Yashiro-Ohtani et al., Notch1 regulation by pre-TCR signals
Supplemental Data
Pre-TCR signaling inactivates Notch1 transcription by antagonizing E2A
Yumi Yashiro-Ohtani et al.
Supplemental Experimental Procedures
Cell preparation and Western blotting
5 x 105 Rag2-/- DN3 cells were pre-treated with either DMSO, 2.5µM MG132 (Calbiochem) and/or 10 µM MEKK1/2 inhibitor (U-0126) for 30 min followed by PMA simulation (20ng/ml). Cells were collected 2h after treatment and 20 µg of protein lysate was subjected to Western blotting. Antibodies against the Notch1 TAD/PEST domain, phospho-ERK (Cell Signaling) and b-actin (Sigma) were used.
Luciferase Reporter assay
The Hes1 reporter (Jarriault et al. 1995) and E2A reporter (Carter et al. 1997) were used. Id3, E47, and Notch1 intracellular domain (ICN1) cDNAs were cloned into the MigR1 retroviral vector. NIH3T3 cells were transduced with MigR1 or MigR1-ID3 retroviral supernatants and GFP+ cells were sorted. These GFP+ cells were transfected with pGL3-Hes1 (200ng) or pGL3-E2A (200ng) and pRL-TK (25ng) by Lipofectamin-2000 (Invitrogen). MigR1, MigR1-ICN1, or MigR1-E47 (25ng or 100ng) were cotransfected. Cells were collected at 28h post-transfection and luciferase activity was measured with the Dual-Luciferase reporter assay kit (Promega).
Supplemental Table 1. Primers used for RT-PCR, ChIP assay, Notch1 promoter cloning
1. RT-PCR
Targets / Forward Primers / Reverse PrimersEF1 alpha / 5'-CACTTGGTCGCTTTGCTGTT-3' / 5'-GGTGGCAGGTGTTAGGGGTA-3'
18S rRNA / 5'-GCGCCGCTAGAGGTGAAAT-3' / 5'-GGCGGGTCATGGGAATAAC-3'
Id1 / 5'-TACTTGGTCTGTCGGAGCAA-3' / 5'-GATCAAACCCTCTACCCACT-3'
Id2 / 5'-CCTGGACTCGCATCCCACTA-3' / 5'-TGCTATCATTCGACATAAGCTCAGA-3'
Id3 / 5'-ATCCTGCAGCGTGTCATAGACT-3' / 5'-AGGCGTTGAGTTCAGGGTAAGT-3'
Notch1 primary RNA / 5'-AGATCGACAACCGGCAATGT-3' / 5'-ATCCCTCCAGCCCCTGATTA-3'
Notch1 mRNA / 5'-AGATCGACAACCGGCAATGT-3' / 5'-CCCACAGCCCACAAAGAAC-3'
2. ChIP assay
Targets / Forward Primers / Reverse PrimersNotch1-10 kb / 5'-TAGGGAGTGCCCACAAAA-3' / 5'-AACCTCCAGCCATCCTTCTT-3'
Notch1 -6 kb / 5'-CTAAGGCCCCATTCCTTCTC-3' / 5'-CATTGCTCCATCCTCCATTT-3'
Notch1 -5 kb / 5'-AAATGGCCCTGAGCAAGACT-3' / 5'-CCGCACATCTGTGAGCTATTT-3'
Notch1 -1 kb / 5'-AGCATGAGAGGCTGTGTTGA-3' / 5'-AGGGAACTCCCCAAGGACTA-3'
Notch1 intron1 / 5'-CTCCACCCTTGCCTCAGTTC-3' / 5'-ACAATGGGCCGCTCTGATT-3'
Notch1 -5.5 kb / 5'-CCAGAGAGCCCCTTCAGAGA-3' / 5'-GGCCTCTGTGATCCCCTACA-3'
Notch1 -4.4 kb / 5'-GGTAAAGCAACAAGAGCTTGAACA-3' / 5'-CAACCCTCGGCAGTGTCTCT-3'
Notch1 -3 kb / 5'-AGGCCCTGCTGTTCTCCTACT-3' / 5'-AAGGCCATCTGTGGGATGAC-3'
Notch1 -0.3 kb / 5'-AAGGAGGAATGGAGGGGTGTA-3' / 5'-TTTCCCACAAGACCCGTACC-3'
Hes1 promoter / 5'-CGTGTCTCTTCCTCCCATTG-3' / 5'-CCAGGACCAAGGAGAGAGGT-3'
TCR beta Vb5.1 / 5'-CCCAGCAGATTCTCAGTCCAACAG-3' / 5'-GTTTAATGGCATCACCCAACC-3'
3. Cloning primers
Target / Forward Primers / Reverse PrimersNotch1 promoter / 5’-AACGCGTCCCTCACCTGCTTGGTTCTC-3’ / 5’-AAGCTTGCCTGCTCGCCAGCTGCC-3’
HEB / 5'-GACTCGAGCACCATGAATCCCCAGCAGCA-3' / 5'-CAGATATCTTACAGATGACCCATAGG-3'
Figure S1. Notch1 downregulation by pre-TCR stimulation in DN3 cells
DN3 cells were purified from Rag2-/- mice and were treated with DMSO and/or the MEK inhibitor, U-0126 and/or the protease inhibitor, MG132 for 1 hour before PMA stimulation (20ng/ml). Cells were collected 2 hours after stimulation and Notch1 protein expression and ERK phosphorylation were detected by Western blot analysis (A). qPCR was performed and Notch1 mRNA expression relative to EF1-alpha was shown relative to the control sample (B). Data are representative of three independent experiments. UT-untreated sample.
Figure S2. Scid-adh phenotype after pre-TCR signals.
Scid-adh cells were stimulated with PMA (20ng/ml) for 6h, 24h and 48h. Cells were then stained with aCD44 and aCD25. Analytical flow cytometry was performed on a FACS Calibur (Becton Dickinson) and analyzed with FlowJo software (Treestar).
Figure S3. Notch1 expression in ICN1 and/or Id3 expressing T-ALL cells
G4A2 cells were co-transduced with the GFP (MigR1), Mig-ICN1, NGFR and NGFR-Id3 retroviruses and GFP+NGFR+ cells were sorted at day 1 post-transduction. GFP+NGFR+ cells were cultured for 2 days. Notch1 expression relative to EF1a is shown as the mean of values from triplicate wells ± SD. Data are representative of three independent experiments.
Figure S4. Id3 does not block ICN1-induced transcriptional activation of the Hes1 promoter.
NIH3T3 cells were transduced with either MigR1 (control) or MigR1-Id3. Transduced cells (GFP+) were sorted and transiently cotransfected with the promoter construct and either empty vector, E47 or ICN1. Luciferase activity relative to Renilla luciferase activity is shown as a mean of values from triplicate wells ± SD.
References:
Carter, R.S., Ordentlich, P., and Kadesch, T. 1997. Selective utilization of basic helix-loop-helix-leucine zipper proteins at the immunoglobulin heavy-chain enhancer. Mol Cell Biol 17: 18-23.
Jarriault, S., Brou, C., Logeat, F., Schroeter, E.H., Kopan, R., and Israel, A. 1995. Signalling downstream of activated mammalian Notch. Nature 377: 355-358.
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