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Blood, Vol. 109, Issue 9, 4023-4027, May 1, 2007
Concurrent transcriptional deregulation of AML1/RUNX1 and GATA factors by the AML1-TRPS1 chimeric gene in t(8;21)(q24;q22) acute myeloid leukemia
Blood Asou et al.
109: 4023
Supplemental materials for: Asou et al, Vol 109, Issue 9, 4023-4027
Files in this Data Supplement:
- Figure S1. Specificity of AML1, AML1-TRPS1, or TRPS1 binding to the AML1 or GATA site (PDF, 45.2 KB) -
(A) Sequence-specific binding of AML1 and AML1-TRPS1 to the AML1 site. Whole-cell extracts of COS7 cells transfected with the expression vectors for AML1 and AML1-TRPS1 were subjected to EMSA. All lanes, except for the end left lane (–), were supplemented with a 50-fold molar excess of mutant probe (M) to eliminate the non-specific band indicated by the asterisk. The binding of AML1 or AML1-TRPS1 was competed out by 50-fold molar excess of unlabeled wild type competitor (lanes M+C). The sequences of the wild-type and mutant probes for the AML1 site have been described previously.12 (B-C) Specificity of GATA-1, AML1-TRPS1 and TRPS1 binding to the GATA site. Whole-cell extracts of COS7 cells transfected with the expression vectors for GATA-1, AML1-TRPS1, or FLAG-tagged TRPS1 (FL-TRPS1) were subjected to EMSA. The binding of these 3 factors to the GATA probe lanes (–) was competed out by a 50-fold molar excess amount of unlabeled wild-type competitor (C), but not by the 50-fold molar excess of mutant probe (M), in both panels B and C. The sequences of the wild-type and mutant probes for GATA1 were 5′-ctggggacagataagctacagc-3′, and 5′-ctggggacagacaagctacagc-3′, respectively. The GATA site is underlined. The FL-TRPS1 and DNA complex was supershifted in the presence of anti-FLAG antibody (M2 monoclonal antibody; Sigma) (C; lane Ab).
- Figure S2. AML1 or GATA site-dependent activation of pBXH2-luc or pRBGP3-MαP reporter (PDF, 24.8 KB) -
(A) AML1 site-dependent activation of pBXH2-LTR-luc reporter. AML1 and AML1-TRPS1 were cotransfected with pBXH2-LTR-luc or mutant pBXH2-LTR-luc (Mut LTR) reporters, at the indicated doses, into HL-60 cells. Luciferase activity is expressed as fold changes relative to the control. The Mut LTR reporter carrying AML1 site mutation was generated by PCR-based mutagenesis using 5′-aacaggatatgtgtCgtcaagcactagggc-3′ as a primer. The substituted nucletotide in the AML1 site underlined is capitalized. (B) Repression of GATA-1–mediated transcription by TRPS1 per se. GATA-1, AML1-TRPS1, or FLAG-tagged TRPS1 (FL-TRPS1) were cotransfected with M P or mutant MP (Mut MP) reporters at the indicated doses, into NIH3T3 cells. Luciferase activity is expressed as fold changes relative to the control. Repression of GATA-1–mediated transcription by TRPS1 was statistically significant (P = .007, unpaired student t test). Protein expression level of FL-TRPS1 and AML1-TRPS1 is shown in the lower panel. Western blotting was carried out using goat polyclonal anti-TRPS1 antibody (N-18; Santa Cruz Biotechnology). Since the protein expression of FL-TRPS1 was lower than that of AML1-TRPS1, the repression of FL-TRPS1 might has been comparable to that of AML1-TRPS1. The Mut MP reporter carrying GATA site mutation was generated by PCR-based mutagenesis using 5′-tctccggcaactgaCaaggattccctgga-3′ as a primer. The substituted nucletotide in the underlined GATA site is capitalized. No activity in the Mut MP reporter suggests that various changes in transcriptional activities observed in this assay, namely activation by GATA-1 and repression by AML1-TRPS1 and TRPS1 per se, are all GATA site dependent.
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