Fig 1: HBZ associates with MAREs in an enhancer upstream of HMOX1.(A) The HMOX1 promoter contains two small-Maf-binding regions (Distal and Proximal). The schematic generated from the UCSC genome browser (http://genome.ucsc.edu/, assembly GRCh37/hg19) shows peaks of MafK enrichment within the HMOX1 promoter in HeLa cells (http://genome.ucsc.edu/; MafK, assembly, GRCh37/hg19). The Distal MafK-binding peak in HeLa cells overlaps with a peak of HBZ enrichment in ATL cells (http://genome.ucsc.edu/; HBZ assembly, NCBI36/hg18 using public data sets GSM2481678 and GSM2481679). Distal and Proximal peak regions and a downstream region used as a ChIP control are shown in the schematic. (B-D) HBZ is recruited to the region of the HMOX1 promoter corresponding to the distal peak of MafK-binding. ChIP assays were performed on chromatin prepared from the indicated HeLa cell lines using antibodies against MafG, Nrf2 and the C-terminal His tag of HBZ. Data are presented as fold enrichment relative to enrichment at the downstream control region in the empty vector (pcDNA) cell line. Data are an average of three independent experiments. Error bars represent SEM (two-tailed Student’s t-test, *p<0.05, **p<0.01, ***p<0.001). (E) In TL-Om1 cells, MafG binds to the Distal peak region that is also bound by HBZ. ChIP assays were performed on chromatin prepared from TL-Om1 cells using antibodies against MafG. Data are presented as fold enrichment relative to enrichment at the downstream control region. Data are an average of 5 independent experiments. Error bars represent SEM (two-tailed Student’s t-test, *p<0.05, **p<0.01, ***p<0.001).
Fig 2: HBZ augments formation of the MafG complex on all three distal MAREs in vitro.In EMSAs, the indicated combinations of recombinant, purified MafG-His (0.5 nM), GST-HBZ (50 or 500 nM), and T7-HBZ-bZIP (50 or 500 nM) were combined with the indicated DNA probes (sequences shown below) prior to electrophoretic separation of complexes. The arrow denotes the shifted protein/DNA complex, the asterisk denotes unbound probe.
Fig 3: HBZ/small Maf heterodimers activate transcription from T-MAREs.(A) The schematic shows the pGL-4xT-MARE-Luc plasmid used in reporter assays. Four tandem repeat sequences containing a T-MARE were cloned into pGL4.26 upstream of the minimal promoter (minP). (B) HBZ activates transcription from T-MAREs. Jurkat cells were co-transfected with increasing amounts of pSG-HBZ-Myc and with pGL4.26 or PGL-4xT-MARE-Luc. The graph shows data calculated by subtracting pGL4.26 luminescence values from expression-matched pGL-4xT-MARE-Luc luminescence values. Data are an average of two independent experiments, each of which was performed in triplicate. Error bars represent SEM (two-tailed Student’s t-test, **p≤0.01, ***p≤0.001). (C) HBZ activates transcription from T-MAREs, which is dependent on the ZIP domain. Jurkat cells were co-transfected with pSG-HBZ-Myc or pSG-HBZ-MutZIP and pGL4.26 or pGL-4xT-MARE-Luc. The graph shows data calculated by subtracting pGL4.26 luminescence values from expression vector-matched pGL-4xT-MARE-Luc luminescence values. Data are an average of three independent experiments, each of which was performed in triplicate. Error bars represent SEM (two-tailed Student’s t-test, ***p≤0.001). (D) The schematic shows full-length Nrf2 with its characterized domains and the region of Nrf2 comprising the dominant negative deletion mutant (Nrf2 DN). The Keap1 binding site, activation domain (AD) and Cap’n’Collar (CNC) bZIP domain encompass Nrf2-ECH homology domain 2 (Neh2), Neh4/5 and Neh1, respectively. (E) A dominant negative mutant of Nrf2 suppresses HBZ-mediated transcriptional activation from T-MAREs. Jurkat cells were co-transfected with pSG-HBZ-Myc and/or pcDNA-Nrf2-DN-Myc and pGL4.26 or pGL-4xT-MARE-Luc. The graph shows data calculated by subtracting pGL4.26 luminescence values from expression vector-matched pGL-4xT-MARE-Luc luminescence values. Data are an average of three independent experiments, each of which was in performed in triplicate. Error bars represent SEM (two-tailed Student’s t-test, **p≤0.01, ***p≤0.001). (F) Jurkat cells were co-transfected with pSG-HBZ-Myc, increasing amounts of pcDNA-Nrf2-DN-Myc, and pGL4.26 or pGL-4xT-MARE-Luc. The graph shows data calculated by subtracting pGL4.26 luminescence values from expression vector-matched pGL-4xT-MARE-Luc luminescence values. Data shown are representative of three independent experiments, each performed in triplicate. Error bars represent standard deviation. (G) A dominant negative mutant of Nrf2 suppresses transcription of endogenous HMOX1 in Hela cells stably expressing HBZ. HeLa cells stably expressing HBZ-Myc-His were transfected with pMACS CD4 and pcDNA or pcDNA-Nrf2-DN-Myc. Successfully transfected cells were isolated according to ectopic expression of the CD4 surface marker and aliquoted for RNA extraction and, separately, whole cell extract preparation. Relative mRNA levels in control- and Nrf2-DN-transfected cells were quantified by qRT-PCR. Levels of the indicated proteins in 40 μg of whole cell extract were evaluated by Western blot using the antibodies indicated. qRT-PCR data are an average of four independent experiments. Error bars represent SEM (two-tailed Student’s t-test, *p≤0.05). (H) HBZ modulates binding of p300/CBP to HBZ/small Maf complexes. HEK 293T cells were transfected with 1 μg pCMV-MafG-FLAG and/or 6 μg pcDNA-HBZ-Myc-His as indicated (adjust to 12 μg with the empty vectors). Proteins were immunoprecipitated using an anti-FLAG antibody to bind MafG. Immunoprecipitates and 10% the whole cell extract inputs were analyzed by Western blot using the antibodies indicated.
Fig 4: HBZ interacts with the small Mafs to form a DNA-bound complex at MAREs.(A) A proteomic analysis identified all three small Mafs (MafF, MafG, and MafK) as potential HBZ-binding partners. A FLAG antibody was used to immunoprecipitate proteins from whole cell extracts prepared from a HeLa clonal cell line expressing HBZ with a C-terminal FLAG tag or a cell line containing the empty vector. Immunoprecipitates were analyzed by LC-MS/MS. Protein identifications were accepted at a false discovery rate of <1%, and a probability of ≥95%, and a maximum of one missed cleavage [68]. The number of unique peptides corresponding to each small Maf identified are shown. Data were obtained from a single experiment. (B and C) HBZ interacts with the small Mafs in transfected HEK 293T cells. Cells were transfected with 6 μg pcDNA-HBZ-Myc-His and/or 6 μg pCMV-MafG-FLAG or pCMV-MafK-FLAG (adjusted to 12 μg of total plasmid with the empty vector). HBZ and the small Mafs were immunoprecipitated using anti-Myc (IP: Myc) and anti-FLAG (IP: FLAG) antibodies, respectively. Immunoprecipitates and ten percent of the whole cell extract inputs were analyzed by Western blot using the antibodies indicated. (D) Endogenous MafG and HBZ interact in ATL cells. MafG (IP:MafG) was immunoprecipitated from CEM or ATL-2s whole cell extracts. Mouse serum (IP:MS) was used as a negative control for immunoprecipitation. Immunoprecipitates were analyzed by Western blot using the antibodies indicated. (E) The small Maf/HBZ interaction requires the ZIP domain of HBZ. HEK 293T cells were transfected with 1 μg pCMV-MafG-FLAG and/or 6 μg pcDNA-HBZ-Myc-His or 6 μg pcDNA-HBZ-MutZIP-Myc-His (adjusted to 12 μg of total plasmid with the empty vector). HBZ/MutZIP and the small Mafs were immunoprecipitated using anti-Myc (IP: Myc) and anti-FLAG (IP: FLAG) antibodies, respectively. Immunoprecipitates and ten percent of the whole cell extract inputs were analyzed by Western blot using the antibodies indicated. (F) HBZ augments formation of a MafG/MARE complex in vitro. In EMSAs, the indicated combination of recombinant, purified MafG-His (0.5 nM or 1 nM), GST-HBZ (50 nM), and GST-HBZ-AD (50 nM) were combined with a DNA probe containing the consensus T-MARE sequence or one containing AA mutations in the flanking GC boxes (MARE MT; sequences shown below) prior to electrophoretic separation of complexes. The panels shown are from the same gel/same scanned image (identical threshold adjustment). The arrow denotes the shifted protein/DNA complex; the asterisk denotes unbound probe. (G) In the presence of MafG, HBZ binds DNA containing the T-MARE sequence. Nuclear extracts prepared from HEK 293T cells transfected with pcDNA-HBZ-Myc-His and/or pCMV-MafG-FLAG were combined with a biotinylated DNA probe containing the T-MARE sequence coupled to streptavidin beads or with streptavidin beads alone (Beads). Bound proteins and 10% of the nuclear extract inputs were analyzed by Western blot using the antibodies indicated.
Supplier Page from DNASU for MAFG (Homo sapiens) in pDNR-Dual (Creator donor/master vector)