Fig 1: Brf2/DNA Sequence-Specific Interactions(A) Close-up view of the TATA box (yellow), downstream flanking region and sequence-specific interactions with Brf2. DNA template and non-template strands are in blue and cyan respectively.(B) Substitutions at positions +3 and +4 of the wild-type (circled in red) U6-2 promoter decrease binding of a R110A mutant, in particular when a T nucleobase is present at position +3 on the non-template strand (in cyan). R110A versus wild-type (WT) is the ratio between the percentage of binding of the mutant versus wild-type Brf2 proteins.(C) Close-up view of the TATA box (yellow), upstream flanking region and sequence-specific interactions with Brf2. DNA template and non-template strands are in blue and cyan respectively.(D) Substitutions at positions −3 and −4 of the wild-type (circled in red) U6-2 promoter reveal more efficient complex formation with a pyrimidine nucleobase and a C nucleobase at positions −3 and −4 of the template strand, respectively. (B and D) The intensity of the complex formed with TBP, U6-2 non mutated sequence and wild-type Brf2 (lane 1) was used as a reference for relative quantification. ∗Indicates samples that were quantified relative to a distinct wild-type sequence reference not shown on the figure. Representative gels of three independent experiments. The data shown are the mean values and SE of three independent experiments. In the insets, 10 μl of a typical binding reaction (25 μl total) with Brf2 wild-type or Brf2 mutants were loaded on a SDS-PAGE gel and stained with Coomassie-blue, confirming that equal amounts of protein of comparable quality were used for EMSA assays.See also Figures S2 and S7.
Fig 2: Selenoproteins Levels and Resistance to Oxidative Stress Are Regulated in a Brf2-Dependent Manner(A) Manipulation of Brf2 protein levels affects selenoproteins expression levels during oxidative stress in MRC5 and A549 cells. In the upper insets, a western blot analysis of Nrf2 confirms induction of oxidative stress with 50 μM and 100 μM t-BHP in MRC5 and A549 cells, respectively. A western blot analysis of Brf2 immunoprecipitation from 107 MRC5 or A549 cells is shown in the lower insets (IP).(B) Overexpression of Brf2 in MRC5 cells challenged with t-BHP results in decreased apoptosis as measured by FACS analysis via annexin V-FITC/PI staining. The y axis represents the % of apoptotic cells, including both cells in early (annexin V-positive and PI-negative) and late (annexin V-positive and PI-positive) apoptosis.(C) Effects of overexpression of Brf2 wild-type and mutants on acquired resistance to apoptosis in MRC5 cells as measured by FACS analysis via annexin V-FITC/PI staining. The y axis represents the % of apoptotic cells, including both cells in early (annexin V-positive and PI-negative) and late (annexin V-positive and PI-positive) apoptosis.(D) Lowering Brf2 protein levels by siRNA in A549 cells challenged with t-BHP results in an increased cellular commitment to apoptosis as measured by FACS analysis via annexin V-FITC/PI staining. Inset: a western blot analysis of Brf2 immunoprecipitation from 107 A549 cells shows siRNA-induced Brf2 protein level reduction. The y axis represents the % of apoptotic cells, including both cells in early (annexin V-positive and PI-negative) and late (annexin V-positive and PI-positive) apoptosis.See also Figures S5 and S7.
Fig 3: Mechanism of the Redox-Dependent Brf2 Blockade during Oxidative Stress and Carcinogenesis, Related to Figures 1, 2, 3, 4, 5, 6, and 7During normal growth conditions (A) Brf2- and Nrf2-dependent transcripts are synthesized at basal levels. Upon moderate oxidative stress (B), Nrf2 is activated and Nrf2-dependent transcripts upregulated. Concomitantly, Brf2-dependent transcription, including SeCys tRNAs, is rapidly downregulated via redox-dependent modifications of Brf2. The pre-existing pool of SeCys tRNA is sufficient to sustain synthesis of selenoproteins. Upon prolonged oxidative stress (C), SeCys tRNA levels become limiting while, simultaneously, selenoprotein’s mRNAs continue to be highly upregulated by Nrf2. In this scenario, compromised synthesis of selenoproteins drives the cells into apoptosis. In cancer cells (D), the Nrf2 pathway is constitutively activated and contributes to the observed resistance of cancerous cells to higher than normal concentrations of reactive oxygen species. Under these circumstances, Brf2 overexpression is required to overcome the innate redoxdependent blockade, ensuring elevated synthesis of SeCys tRNAs and, ultimately, enabling cancer cells to evade apoptosis under prolonged oxidative stress.
Fig 4: Sequence Alignments of Brf2 Promoters and Protein, Related to Figures 1 and 3(A) Brf2-dependent promoters (40 nucleobase long centered around putative TATA boxes) were aligned using MEME (Bailey et al., 2009) by searching for a 16 nucleobase long consensus motif.(B) Sequence and domain conservation between Brf2, Brf1 and TFIIB. Color-coding is as in Fig. 1. Brf2-Brf1 alignments are based on sequence conservation, while Brf2-TFIIB is based on a structural alignment.(C) Two views of the specific interaction of Brf2 R110 and A108 with a TG (U6-2 in pale green) and TC (RPPH1 in light grey) dinucleotide step. In presence of the TG sequence, the T on the nontemplate strand (in red) is left unstacked at its downstream edge. In presence of a TC sequence, no local distortions of the DNA are observed. The two structures were superimposed by structurally aligning the Brf2 N-terminal cyclin repeats.
Fig 5: Structure of the Brf2-TBP/DNA Ternary Complex(A) Overview of the Brf2-TBP/U6 promoter structure. DNA template and non-template strands are in blue and cyan respectively. Dashed lines represent disordered regions or regions not present in the crystallization construct.(B) Schematic of Brf2 domain organization.See also Figures S1, S2, and S7 and Tables S1 and S2.
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