1. Regulation of the Sre1 Hypoxic Transcription Factor by Oxygen-Dependent Control of DNA Binding 
Chih-Yung S. Lee, Tzu-Lan Yeh, Bridget T. Hughes, Peter J. Espenshade 
Molecular Cell 
Volume 44, Issue 2, Pages (October 2011)
DOI: /j.molcel
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2. Molecular Cell 2011 44, 225-234DOI: (10.1016/j.molcel.2011.08.031)
Copyright © 2011 Elsevier Inc. Terms and Conditions

3. Figure 1 Sre1N Degradation Requires Rhp6 and Ubr1
(A) Wild-type and ubr1Δ cells were grown in the absence of oxygen for 6 hr. At t = 0, cycloheximide (200 μg/ml) was added and cells were shifted to the presence of oxygen. Samples were collected at the indicated times.
(B) Wild-type and rhp6Δ cells were grown and processed as in (A). P denotes Sre1 precursor and N denotes the Sre1 nuclear form.
(C) sre1N, sre1N ubr1Δ, and sre1N ubr11Δ cells were grown in the absence of oxygen for 3 hr. At t = 0, cycloheximide (200 μg/ml) was added and cells were shifted to the presence of oxygen. Samples were collected at the indicated times. (A–C) Whole-cell extracts were subjected to western blot analysis using anti-Sre1 IgG. Both long and short exposures to film are shown. The percentage of Sre1N remaining at each time point relative to t = 0 is quantified to the right. Other mutant strains tested are listed in the Strain Table (Table S1).
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4. Figure 2 Ofd1 Acts Upstream of Ubr1 in Sre1N Degradation
(A) Diagram of sre1N promoter showing DNA elements required for positive feedback regulation.
(B and C) sre1N-MP, sre1N-MP ofd1Δ, sre1N-MP ubr1Δ, sre1N-MP ubr1Δ ofd1Δ and sre1N-MP ubr1Δ nro1Δ cells were cultured in the presence or absence of oxygen for 3 hr (B) or with either 1% DMSO or 20 mM DMOG for 6 hr (C). Whole-cell extracts (40 μg) were subjected to western blot analysis using indicated antibodies and total RNA (5 μg) was subjected to northern analysis with the indicated 32P-labeled probes. Actin protein and α-tubulin (tub1+) RNA served as loading controls. Northern blots were stripped and reprobed for tub1+.
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5. Figure 3 Ofd1 Regulates Sre1N Independently from Degradation
(A and B) sre1N, sre1N ofd1Δ, sre1N ubr1Δ, and sre1N ubr1Δ ofd1Δ cells were cultured in the absence of oxygen for designated time (A) or with either 1% DMSO or 20 mM DMOG for 3 hr (B). Western blot and northern blot analysis were performed as described in Figure 2 and Experimental Procedures. See also Figure S1.
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6. Figure 4
Ofd1-Nro1 Regulate Oxygen-Dependent Sre1N Transcriptional Activity
(A) Diagram of the integrated 7xSRE-lacZ reporter gene in sre1N-MP ubr1Δ strain.
(B and C) sre1N-MP ubr1Δ, sre1N-MP ubr1Δ ofd1Δ, sre1N-MP ubr1Δ nro1Δ, and ubr1Δ sre1Δ cells with 7xSRE-lacZ reporter integrated were cultured in rich medium with either 1% DMSO or 20 mM DMOG for 6 hr (B) or with or without oxygen for 3 hr (C). Cells were assayed for β-galactosidase activity as described in Experimental Procedures. Data are the mean of six replicates and error bars indicate standard error. ∗ Indicates significant difference from sre1N-MP ubr1Δ (WT) control samples, either –DMOG or + oxygen (p value < ). ∗∗ Indicates significant difference from ofd1Δ + oxygen samples (p value = 0.001). Whole-cell extracts (40 μg) were subjected to western blot analysis using indicated antibodies. See also Figure S2.
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7. Figure 5 Oxygen Regulates Sre1N DNA Binding through Ofd1
(A) sre1N-MP ubr1Δ 4xSRE-lacZ and sre1N-MP ubr1Δ ofd1Δ 4xSRE-lacZ cells were grown with either 1% DMSO or 20 mM DMOG for 6 hr and subjected to chromatin immunoprecipitation using anti-Sre1 IgG or rabbit IgG. Binding of Sre1N to lacZ promoter regions was assayed by real-time PCR. Bound DNA was expressed as the fold change normalized to ofd1+ treated with 1% DMSO. Recovery of Sre1N bound DNA ranged from 0.35% to 1.1% of input gDNA for untreated sre1N-MP ubr1Δ cells.
(B) sre1N-MP ubr1Δ 4xSRE-lacZ and sre1N-MP ubr1Δ ofd1Δ 4xSRE-lacZ cells were grown in the presence or absence of oxygen for 3 hr and subjected to chromatin immunoprecipitation as in (A). Bound DNA was expressed as the fold change normalized to ofd1+ grown with oxygen. Recovery of Sre1N bound DNA ranged from 0.22% to 0.72% of input gDNA for sre1N-MP ubr1Δ cells grown plus oxygen. For (A) and (B), data are the mean of 4 biological replicates, and error bars denote standard error among biological replicates. Nonspecific DNA binding to rabbit IgG ranged from % to 0.005% of input DNA and is not shown.
(C) sre1N-MP ubr1Δ and sre1N-MP ubr1Δ ofd1Δ cells were cultured in rich medium to exponential phase. Total RNA (5 μg) was subjected to northern blot analysis with the indicated 32P-labeled probes.
(D) sre1N-MP ubr1Δ, sre1N-MP ubr1Δ ofd1Δ, sre1N-MP ubr1Δ nro1Δ, and sre1N-MP ubr1Δ sre1Δ cells were grown in the presence or absence of oxygen for 3 hr and subjected to chromatin immunoprecipitation using anti-Sre1 IgG. Binding of Sre1 to different promoter regions was assayed by real-time PCR. Bound DNA was expressed as the fold change normalized to sre1N-MP ubr1Δ cells (WT) grown with oxygen for each primer pair. Recovery of Sre1N bound DNA relative to input gDNA for sre1N-MP ubr1Δ cells grown plus oxygen was: 0.1% for hem13+; 0.22% for erg3+; 0.019% for tf2-3+; and 0.079% for osm1+. Data are the mean of three biological replicates, and error bars denote standard error among biological replicates. ∗ Indicates significant difference from sre1N-MP ubr1Δ (WT) plus oxygen samples (p value < 0.02). ∗∗ Indicates significant difference from ofd1Δ plus oxygen sample (p value = 0.04).
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8. Figure 6 Low Oxygen Rapidly Stimulates Sre1N DNA Binding
(A) sre1N-MP ubr1Δ and sre1N-MP ubr1Δ nro1Δ cells were grown in the absence of oxygen for the indicated times and were treated with 0.5 mM DSP crosslinker in PBS for 5 min. Detergent-solubilized whole-cell extracts were subjected to immunoprecipitation with anti-Nro1 IgG. Bound (10-fold overloaded) and unbound fractions were analyzed by western blot using anti-Nro1–HRP and anti-Ofd1–HRP antibodies.
(B) sre1N-MP ubr1Δ and sre1N-MP ubr1Δ sre1Δ cells were grown in the absence of oxygen for the indicated times and subjected to chromatin immunoprecipitation using anti-Sre1 IgG. Sre1N promoter occupancy was assayed by real-time PCR using hem13+ primer pair. Bound DNA was expressed as the fold change normalized to sre1N-MP ubr1Δ cells grown with oxygen. Recovery of Sre1N bound DNA to input gDNA in sre1N-MP ubr1Δ samples ranged from 0.12% to 0.39%. Data are the mean of six biological replicates, and error bars denote standard error among biological replicates.
(C) Upper panel: sre1N-MP 7xSRE lacZ cells were grown in the absence of oxygen for the indicated times and subjected to chromatin immunoprecipitation using anti-Sre1 IgG, and western blot analysis using anti-Sre1 and anti-Actin antibodies. Sre1N promoter occupancy was assayed by real-time PCR using hem13+ primer pair. Bound DNA was expressed as the fold change normalized to sre1N-MP cells grown with oxygen. Recovery of Sre1N bound DNA to input gDNA in sre1N-MP samples ranged from 0.04% to 0.26%. Lower panel: Sre1N protein levels were quantified and normalized to Actin. Sre1N / Actin ratio was expressed as the fold change normalized to samples grown with oxygen. Data are the mean of four biological replicates for sre1N-MP and two biological replicates for control sre1Δ cells. Error bars denote standard error among biological replicates. ∗ Indicates significant difference from samples grown in the presence of oxygen (p value < 0.05). See also Figure S3.
(D) sre1N-MP, sre1N-MP ubr1Δ, and sre1N-MP ubr1Δ ofd1-H142A cells were grown in the presence or absence of oxygen for 3 hr. Western blot and northern blot analysis were performed as described in Figure 2 and Experimental Procedures.
(E) sre1N-MP ubr1Δ, sre1N-MP ubr1Δ ofd1-H142A, and sre1N-MP ubr1Δ nro1Δ cells were grown with or without oxygen for 20 min and then were treated with 0.5 mM DSP crosslinker in PBS for 5 min. Immunoprecipitation and western blot were performed as described in (A).(F) sre1N-MP ubr1Δ, sre1N-MP ubr1Δ ofd1-H142A, and sre1N-MP ubr1Δ sre1Δ cells were grown with or without oxygen for 20 min. Cells were subjected to chromatin immunoprecipitation using anti-Sre1 IgG. Promoter occupancy by Sre1N was assayed by real-time PCR using hem13+ primer pair. Bound DNA was expressed as the fold change normalized to sre1N-MP ubr1Δ cells (WT) grown with oxygen. Recovery of Sre1N bound DNA in sre1N-MP ubr1Δ samples ranged from 0.14% to 0.5% of input gDNA. Data are the mean of four biological replicates, and error bars denote standard error among biological replicates. ∗ Indicates significant difference from sre1N-MP ubr1Δ (WT) plus oxygen samples (p value < 0.02).
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9. Figure 7 Ofd1 CTD Binds Sre1N and Inhibits DNA Binding
(A) Yeast two-hybrid assay showing the in vivo interaction between Sre1N and Ofd1CTD. Cells containing an ofd1CTD plasmid were transformed with plasmids expressing different truncation mutants of Sre1N or empty vector. Cells were plated on selection medium.
(B) sre1N-MP ubr1Δ ofd1Δ cells containing empty vector or ofd1CTD plasmid were cultured to exponential phase. Whole-cell extracts (40 μg) were subjected to western blot analysis using indicated antibodies (upper panels). sre1N-MP ubr1Δ ofd1Δ cells containing empty vector or ofd1CTD, and sre1N-MP ubr1Δ sre1Δ cells containing empty vector, were subjected to chromatin immunoprecipitation using anti-Sre1 IgG. Promoter occupancy by Sre1N was assayed by real-time PCR using hem13+ primer pair. Bound DNA was expressed as the fold change normalized to sre1N-MP ubr1Δ ofd1Δ containing vector. Recovery of Sre1N bound DNA in sre1N-MP ubr1Δ ofd1Δ samples ranged from 0.24% to 0.94% of input gDNA. Data are the mean of six biological replicates for sre1N-MP ubr1Δ ofd1Δ containing ofd1CTD plasmid and four biological replicates for others. Error bars denote standard error among biological replicates. ∗ Indicates significant difference from sre1N-MP ubr1Δ ofd1Δ control samples (p value = 2.1 × 10−5).
(C) Extracts from sre1N ofd1Δ nro1Δ ubr1Δ cells were mixed with Sre1N specific 32P-labeled DNA probes in the presence of indicated concentrations of Ofd1CTD, Nro1, or Nro1 (aa 59–393) and DNA binding was assayed. Monoclonal antibody to Sre1N (4 μg) was added to the reaction in lane 10. See also Figure S4.
(D) Model for oxygen-dependent regulation of Sre1N DNA binding. The dioxygenase domain of Ofd1 (N-Reg) regulates oxygen-dependent binding of Nro1 to Ofd1. In the presence of oxygen, Nro1 does not bind Ofd1CTD, allowing Ofd1CTD to bind Sre1N, leading to Sre1N degradation and transcriptional inhibition. In the absence of oxygen, Nro1 binds Ofd1CTD, thereby allowing Sre1N to bind DNA and activate its own expression.
Molecular Cell  , DOI: ( /j.molcel )
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