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Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess
Abhijit Saha1, Seiichiro Kizaki1, Debojyoti De2
1Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-Ku, Kyoto 606-8502, Japan.
Abstract:
Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including mammalian development. However, their molecular interaction during the cooperative binding cannot be precisely understood due to lack of efficient tools for the analyses of protein-DNA interaction in the transcription complex. Here, we demonstrate that photoinduced excess electron transfer assay can be used for analysing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. In this assay, (Br)U-labelled DC5 was introduced for the efficient detection of transferred electrons from Sox2 and Pax6 to the DNA, and guanine base in the complementary strand was replaced with hypoxanthine (I) to block intra-strand electron transfer at the Sox2-binding site. By examining DNA cleavage occurred as a result of the electron transfer process, from tryptophan residues of Sox2 and Pax6 to DNA after irradiation at 280 nm, we not only confirmed their binding to DNA but also observed their increased occupancy on DC5 with respect to that of Sox2 and Pax6 alone as a result of their cooperative interaction.
Insights
Researchers developed a novel photoinduced electron transfer assay to study how transcription factors cooperatively bind to DNA. This method precisely analyzes protein-DNA interactions, revealing enhanced binding when Pax6 and Sox2 work together.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Transcription factor cooperativity is crucial for cellular processes like mammalian development.
- Understanding molecular interactions in transcription complexes is challenging due to limited analytical tools.
- Precise analysis of protein-DNA interactions is needed to understand cooperative binding.
Purpose of the Study:
- To demonstrate a new photoinduced excess electron transfer assay for analyzing protein cooperativity in transcription complexes.
- To investigate the cooperative binding of transcription factors Pax6 and Sox2 on the DC5 enhancer DNA element.
Main Methods:
- Utilized a photoinduced excess electron transfer assay.
- Employed (Br)U-labeled DC5 DNA and hypoxanthine (I) substitution to block intra-strand electron transfer.
- Analyzed DNA cleavage resulting from electron transfer from tryptophan residues of Sox2 and Pax6 to DNA upon 280 nm irradiation.
Main Results:
- Confirmed the binding of Pax6 and Sox2 to the DC5 enhancer DNA.
- Observed increased DNA occupancy on the DC5 enhancer when Pax6 and Sox2 bind cooperatively compared to individual binding.
- Demonstrated the utility of the assay for detecting cooperative protein-DNA interactions.
Conclusions:
- The photoinduced excess electron transfer assay is an effective tool for analyzing transcription factor cooperativity.
- Cooperative binding of Pax6 and Sox2 leads to enhanced occupancy on the regulatory DNA element.
- This assay provides a precise method for studying molecular interactions within transcription complexes.
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