Quantification of transcription factor-DNA binding affinity in a living cell

Area of Science:

• Molecular Biology
• Genetics
• Endocrinology

Background:

• Nuclear receptors like glucocorticoid receptor (GR) and androgen receptor (AR) regulate gene expression.
• Understanding their DNA binding dynamics in vivo is crucial for deciphering transcriptional control.

Purpose of the Study:

• To determine the in vivo DNA binding affinity of GR and AR in Xenopus oocytes.
• To investigate the role of FoxA1 in modulating GR and AR DNA binding.

Main Methods:

• Quantification of nuclear receptor concentration using radiolabeled hormone.
• In vivo DNA footprinting using dimethylsulphate to assess receptor-DNA interactions.
• Nuclear microinjection of DNA into oocytes to form chromatin.

Main Results:

• FoxA1 enhanced GR DNA binding with an apparent Kd of ~1 μM.
• FoxA1 dramatically stimulated AR DNA binding with an apparent Kd of ~0.13 μM.
• FoxA1 exhibited cooperative binding with AR, highlighting its licensing function.

Conclusions:

• FoxA1 significantly enhances androgen receptor DNA binding affinity.
• FoxA1 plays a critical licensing role in regulating hormone receptor binding to DNA.
• In vivo footprinting provides insights into nuclear receptor-DNA interactions.