was isolated. S1 nuclease protection assay was performed on FET1 and FTR1, with CMD1 as a positive control.