Diagenode

An investigation of the affinities, specificity and kinetics involved in the interaction between the Yin Yang 1 transcription factor and DNA.


Golebiowski FM, Górecki A, Bonarek P, Rapala-Kozik M, Kozik A, Dziedzicka-Wasylewska M

Human transcription factor Yin Yang 1 (YY1) is a four-zinc-finger protein that regulates a large number of genes with different biological functions in processes such as development, carcinogenesis and B-cell maturation. The natural binding sites of YY1 are relatively unconserved and have a short core sequence CCAT. We were interested in determining how YY1 recognises its binding sites and achieves the necessary sequence selectivity in the cell. Using fluorescence anisotropy, we determined the equilibrium dissociation constants for the selected, naturally occurring YY1 binding sites that have different levels of similarity to the consensus sequence. We found that recombinant YY1 interacts with its specific binding sites with relatively low affinities from a high nanomolar to a low micromolar range. By using a fluorescence anisotropy competition assay, we determined the affinity of YY1 for nonspecific DNA to be between 30 and 40 μM, which results in low specificity ratios of between 3 and 220. Additionally, surface plasmon resonance measurements showed rapid association and dissociation rates, which suggested that the binding strength is regulated through changes in both k(a) and k(d) . In conclusion, we propose that in the cell, YY1 may achieve a higher specificity by associating with co-regulators or as a part of multi-subunit complexes.

Tags
DNA shearing
Bioruptor

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Published
July, 2012

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