Fan and Gélinas, Rel’s TADs and cell transformation
Supplementary material
“An optimal range of transcription potency is necessary for efficient cell transformation by c-Rel to ensure optimal nuclear localization and gene-specific activation”
Yongjun Fan and Céline Gélinas
Plasmids and Mutagenesis
Rel cDNAs were expressed from the cytomegalovirus (CMV) immediate-early promoter of pJDCMV19SV (Dougherty et al., 1989) for IL6kB-LUC activation assays. Rel cDNAs were expressed from the long terminal repeat (LTR) promoter of spleen necrosis virus (SNV)-derived retroviral vector pJD214 (Dougherty & Temin, 1986) for DNA binding and lymphoid cell transformation assays, immunofluorescence, western blot, co-immunoprecipitation and RT-PCR analyses. SW253 encodes a replication-competent RevA helper virus (Watanabe & Temin, 1983). cc-rel cDNA was a gift from T. Gilmore (Boston Univ., MA) and was subcloned into the Xba 1 site of pAlter-1 (Promega, WI) for mutagenesis with QuickChange (Stratagene). Mutant c-rel cDNAs were recovered by restriction with Hpa II and subcloned in the Acc 1 site of pJDCMV19SV or the Cla 1 site of pJD214 for expression in vivo. Wild-type or mutant v-Rel or c-Rel TADs were fused in frame to a yeast GAL4 DNA binding domain by PCR using 5’ and 3’ oligonucleotide primers containing Sty 1 and BamH I sites, respectively, followed by subcloning into plasmid pCG147 (Tanaka & Herr, 1990). TAD exchange mutants were generated by two-step overlapping PCR, as described (Fan et al., 2004). All constructs were verified by sequencing (NJMS Molecular Resource Facility, Newark, NJ).
Cell transfection and luciferase assays
Transactivation was measured in 293T cells (1 x 106 per 6-cm dish) co-transfected with GAL4-Rel plasmids (50 ng), a GAL-luciferase reporter (0.5 µg) and pRL-TK Renilla luciferase (10 ng; Promega Corp., Madison, WI) using calcium phosphate. The transcriptional activity of cc-Rel or v-Rel mutants expressed from pJDCMV19SV was determined on pIL6kBLUC in transfected 293T cells. In all cases, the total amount of transfected DNA (3 µg) was kept constant by addition of pUC18 DNA. Extracts were prepared at 36 hours post-transfection and analyzed for protein concentration and dual-luciferase (Promega Corp., Madison, WI). The average of three experiments is shown.
DNA binding assays
The DNA-binding activity of Rel mutants expressed from the SNV-derived retroviral vector pJD214 was determined by gel shift on an IL6kB oligonucleotide probe, using equivalent amounts of nuclear Rel proteins (Fan et al., 2004).
CEF transfection and primary lymphoid cell transformation assays
Cell transformation was analyzed in primary chicken splenic lymphocytes (Charles River SPAFAS, North Franklin, CT) infected with virus harvested from chicken embryo fibroblasts (CEFs) co-transfected with pJD214-rel (5 mg) and RevA helper virus DNA (0.1 mg). Lymphoid cells were seeded into soft agar at day 4 post-infection and colonies were scored after 2 weeks. Three independent experiments performed in duplicate or triplicate are shown.
Immunofluorescence analysis
The subcellular localization of Rel mutants was analyzed in infected CEFs at day 4 post-infection using anti-Rel antibody sc-6955 (Santa-Cruz Biotechnology, Santa Cruz, CA) and photographed at a magnification of 1000x.
Western blot and co-immunoprecipitation assays
The steady-state levels of endogenous IkBa protein were determined by western blot at day 4 post-infection of CEFs with Rel mutants, using anti-Rel RHD antibody #2716 or anti-chicken IkBa antibody #1275 (a gift from N. Rice and M. Ernst, NCI, Frederick MD). Association of Rel mutants with endogenous IkBa was analyzed by co-immunoprecipitation of CEFs extracts (300 µg) prepared at day 4 post-infection with anti-Rel RHD antibody #2716 followed by immunoblotting with anti-Rel and reprobing for IkBa using ReliaBLOT blocking reagent (Bethyl Laboratories, Montgomery, TX). The anti-Rel RHD antibody poorly recognized A4 (Figure 2f-g).
cc-Rel-sw-vTAD showed reduced relative association with endogenous IkBa compared to cc-Rel, despite the fact that it retains the c-Rel-derived amino acids near the nuclear localization signal implicated in preferential interaction with IkBa compared to v-Rel (Figure 2g lane 7; Diehl et al., 1993; Hrdlickova et al., 1995; Rottjakob et al., 1996; Sachdev & Hannink, 1998).
Semi-quantitative RT-PCR
The effects of Rel mutants on the activation of endogenous mip-1b, irf-4 and gapdh transcripts in infected CEFs (day 4) was analyzed by semi-quantitative RT-PCR, using the iscript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA) and Taq DNA polymerase (Invitrogen Corp., Carlsbad, CA).
References for Supplementary Material
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Dougherty JP, Wisniewski R, Yang S, Rhode BW, Temin HM (1989). New retrovirus helper cells with almost no nucleotide sequence homology to retrovirus vectors. J. Virol. 63: 3209-3212.
Fan Y, Rayet B, Gélinas C (2004). Divergent C-terminal transactivation domains of Rel/NF-kB proteins are critical determinants of their oncogenic potential in lymphocytes. Oncogene 23: 1030-1042.
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