GCN5 and deacetylase levels were increased in sba1Δ yeast and p23 null MEF cells. (A) The expression of the HDA1, HOS2 and GCN5 loci in sba1Δ yeast relative to the parental strain was established by real time RT-PCR. Error bars represent the standard error of the mean. (B) The relative enrichment in the steady-state mRNA amounts of the indicated acetylases (grey bars) and deacetylases (black bars) were determined using p23 null and parental MEFs. The steady-state levels of the indicated proteins in p23 null (−/−) (C) or GCN5 null (−/−) (D) MEFs relative to the respective parental (+/+) cells were investigated by immunoblot analysis.

GR and HSF1 transcriptional and DNA binding activities were inhibited by p23 or GCN5 but unaltered upon co-overexpression of p23 and GCN5. The effect of p23 and/or GCN5 overexpression on endogenous genes regulated by either activated GR (A) or HSF1 (B) was determined (graphs). Error bars represent the standard error of the mean. In addition, the steady-state levels of the indicated proteins were determined by immunoblot analysis (right panels) along with the loading control protein HSP90. The impact of increased p23 and/or GCN5 on hormone-induced GR DNA binding (C) or heat-stimulated HSF1 DNA binding (D) was investigated in p23 null MEFs by EMSA using GRE or HSE oligonucleotides, respectively. (E) The influence of p23 and GCN5 overexpression on the HSF1 heat-stress DNA binding attenuation period was tested in transiently transfected human cells by EMSA.

GCN5 relies on p23 to modify a DNA-bound transcription factor. (A) The effect of GCN5 and p23 on the acetylation status of HSF1 was investigated in vitro. Myc-HSF1 wild type or K80R was supplemented with p23, GCN5 or GCN5 E575Q and the reactions were incubated at 37°C (−HS) or 43°C (+HS), as marked. The Myc-HSF1 or acetyl lysine was detected by immunoblot analysis. (B) Aliquots of in vitro translated HSF1 were incubated at 37°C (−HS) or 43°C (+HS) for 30 min with radiolabeled HSE oligonucleotide and reactions were either mock treated or p23-immunodepleted (IP α-p23). GCN5, GCN5 E575Q and/or p23 were added, as marked, and the samples were analyzed by EMSA. (C) The stability of preformed HSF1:HSE (radiolabeled oligonucleotide) complexes before or after immunodepletion of rabbit p23 were tested by the addition of either p23 (2x normal levels) or unlabeled HSE oligonucleotide. The reactions were resolved by native gel electrophoresis at the indicated time points following addition of either challenger. (D) The rabbit p23 was immunodepleted from preformed HSF1-HSE complexes, GCN5 alone or GCN5 and recombinant p23 was added, as indicated, and the Myc-HSF1 or acetyl lysine levels were determined by immunoblot analysis. (E) p23 was in vitro cotranslated with Myc-HSF1, Flag-GCN5 or both Myc-HSF1 and Flag-GCN5 (left panel represents 10% of IP input). The reactions were subjected to immunoprecipitation (right panel) using α-IgG (NS), α-Myc (HSF1) and/or α-Flag (GCN5) antibodies and the ³⁵S-labeled proteins were visualized following resolution by denaturing gel electrophoresis. (F) p23 was immunoprecipitated from nuclear extract prepared from HeLa, parental MEF or GCN5 null MEF cells. The presence of p23, GCN5, SPT3 or HSP90 in the pull-downs was determined by immunoblot analysis, as marked.

Yeast p23 and GCN5 are DHSs maintenance factors. (A) DHSs were identified by deep-sequencing samples following limited DNase I digestion of chromatin within nuclei prepared from wild type, sba1Δ and gcn5Δ yeast. The green bars represent DHSs unique to wild type, blue bars are DHSs specific to gcn5Δ, red bars are select to sba1Δ and grey bars are the remaining sites with the number of overlaps indicated in parentheses. (B) The typical size of DHSs increased in sba1Δ relative to wild type or gcn5Δ. In the top graph, the yellow bars represent the 25–75% quantiles (bottom to top, respectively), the error bars mark the maximal and minimal sizes, and the closed circles represent atypical points. In the bottom graph, the size distribution of DHSs in wild type (grey), sba1Δ (red) and gcn5Δ (blue) yeast is shown. The size distribution of smaller (<500 bp) and larger (>500 bp) DHSs is marked with single and double asterisks, respectively.

Transcription factor DNA residency increases in the sba1Δ yeast. (A) The number of DNA footprints associated with numerous transcription factors is shown. The average occupancy (per 10⁶ bp) of each motif has been categorized according the type of DHSs including WT specific (black), sba1Δ specific (white), increased in sba1Δ (light grey) and decreased in sba1Δ (dark grey). (B) Shown is the DNase I cleavage densities from the WT and sba1Δ data sets across two 100-kilobase regions of chromosome X (coordinates 245345–345345) with magnification of 5 kilobase sectors, followed by further enlargement of 500 base pair sections down to 50 base segments that visualize the positions and frequencies of the DNase I cleavage events, which reveal the individual DNase I footprints. The identities of the underlying motifs within the DNA footprints (red bar) are indicated.

Yeast p23 (Sba1) regulates the Mcm1 transcription factor. (A) DNA binding of purified Mcm1 (250 nM) was measured by fluorescence anisotropy and a fluorescein-labeled oligonucleotide representing the Mcm1 response element (MRE) from the MFA1 promoter. Reactions were supplemented with recombinant Sba1 (0, 0.5, 1.0, 2.0, 4.0 or 8.0 μM), as indicated. (B) Sba1 regulates the activity of the Mcm1-controlled promoter MFA1 in vitro. As controls, the activity of MFA1 promoter was checked using nuclear extract (NE) prepared from WT a-mating type (a), WT α-mating type (α) and sba1Δ a-mating type yeast. To check that the products were RNA polymerase-dependent, NEs were supplemented with α-amanitin (10 μg mL⁻¹ final), as marked. The impact of Sba1 on MFA1 promoter activity was determined by supplementing the Δsba1 extracts with 0.01, 0.1 or 1.0 μg of recombinant Sba1 corresponding to 0.1x, 1x and 10x the WT levels of Sba1. For all reactions, the MFA1 RNA signal was normalized to the levels of ENO2 RNA produced from an independent ENO2 DNA template. Error bars represent the standard error of the mean.

The p23 molecular chaperone and GCN5 acetyltransferase cooperate to modulate the stabilities of a wide range of protein-DNA complexes. The presented data support a model in which 1) a DNA binding protein (purple) forms an intrinsically long-lived DNA-bound complex; 2) p23 (green) disassembles the protein-DNA structure; 3) GCN5 (blue) acetylates the released protein; 4) acetylation (red) of a lysine within the DNA binding cleft of the target prevents rebinding; 5) p23 impedes GCN5 to avoid constitutive acetylation; 6) a promoter engaged deacetylase (grey) (e.g., HDAC1 or Hda1) removes the inhibitory acetyl group to allow the target to rebind its cognate DNA site.