Combinatorial control mechanisms in gene regulation by Sox and POU transcription factors

• Main Author: Ng, Calista Keow Leng
• Other Authors: School of Biological Sciences
• Format: Theses and Dissertations
• Language: English
• Published: 2014
• Subjects: DRNTU::Science::Biological sciences::Biochemistry
• Online Access: http://hdl.handle.net/10356/55777
• Institution: Nanyang Technological University

The Sox and POU transcription factor families have been known to physically and functionally cooperate to regulate gene expression in a tissue/developmental stage-specific manner through differential cooperation between its member pairs. Since cooperation between transcription factors can affect biological outcomes, the ability to quantify biophysical cooperativity could give insights into how Sox-POU partnerships are established on tissue/developmental stage specific cis-regulatory modules. Here, I developed a method to comprehensively assess the biophysical cooperativity of several Sox-POU pairings. As a result, I generated a cooperativity dataset for the heterodimer complex formation of eleven Sox proteins paired with a POU member, Oct4, on a range of Sox-Oct DNA elements. In the light of existing structural models of Sox-POU transcription factors, the cooperativity data was used to rationally engineer functional outcomes of Sox17, which originally is an inducer of endodermal development, can now be turned into a highly efficient induced pluripotency factor while Sox2, which originally is a pluripotency factor, can now be turned into an inducer of endodermal development. Whilst the cooperativity dataset had highlighted the importance of critical amino acids of Sox proteins at the Sox-POU interface which determines how well a particular Sox protein is selected to pair with a POU member, I could not find such amino acids in the POU family to explain how individual POU factors remained functionally unique when most of them appeared equally capable of cooperating with the Sox2 protein. To follow up, I studied on another DNA motif, MORE, which POU also recognizes apart from the Sox-Oct motif. While POU members recognize the MORE motif, it turns out that they bind MORE as a POU-POU homodimer rather than as a Sox-POU heterodimer. By comparing how well each POU member binds on both types of motifs, Oct4 was found to be the least effective POU in homodimerizing on the MORE motif yet the most efficient POU that heterodimerizes with Sox2 on Sox-Oct motif. By studying our published crystal structure model of Oct6 homodimer on a MORE motif DNA, I introduced rational mutations to generate Oct4MORE and Oct6MORE mutants. While the exchange of a single amino acid between Oct4 and Oct6 proteins maintained heterodimerization efficiency on Sox-Oct motif, I observed a swap of homodimerization efficiency on the MORE motifs. Oct6MORE no longer homodimerize well while Oct4MORE homodimerizes very efficiently. Even though themutants are not affected in their efficiency to heterodimerize on Sox-Oct motif, when individual POUs are allowed to “pick” the more preferred DNA motifs out of the two, Oct4MORE, which originally selects strongly for Sox-Oct motif, now binds very weakly. On the other hand, Oct6MORE, which originally selects strongly for the MORE motif, now “picks” for the Sox-Oct motif. Knowing their original functions of inducing cells either towards pluripotency or neuronal pathways in Oct4 and Oct6, respectively, I then test for the change of pluripotency ability in the re-engineered POU factors by using pluripotent induction assay. Results indicate that the unique biological functions of POU proteins may be highly dependent on the type of DNA motifs that each POU member eventually selects for binding. This hypothesis is particularly evident from the cell based assays of Oct4MORE mutant where any disturbance to the shifting of the balance of motif selection affects the pluripotency inducing ability of Oct4. This way, we have successfully generated a pluripotency incompetent Oct4 that may potentially gain an Oct6-like neuroinducing capability.