Identification and characterization of specific DNA-binding complexes containing members of the Myc/Max/Mad network of transcriptional regulators
In the past, eukaryotic cell-derived complexes of the Myc/Max/Mad network of transcriptional regulators have largely been refractory to DNA binding studies. We have developed electrophoretic mobility shift assay conditions to measure specific DNA binding of Myc/Max/Mad network complexes using a COS7 cell-based overexpression system. With the established protocol, we have measured on- and off-rates of c-Myc/Max, Max/Max, and Mad1/Max complexes and determined relative affinities. All three complexes appeared to bind with comparable affinity to a Myc E-box sequence. Furthermore, our data derived from competition experiments suggested that the Mad3/Max and Mad4/Max complexes also possess comparable DNA binding affinities. The conditions established for COS7 cell-overexpressed proteins were then used to identify c-Myc/Max, Max/Max, and Mnt/Max complexes in HL-60 cells. However, no Mad1/Max could be detected, despite the induction of Mad1 expression during differentiation. Whereas the DNA binding activity of c-Myc/Max complexes was down-regulated, Max/Max binding increased, and Mnt/Max binding remained unchanged. In addition, we have also tested for upstream stimulatory factor (USF) binding and observed that, in agreement with published data, USF comprises a major Myc E-box-binding factor that is more abundant than any of the Myc/Max/Mad network complexes. Similar to the Mnt/Max complex, the binding activity of USF remained constant during HL-60 differentiation. Our findings establish conditions for the analysis of DNA binding of Myc/Max/Mad complexes and indicate posttranslational regulation of the Max/Max complex.
Medienart: |
Artikel |
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Erscheinungsjahr: |
1998 |
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Erschienen: |
1998 |
Enthalten in: |
Zur Gesamtaufnahme - volume:273 |
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Enthalten in: |
The Journal of biological chemistry - 273(1998), 12 vom: 20. März, Seite 6632-42 |
Sprache: |
Englisch |
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Beteiligte Personen: |
Sommer, A [VerfasserIn] |
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Anmerkungen: |
Date Completed 16.04.1998 Date Revised 09.02.2021 published: Print Citation Status MEDLINE |
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Förderinstitution / Projekttitel: |
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PPN (Katalog-ID): |
NLM09448676X |
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245 | 1 | 0 | |a Identification and characterization of specific DNA-binding complexes containing members of the Myc/Max/Mad network of transcriptional regulators |
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500 | |a Date Completed 16.04.1998 | ||
500 | |a Date Revised 09.02.2021 | ||
500 | |a published: Print | ||
500 | |a Citation Status MEDLINE | ||
520 | |a In the past, eukaryotic cell-derived complexes of the Myc/Max/Mad network of transcriptional regulators have largely been refractory to DNA binding studies. We have developed electrophoretic mobility shift assay conditions to measure specific DNA binding of Myc/Max/Mad network complexes using a COS7 cell-based overexpression system. With the established protocol, we have measured on- and off-rates of c-Myc/Max, Max/Max, and Mad1/Max complexes and determined relative affinities. All three complexes appeared to bind with comparable affinity to a Myc E-box sequence. Furthermore, our data derived from competition experiments suggested that the Mad3/Max and Mad4/Max complexes also possess comparable DNA binding affinities. The conditions established for COS7 cell-overexpressed proteins were then used to identify c-Myc/Max, Max/Max, and Mnt/Max complexes in HL-60 cells. However, no Mad1/Max could be detected, despite the induction of Mad1 expression during differentiation. Whereas the DNA binding activity of c-Myc/Max complexes was down-regulated, Max/Max binding increased, and Mnt/Max binding remained unchanged. In addition, we have also tested for upstream stimulatory factor (USF) binding and observed that, in agreement with published data, USF comprises a major Myc E-box-binding factor that is more abundant than any of the Myc/Max/Mad network complexes. Similar to the Mnt/Max complex, the binding activity of USF remained constant during HL-60 differentiation. Our findings establish conditions for the analysis of DNA binding of Myc/Max/Mad complexes and indicate posttranslational regulation of the Max/Max complex | ||
650 | 4 | |a Journal Article | |
650 | 4 | |a Research Support, Non-U.S. Gov't | |
650 | 7 | |a Basic Helix-Loop-Helix Leucine Zipper Transcription Factors |2 NLM | |
650 | 7 | |a Basic-Leucine Zipper Transcription Factors |2 NLM | |
650 | 7 | |a DNA-Binding Proteins |2 NLM | |
650 | 7 | |a MAX protein, human |2 NLM | |
650 | 7 | |a MXD1 protein, human |2 NLM | |
650 | 7 | |a Myc associated factor X |2 NLM | |
650 | 7 | |a Proto-Oncogene Proteins c-myc |2 NLM | |
650 | 7 | |a Repressor Proteins |2 NLM | |
650 | 7 | |a Transcription Factors |2 NLM | |
650 | 7 | |a DNA |2 NLM | |
650 | 7 | |a 9007-49-2 |2 NLM | |
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700 | 1 | |a Austen, M |e verfasserin |4 aut | |
700 | 1 | |a Lüscher, B |e verfasserin |4 aut | |
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