The Mediator Subunit MED16 Transduces NRF2-activating Signals into Antioxidant Gene Expression

56 Pages·2015·2.58 MB·English

MCB Accepted Manuscript Posted Online 16 November 2015
Mol. Cell. Biol. doi:10.1128/MCB.00785-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Sekine et al.
1 The Mediator Subunit MED16 Transduces NRF2-activating Signals into
2 Antioxidant Gene Expression
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4 Hiroki Sekine,a,b Keito Okazaki,b Nao Ota,b Hiroki Shima,c,d Yasutake Katoh,e Norio Suzuki,f
5 Kazuhiko Igarashi,c Mitsuhiro Ito,g Hozumi Motohashi,b# Masayuki Yamamotoa,e#
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7 Department of Medical Biochemistrya, Department of Biochemistryc, and Division of D
8 Interdisciplinary Medical Sciencef, Tohoku University Graduate School of Medicine, Sendai, ow
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9 Japan; Department of Gene Expression Regulation, Institute of Development, Aging and Cancer,
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10 Tohoku University, Sendai, Japanb; CREST, AMED, Sendai, Japand; Tohoku Medical Megabank a
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11 Organization, Tohoku University, Sendai, Japane; Laboratory of Hematology, Division of Medical e
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12 Biophysics, Kobe University Graduate School of Health Sciences, Kobe, Japang f
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14 Running Head: NRF2-MED16 pathway for antioxidant response h
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17 #Address correspondence to Hozumi Motohashi, [email protected] and Masayuki c
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18 Yamamoto, [email protected]. .a
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19 Hozumi Motohashi, M.D., Ph.D., m
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20 Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku o
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21 University.
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22 4-1 Seiryo-cho, Aoba-ku, Sendai, Miyagi, 980-8575, Japan. n

23 Phone: [phone] A
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24 Fax: [phone] r
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25 E-mail: [email protected]. 3
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26 2
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27 Masayuki Yamamoto, M.D., Ph.D., 1
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28 Department of Medical Biochemistry, Graduate School of Medicine, Tohoku University. b
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29 2-1 Seiryo-cho, Aoba-ku, Sendai, Miyagi, 980-8575, Japan. g
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30 Phone: [phone] e
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31 Fax: [phone] t
32 E-mail: [email protected].
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34 Word count for the Materials and Methods section; 2,718 words
35 Combined word count for the Introduction, Results, and Discussion sections; 4,256 words
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37 Abstract
38 The KEAP1-NRF2 system plays a central role in cytoprotection. NRF2 is stabilized in response to
39 electrophiles and activates transcription of antioxidant genes. Although robust induction of NRF2
40 target genes confers resistance to oxidative insults, how NRF2 triggers transcriptional activation
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41 after binding to DNA has not been elucidated. To decipher the molecular mechanisms underlying
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42 NRF2-dependent transcriptional activation, we purified NRF2 nuclear protein complex and lo
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43 identified the Mediator subunits as NRF2 cofactors. Among them, MED16 directly associated e
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44 with NRF2. Disruption of Med16 significantly attenuated the electrophile-induced expression of ro
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45 NRF2 target genes but did not affect hypoxia-induced gene expression, suggesting a specific h
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46 requirement for MED16 in NRF2-dependent transcription. Importantly, we found that 75% of /
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47 NRF2-activated genes exhibited blunted inductions by electrophiles in Med16-deficient cells b
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48 compared with wild-type cells, which strongly argues that MED16 is a major contributor m
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49 supporting the NRF2-dependent transcriptional activation. NRF2-dependent phosphorylation of g
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50 RNA polymerase II C-terminal domain was absent in Med16-deficient cells, suggesting that

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51 MED16 serves as a conduit to transmit NRF2-activating signals to RNA polymerase II. MED16 ril
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52 indeed turned out essential for cytoprotection against oxidative insults. Thus, the 2
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53 KEAP1-NRF2-MED16 axis has emerged as a new regulatory pathway mediating the antioxidant 9

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54 response through the robust activation of NRF2 target genes. g
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55 t
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56 Introduction
57 The KEAP1 (Kelch-like ECH-associated protein 1)-NRF2 (nuclear-factor erythroid 2-related
58 factor 2) system is the major regulatory pathway for cytoprotection from oxidative insults (1, 2).
59 NRF2 is a potent transcriptional activator, coordinately regulating the cytoprotective genes whose
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60 products are involved in glutathione synthesis, elimination of reactive oxygen species (ROS) and
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61 detoxification of xenobiotics. In unstressed conditions, NRF2 is trapped by KEAP1, ubiquitinated lo
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62 and degraded by proteasomes. KEAP1 is inactivated upon exposure to electrophilic chemicals, e
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63 resulting in the stabilization of NRF2 and the activation of NRF2 target genes. NRF2 ro
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64 heterodimerizes with small Maf proteins (sMAF) and binds to the antioxidant response element h
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65 (ARE) [(A/G)TGA(G/C)NNNGC], which is commonly found in the regulatory regions of the /
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66 cytoprotective genes activated by NRF2. Recent analyses of the genome-wide distribution of b
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67 NRF2 have characterized the NRF2 cistrome, in which the ARE sequence has been selected as the m
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68 most highly enriched motif (3-5). g
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69 Mediator is an evolutionarily conserved multi-protein complex consisting of

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70 approximately 30 subunits and required for transcription driven by RNA polymerase II (RNAP II) ril
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71 (6,7). Mediator plays a canonical role connecting DNA-binding transcription factors with the basal 2
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72 transcription machinery containing RNAP II. Biochemical and structural approaches have 9

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73 revealed four modules in the Mediator complex, i.e., the head, middle, tail and kinase modules. g
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74 The head and middle modules are involved in the association with the basal transcription s
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75 machinery, whereas the tail module is targeted by various signal-specific transcription factors (6).
76 For instance, SREBP (sterol regulatory element binding protein) responding to lipid metabolism
77 status and ELK responding to MAPK signaling directly interact with MED15 and MED23,
78 respectively, in the tail module. Therefore, Mediator functions as a “hub”, receiving and
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79 integrating various regulatory signals for the inducible transcription of signal-specific genes.
80 Currently, a Mediator subunit that receives electrophilic stress signals has not been identified.
81 Six Neh (NRF2-ECH homology) domains have been defined in NRF2 based on species
82 conservation (8). The Neh1 domain contains the basic region-leucine zipper motif, mediating
83 DNA binding and dimerization. The Neh2 and Neh6 domains contain degrons responsible for D
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84 KEAP1-CUL3-dependent and βTrCP (beta-transducin repeat-containing E3 ubiquitin protein n
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85 ligase)-CUL1-dependent degradation, respectively (9, 10). The other domains, namely Neh4, d
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86 Neh5 and Neh3, are considered trans-activation domains. CBP (CREB binding protein) and BRG1 fr
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87 (SMARCA4) associate with the Neh4 and Neh5 domains (11, 12), and CHD6 (chromodomain h
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88 helicase DNA binding protein 6) associates with the Neh3 domain (13). However, the functional :/
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89 significance of the interaction with these factors for NRF2-mediated cytoprotection against b
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90 oxidative insults has not been evaluated. Moreover, a general understanding of how NRF2 m
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91 conveys the activation signal to the basal transcription machinery and triggers the robust induction rg
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92 of its target genes after binding to ARE has not been established. n

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93 To identify an essential transcription cofactor of NRF2 for the antioxidant response, we r
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94 purified and characterized the NRF2 nuclear protein complex. We identified several Mediator
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95 subunits as novel NRF2 associating proteins. Of these, MED16 directly interacted with NRF2 and 9

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96 was essential for the inducible expression of the majority of the NRF2 target genes. Importantly,
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97 MED16 deficiency remarkably sensitized cells to oxidative insults. Thus, this study has clarified a s
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98 missing piece between NRF2 and the basal transcription machinery and revealed the critical
99 contribution of the KEAP1-NRF2-MED16 axis to the defense mechanism from oxidative insults.
100
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101 Materials and Methods
102 Cell culture. 293F, Hepa1c1c7, and Hep3B cells were maintained in low glucose DMEM (Wako)
103 supplemented with 10% fetal bovine serum (Sigma) and penicillin/streptomycin (Gibco) under
104 5.0% CO at 37°C.
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106 Mice. Ptenflox/flox and Keap1flox/flox mice were previously described (14, 15). Ptenflox/+ line of mice lo
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107 was a kind gift from Dr. Akira Suzuki (Kyushu University). The Albumin-Cre transgenic mouse e
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108 was purchased from The Jackson Laboratory (Bar Harbor, ME, USA) (16). ro
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109 Ptenflox/flox::Keap1flox/flox::Albumin-Cre mice were obtained from the mating between h
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110 Ptenflox/flox::Keap1flox/flox and Ptenflox/flox::Keap1flox/+::Albumin-Cre mice, and sacrificed for liver //
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111 protein preparation. The mice were provided water and rodent chow ad libitum. All mice were b
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112 maintained under specific-pathogen-free conditions and treated according to the regulations of The m
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113 Standards for Human Care and Use of Laboratory Animals of Tohoku University and Guidelines g
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114 for Proper Conduct of Animal Experiments of the Ministry of Education, Culture, Sports, Science,
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115 and Technology of Japan. All the animal experiments were approved by The Tohoku University il
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116 Committee for Laboratory Animal Research. 2
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117

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118 Chemicals. Diethyl maleate (DEM) and dimethyl sulfoxide (DMSO) were purchased from Wako g
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119 Pure Chemicals. Menadione was purchased from Sigma-Aldrich. Dithiobis succinimidyl t
120 propionate (DSP) was purchased from Pierce.
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122 Plasmids. pQC-FLAG-hNRF2 T80R and FLAG-hMED10 were generated by inserting
123 FLAG-hNRF2 T80R and FLAG-hMED10 cDNA fragments into the pQCXIP vector (Clontech),
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124 respectively. pGEX4T-1 mNRF2 mutant vectors were generated by inserting cDNAs encoding
125 various mNRF2 truncations into pGEX4T-1. pcDNA3-FLAG-hMED24,
126 pcDNA3-FLAG-hMED23, pcDNA3-FLAG-hMED16 and pcDNA3-FLAG-hMED14 were
127 generated by inserting hMED24, hMED23, hMED16 and hMED14 cDNAs, respectively, into the
128 pcDNA3FLAG vector. pcDNA3-FLAG-hMED16-A, -B, -C and -D were generated by inserting D
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129 truncated hMED16 (amino acids 2-212), (213-434), (435-650) and (651-877) cDNAs, n
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130 respectively, into the pcDNA-3FLAG vector. The pX330-mMED16 gRNA vector was created by d
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131 inserting annealed oligonucleotide pairs (5’-caccGGCCATCACCTGCCTGGAGT-3’ and fr
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132 5’-aaacACTCCAGGCAGGTGATGGCC-3’) into the BpiI sites of pX330.
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133 :
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134 Generation of stable transformant cell lines. To obtain retroviruses for the establishment of c
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135 FLAG-hNRF2 T80R-expressing cells, FLAG-hMED10-expressing cells and cells harboring a m
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136 vacant vector, pQC-FLAG-hNRF2 T80R, pQC-FLAG-hMED10 and pQCXIP were transfected r
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137 into PLAT-A cells, respectively. Medium was changed after 24 hrs of transfection, and the cells n

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138 were cultured for additional 24 hrs in fresh medium. The retrovirus particles were produced in the r
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139 medium, which was used for the transduction of 293F cells (Life Technologies), Hepa1c1c7 cells, ,
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140 and Med16-deficient Hepa1c1c7 cells (Med16 KO #1, see below) cells. These cells were 9

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141 transduced with the respective retroviruses in a suspension with 12 μg/mL of polybrene. One day y
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142 after infection, infected cells were replated and incubated in a selection medium containing 2 s
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143 μg/mL of puromycin (Sigma). For the establishment of stable cell lines expressing
144 FLAG-hMED24, FLAG-hMED23, FLAG-hMED16 and FLAG-hMED14, 293F cells were
145 transfected with pcDNA3-FLAG-hMED24, pcDNA3-FLAG-hMED23,
146 pcDNA3-FLAG-hMED16 and pcDNA3-FLAG-hMED14, respectively. After transfection, the
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147 cells were replated and incubated with selection medium containing 1.5 mg/ml Geneticin (Nacalai
148 Tesque).
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150 Identification of NRF2-interacting proteins in 293F cells. Nuclear extract was prepared from
151 FLAG-hNRF2 T80R-expressing 293F cells. Nuclear extract was subjected to anti-FLAG affinity D
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152 purification. The FLAG-hNRF2 T80R complex was eluted by FLAG peptide according to the n
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153 manufacturer’s protocol (Sigma). The eluate was subjected to nano-high performance liquid d
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154 chromatography-tandem mass spectrometry (nano LC-MS/MS) analysis, and NRF2-associated fr
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155 proteins were identified through the protein sequence database searching.
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156 :
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157 Nano LC-MS/MS analysis and protein sequence database searches. Trypsin-digested peptides c
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158 were dissolved in sample solution [5% acetonitrile and 0.1% trifluoroacetic acid (TFA)]. Each m
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159 sample was injected into an EasynLC-1000 system (Thermo Scientific), which was connected to an r
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160 EASY-Spray column (25 cm length x C18 ODS 75 µm, Thermo). Peptides were eluted with a 120 n

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161 min gradient of 4% to 35% solvent B (0.1% formic acid in acetonitrile, v/v) in solvent A (0.1% r
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162 formic acid in water, v/v) at a flow rate of 300 nl/min. Peptides were then ionized and analyzed by ,
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163 a Q-Exactive mass spectrometer (Thermo Scientific) using a nano-spray source. High-resolution 9

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164 full scan MS spectra (from m/z 380-1800) were acquired in the Orbitrap with resolution R=70,000
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165 at m/z 400 and lockmass enabled (m/z at 445.12003, and 391.28429), followed by MS/MS s
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166 fragmentation of the 10 most intense ions in the linear ion trap with high collisionally activated
167 dissociation (HCD) energy of 35%. The exclusion duration for the data-dependent scan was 0 s,
168 and the isolation window was set at 2.0 m/z.
169 The MS/MS data were analyzed by sequence alignment using variable and static
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170 modifications with Mascot algorithms. The protein database utilized was Swiss-Plot, which
171 considers each peptide sequence in Trypsin-digested fragment patterns. The specific parameters
172 for protein sequence database searching included oxidation (M), deamination (N, Q), acetylation
173 (N-term.), and pyroglutamation (E) as variable modifications and carbamidomethylation (C) as a
174 static modification. Other parameters used in data analysis were: two allowed missing cleavages, D
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175 the mass error of 10 ppm for precursor ions, and 0.02 Da for fragment ions. Charge states of +2 to n
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176 +4 were considered for parent ions. If more than one spectrum was assigned to a peptide, only the d
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177 spectrum with the highest Mascot score was selected for manual analysis. fr
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178
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179 Generation of Nrf2 knockdown cell lines. Lentiviral particles expressing control shRNA and :
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180 mNrf2 shRNAs (TRCN54659 and TRCN54658) were purchased from Sigma-Aldrich. Hepa1c1c7 c
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181 cells were infected for 24 hrs with the lentivirus at a multiplicity of infection of 10. Infected cells m
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182 were washed with PBS, followed by 12 hrs of incubation. The cells were replated and incubated in r
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183 a selection medium containing 2 μg/mL of puromycin (Sigma). n

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184 r
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185 Generation of Med16 knockout cell lines. Hepa1c1c7 cells (1.5x105 cells/well) were plated in ,
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186 6-well plates 24 hrs prior to transfection. Cells were co-transfected with 2 μg pX330-mMED16 9

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187 gRNA and 0.2 μg pcDNA3 using Lipofectamine 2000 (Life Technologies). pcDNA3 was included g
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188 to confer the Geneticin resistance as a selection marker. Medium was changed after 24 hrs of s
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189 transfection. After another 24 hrs of incubation, the cells were replated in 10-cm dishes and
190 incubated with selection medium containing 1.5 mg/ml Geneticin (Nacalai Tesque). Among the
191 Geneticin-resistant healthy clones, two clones, Med16 KO #1 and Med16 KO #2, were arbitrarily
192 selected and used for further analyses. Disruption of Med16 gene was verified by sequencing DNA
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193 surrounding the target site of the gRNA. Genomic DNA was purified from the two clones and used
194 as a template for PCR amplification of the DNA fragment spanning the gRNA target site using a
195 primer set, mMed16 CRISPR seq F (5’-CAG CAT CAG CAG ACA GTA GCC G-3’) and
196 mMed16 CRISPR seq R (5’-GTG GGG ACA CAG GCA CTT CG-3’). The amplified DNA
197 fragments were inserted into EcoRV site of pcDNA3 and used for bacterial transformation. Nine D
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198 and ten bacterial colonies were picked up for MED16 KO #1 and #2, respectively, and purified n
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199 plasmids were sequenced using T7 primer (5’-TAA TAC GAC TCA CTA TAG GG-3’) and d
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200 pcDNA3 3’ primer (5’-TAG AAG GCA CAG TCG AGG-3’). fr
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201
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202 Microarray analysis and definition of NRF2-activated genes. Hepa1c1c7 cells with Nrf2 :
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203 shRNA (Nrf2 KD cells; samples 1 and 2), Hepa1c1c7 cells with control shRNA (sh-control cell; c
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204 samples 3 and 4), Med16 KO Hepa1c1c7 cells (Med16 KO cells; samples 5 and 6) and parent m
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205 Hepa1c1c7 cells (WT cells; samples 7 and 8) were cultured and harvested after 12 hrs of treatment r
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206 with 100 μM DEM (samples 1, 3, 5 and 7) or with DMSO (vehicle) (samples 2, 4, 6 and 8), and n

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207 total RNAs were purified. The total RNAs were processed and hybridized to a 4×44k whole mouse r
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208 genome microarray (Agilent Technologies). The Gene Chip experiments were performed
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209 according to the manufacturer’s protocol. The arrays were scanned using the G2539A Microarray 9

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210 Scanner system (Agilent Technologies).
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211 The resulting data were analyzed using GeneSpring GX software (Agilent technologies). s
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212 The “NRF2-activated genes” were defined as follows. Genes were selected as “DEM-activated
213 genes” if they satisfied both of the following conditions: log [sample 3/ sample 4]
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214 (log2-fold-change of sh-control cells) > 0.5 and log [sample 7/ sample 8] (log2-fold-change of WT
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215 cells) > 0.5. Among the “DEM-activated genes”, “NRF2-dependent DEM-activated genes
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216 (NRF2-activated)” were selected if they satisfied the following condition: log [sample 1/ sample
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217 2] (log2-fold-change of Nrf2 KD cells) < log [sample 3/ sample 4] (log2-fold-change of sh-control
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218 cells). Consequently, 848 genes were defined as “NRF2-activated genes”. Values of log [sample
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219 5/ sample 6] (log2-fold-change of Med16 KO cells) and log [sample 7/ sample 8]
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220 (log2-fold-change of WT cells) of the 848 genes were indicated in a heat map, where the 848 genes D
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221 were aligned in declining order of the values of log [sample 7/ sample 8] (log2-fold-change of WT n
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222 cells) – log [sample 5/ sample 6] (log2-fold-change of Med16 KO cells). d
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223 fr
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224 Immunoblot analysis. Immunoblot analysis was performed as described previously (17). The
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225 antibodies used were as follows: anti-FLAG (F7425; Sigma), anti-MED24 (A301-472A; Bethyl :
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226 lab), anti-MED23 (ab70450; Abcam), anti-MED16 (ab130996; Abcam), anti-MED1 (sc-8998; c
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227 Santa Cruz), anti-MED7 (ab50687; Abcam), anti-hNRF2 (sc-13032; Santa Cruz), anti-Tubulin m
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228 (T9026; Sigma) and anti-mNRF2 (18) antibodies. r
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229 n

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230 Protein preparations from cell lines. GST fusion proteins of various NRF2 mutants were r
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231 expressed in the E. coli strain BL21 (DE3), and soluble lysates were prepared in PBS-T (0.1% ,
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232 Tween 20) by sonication. For Mediator complex purification, a soluble nuclear extract was 9

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233 prepared from 293F cells, Hepa1c1c7 cells, and Med16 KO Hepa1c1c7 cells stably expressing
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234 FLAG-hMED10 (19). The nuclear extract was subjected to anti-FLAG affinity purification. s
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235 FLAG-Mediator complexes were eluted by FLAG peptide according to the manufacturer’s
236 protocol (Sigma). Recombinant FLAG-hMED24, FLAG-hMED23, FLAG-hMED16 and
237 FLAG-hMED14 were expressed in 293F cells and purified by the same procedure as that for the
238 Mediator complex purification. To prepare whole cell extracts containing FLAG-hMED16-A, -B,
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