Why, YY1? The effect of dynamic multimerisation of YY1 and FOXP2 in DNA binding regulation

Abstract

Yin Yang 1 (YY1) is a ubiquitously expressed transcription factor involved in neural development. Depending on the conditions of its own expression, YY1 is capable of either activating or repressing transcription. The oligomeric state of YY1 as well as its association with other regulatory proteins is likely to alter its DNA binding ability and transcriptional activity. Some evidence has suggested that one of YY1’s binding partners may be FOXP2, another transcription factor which is also involved in neural development. The nature of this interaction, however, has not been investigated. The aim of this project was to study the potential binding mechanisms of YY1 by exploring the structure and dynamics of YY1, as well as the interactions it makes with itself, FOXP2 forkhead domain (FHD) and DNA. To do this, the structural integrity of purified recombinant YY1 and FOXP2 FHD was first confirmed using circular dichroism spectroscopy (CD) (secondary structure) and intrinsic tryptophan fluorescence spectroscopy (tertiary structure). In order to determine how YY1 oligomerisation acts as a mechanism of transcriptional control, the quaternary structure of YY1 was observed using both fluorescence anisotropy and size exclusion chromatography (SEC). The specificity of DNA binding to both proteins was studied using electrophoretic mobility shift assays (EMSAs), and the YY1-DNA interaction was further investigated using fluorescence anisotropy. The dynamics of apo and DNA-bound YY1 was determined using hydrogen deuterium exchange mass spectrometry (HDXMS), so as to determine how the YY1 structural integrity changes upon DNA binding, as this will give insight into the binding mechanism. Lastly, the YY1-FHD interaction was confirmed and assessed using fluorescence anisotropy, while supershift EMSAs were used to observe the effect of this interaction on FHD and YY1’s respective interactions with DNA. In this work, YY1 was confirmed to be an intrinsically disordered protein, with large regions of disorder. YY1 was also seen to form heterogeneous mixtures of multimers, which were affected by ionic strength and by protein concentration. These oligomers could bind to DNA with decreased specificity compared to the monomer, but the oligomers could also be disrupted by saturating concentrations of DNA. The general DNA binding mechanism of YY1 involves an increase in disorder in the N-terminal region of the protein upon DNA binding. In addition to binding DNA, it was proved in this study that YY1 is capable of binding to the FOXP2 FHD. Furthermore, YY1 can simultaneously bind to both the FOXP2 FHD and DNA, although the FHD competes with YY1 for FHD specific DNA. Overall, these data indicate that YY1 is a highly dynamic protein with behaviour typical of intrinsic disorder. Its heterogeneous, dynamic multimerisation is likely important for its ability to interact with many other transcription factors, including FOXP2. The interactions that YY1 forms with itself, the FHD and DNA form part of an intricate mechanism of transcriptional regulation by YY1, which is likely dependent on its concentration, localisation and available binding partners. This association is likely to form part of how YY1 and also FOXP2 regulate neural development, where mis-regulated expression of either protein can have detrimental effects resulting in intellectual disorders.