PMC

Effect of SSN6 and NHP6A/B on Aft1-regulated genes. Same blots and analysis for SC and SCBPS RNA samples as described for , using ARN2, FET3, FIT2, FRE3 and CMD1 probes.

Effect of SSN6 and NHP6A/B on Aft1-mediated transcription. (A) β-Galactosidase activity units obtained from the indicated strains cotransformed with FRE2_UAS-HIS3-LacZ and pYX142 or pYX142-AFT1 (AFT1 overexpression) grown in SCBPS. (B) β-Galactosidase activity units obtained from the indicated strains cotransformed with LexAop-HIS3-LacZ and LexA-Aft1 or LexA expression plasmid grown in SC medium. (The activity in SCBPS exceeds measurable levels.) Values in (A) and (B) represent the average obtained from three independent transformants.

Effect of SSN6 and NHP6A/B on metal-regulated transcription. (A) Northern analysis of total RNA extracted from the indicated strains grown in metal-replete (SC), iron-depleted (SCBPS) or copper-depleted (SCBCS) medium using radiolabeled FRE2 and CMD1 (internal control) probes. (B) The same blot using FRE1 and CMD1 probes. Bands were quantified using the PhosphorImager and ImageQuant software, and bars represent the indicated intensity ratios (normalized mRNA levels).

Association of Ssn6 and Nhp6a with Aft1 in vivo. (A) Protein extracts from aft1Δ cells grown in SCBPS and expressing 6xHis or 6xHis-Ssn6 or 6xHis-Nhp6a along with HA-Aft1 were incubated with Ni-NTA beads. Bound proteins were analyzed by SDS–PAGE and immunoblotting using anti-His to detect 6xHis-Ssn6 and 6xHis-Nhp6a. (B) Pulled-down (PD) HA-Aft1 was detected by anti-HA (left) and pulled-down endogenous Tup1 by anti-Tup1 (right). (C) Extracts from nhp6ΔΔ cells grown in SCBPS and expressing 6xHis or 6xHis-Ssn6 along with HA-Nhp6a were incubated with Ni-NTA beads. Pulled-down (PD) and input (I) proteins were analyzed by SDS–PAGE and immunoblotting using anti-HA to detect HA-Nhp6a (left) and anti-Tup1 to detect endogenous Tup1 (right). (D) Extracts from wild-type and nhp6ΔΔ cells grown in SCBPS and expressing 6xHis or 6xHis-Ssn6 along with HA-Aft1 were incubated with Ni-NTA beads. Pulled-down (PD) and flow-through (FT) proteins were analyzed by SDS–PAGE and immunoblotting using anti-HA to detect HA-Aft1.

Interaction of Aft1 with Ssn6 and Nhp6a in vitro. (A) Diagrammatic representation of the Aft1 protein sequence. (B) GST or GST-Ssn6(1-597) or GST-Nhp6a bound on glutathione agarose beads were incubated with purified 6xHis-tagged Aft1 derivatives or N-Tup1 (positive control) and analyzed by SDS–PAGE and immunoblotting using anti-His antibody. The input lane contains 20% of the total amount of each 6xHis-tagged protein incubated with the beads. Multiple bands observed in B-1 correspond to Aft1 degradation products. Top left inset: electrophoretic pattern of the 6xHis-tagged proteins (input amounts).

Model depicting how Nhp6a and Ssn6 distinctly and cooperatively potentiate Aft1-mediated transcription on FRE2 promoter. (A) Upon induction under iron depletion, Nhp6 is in proximity with chromatin and Aft1 interacts with DNA and Nhp6. (B) Structural alterations are induced, resulting in enhanced Aft1 binding to UAS. (C) Ssn6 interaction with Aft1 is favored. (D) The presence of Ssn6 on the promoter further stabilizes Aft1 DNA binding and ensures access to the RNA polymerase II transcription machinery components, resulting in full transcriptional activation.

Low-resolution chromatin analysis of the FRE2 promoter. Wild-type cells under repression (+Fe, SC supplemented with 200 μM FeCl₃ 30 min prior to cell collection) and wild type, nhp6ΔΔ and ssn6Δ under induction (+BPS) conditions were subjected to MNase digestion followed by indirect end-labeling. On the left, nucleosomes seen as protected areas on the repressed FRE2 promoter are depicted as circles. The adjacent map indicates the relative positions of the UAS_Aft1 and the presumptive TATA box. Brackets indicate the corresponding protected regions and asterisks indicate induction-specific MNase sensitivity sites. Lane ND contains a naked genomic DNA digest.

Association of Aft1 and Ssn6 with FRE1 and FRE2 promoters. (A) Wild-type and nhp6ΔΔ cells, carrying chromosomal AFT1-9Myc, grown in SCBPS, were subjected to ChIP with anti-Myc, followed by PCR analysis of the immunoprecipitated (IP) and input DNA using primers specific for FRE1, FRE2 and HTA1 promoters. (B) ssn6Δ* and ssn6Δ* aft1Δ cells, transformed with an HA-Ssn6 expressing plasmid, were grown and analyzed as above using anti-HA and primers specific for FRE1 and FRE2 promoters and ACT1 coding region. (C) ssn6Δ and ssn6Δ nhp6ΔΔ cells were transformed, grown and analyzed as above using anti-HA and primers specific for FRE1, FRE2 and TRP3 promoters. (D) ssn6Δ* cells transformed with an HA-Ssn6 expressing plasmid and grown under high-iron conditions (SC supplemented with 200 μM FeCl₃ 30 min prior to cell collection) were analyzed as above using anti-HA and primers specific for FRE1 and FRE2 promoters and ACT1 coding region. Bands in (A–D) were quantified using the PhosphorImager and ImageQuant software, and numbers express the indicated ratios. The specific recruitment of HA-Ssn6 on FRE1 and FRE2 promoters in ssn6Δ cells of two different genetic backgrounds was comparable.