Abstract
Oligomerization of transcription factors controls their translocation into the nucleus and DNA-binding activity. Here we present a fluorescence microscopy analysis termed pCOMB (pair correlation of molecular brightness) that tracks the mobility of different oligomeric species within live cell nuclear architecture. pCOMB amplifies the signal from the brightest species present and filters the dynamics of the extracted oligomeric population based on arrival time between two locations. We use this method to demonstrate a dependence of signal transducer and activator of transcription 3 (STAT3) mobility on oligomeric state. We find that on entering the nucleus STAT3 dimers must first bind DNA to form STAT3 tetramers, which are also DNA-bound but exhibit a different mobility signature. Examining the dimer-to-tetramer transition by a cross-pair correlation analysis (cpCOMB) reveals that chromatin accessibility modulates STAT3 tetramer formation. Thus, the pCOMB approach is suitable for mapping the impact oligomerization on transcription factor dynamics.
Highlights
Oligomerization of transcription factors controls their translocation into the nucleus and DNA-binding activity
Here we set out to establish and apply an imaging-based approach that could quantify the molecular mobility of the different signal transducer and activator of transcription 3 (STAT3) oligomeric species and map STAT3 dimer versus tetramer DNA binding in a live cell
Instead of performing a higher-order moment or correlation analysis to resolve the dynamics of a heterogeneous oligomeric population within an observation volume[30,31,32], we introduce a spatial component to the correlation function that selectively amplifies the brightest species and filters the different-sized oligomers within this population, based on their distinct arrival times between two locations
Summary
Oligomerization of transcription factors controls their translocation into the nucleus and DNA-binding activity. It is known that the nuclear dimer population can further interact to form STAT3 tetramers on adjacent gamma interferon activation (GAS) elements, which bend the DNA into a conformation that amplifies or represses STAT3 dimer-regulated gene expression[6,7,8] To decipher how these oligomerization events control transcription factor nuclear access, inform target search strategies and confer DNA-binding activity requires a method that can quantify the molecular mobility of each oligomeric state. Pair correlation analysis is a method that spatially correlates fluctuations in fluorescence intensity acquired along a confocal line scan, and we recently demonstrated that this method can be used to quantify the impact of chromatin organization on nuclear protein translocation[27,28,29] This method of analysis, when applied to intensity fluctuations, indicates only the number of molecules that perform a transit and does not discriminate transport based on oligomeric state. Instead of performing a higher-order moment or correlation analysis to resolve the dynamics of a heterogeneous oligomeric population within an observation volume[30,31,32], we introduce a spatial component to the correlation function that selectively amplifies the brightest species and filters the different-sized oligomers within this population, based on their distinct arrival times between two locations
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