TY - JOUR
T1 - Quantitative analysis of transcription factor binding and expression using calling cards reporter arrays
AU - Liu, Jiayue
AU - Shively, Christian A.
AU - Mitra, Robi D.
N1 - Publisher Copyright:
© 2020 The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2020/5/21
Y1 - 2020/5/21
N2 - We report a tool, Calling Cards Reporter Arrays (CCRA), that measures transcription factor (TF) binding and the consequences on gene expression for hundreds of synthetic promoters in yeast. Using Cbf1p and MAX, we demonstrate that the CCRA method is able to detect small changes in binding free energy with a sensitivity comparable to in vitro methods, enabling the measurement of energy landscapes in vivo. We then demonstrate the quantitative analysis of cooperative interactions by measuring Cbf1p binding at synthetic promoters with multiple sites. We find that the cooperativity between Cbf1p dimers varies sinusoidally with a period of 10.65 bp and energetic cost of 1.37 KBT for sites that are positioned 'out of phase'. Finally, we characterize the binding and expression of a group of TFs, Tye7p, Gcr1p and Gcr2p, that act together as a 'TF collective', an important but poorly characterized model of TF cooperativity. We demonstrate that Tye7p often binds promoters without its recognition site because it is recruited by other collective members, whereas these other members require their recognition sites, suggesting a hierarchy where these factors recruit Tye7p but not vice versa. Our experiments establish CCRA as a useful tool for quantitative investigations into TF binding and function.
AB - We report a tool, Calling Cards Reporter Arrays (CCRA), that measures transcription factor (TF) binding and the consequences on gene expression for hundreds of synthetic promoters in yeast. Using Cbf1p and MAX, we demonstrate that the CCRA method is able to detect small changes in binding free energy with a sensitivity comparable to in vitro methods, enabling the measurement of energy landscapes in vivo. We then demonstrate the quantitative analysis of cooperative interactions by measuring Cbf1p binding at synthetic promoters with multiple sites. We find that the cooperativity between Cbf1p dimers varies sinusoidally with a period of 10.65 bp and energetic cost of 1.37 KBT for sites that are positioned 'out of phase'. Finally, we characterize the binding and expression of a group of TFs, Tye7p, Gcr1p and Gcr2p, that act together as a 'TF collective', an important but poorly characterized model of TF cooperativity. We demonstrate that Tye7p often binds promoters without its recognition site because it is recruited by other collective members, whereas these other members require their recognition sites, suggesting a hierarchy where these factors recruit Tye7p but not vice versa. Our experiments establish CCRA as a useful tool for quantitative investigations into TF binding and function.
UR - http://www.scopus.com/inward/record.url?scp=85084920937&partnerID=8YFLogxK
U2 - 10.1093/nar/gkaa141
DO - 10.1093/nar/gkaa141
M3 - Article
C2 - 32133534
AN - SCOPUS:85084920937
SN - 0305-1048
VL - 48
JO - Nucleic acids research
JF - Nucleic acids research
IS - 9
M1 - e50
ER -