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

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.​Doctor of Philosophy (SBS

The Structure of Sox17 Bound to DNA Reveals a Conserved Bending Topology but Selective Protein Interaction Platforms

Sox17 regulates endodermal lineage commitment and is thought to function antagonistically to the pluripotency determinant Sox2. To investigate the biochemical basis for the distinct functions of Sox2 and Sox17, we solved the crystal structure of the high mobility group domain of Sox17 bound to a DNA element derived from the Lama1 enhancer using crystals diffracting to 2.7 Å resolution. Sox17 targets the minor groove and bends the DNA by approximately 80°. The DNA architecture closely resembles the one seen for Sox2/DNA structures, suggesting that the degree of bending is conserved between both proteins and nucleotide substitutions have only marginal effects on the bending topology. Accordingly, affinities of Sox2 and Sox17 for the Lama1 element were found to be identical. However, when the Oct1 contact interface of Sox2 is compared with the corresponding region of Sox17, a significantly altered charge distribution is observed, suggesting differential co-factor recruitment that may explain their biological distinctiveness. © 2009 Elsevier Ltd. All rights reserved.Link_to_subscribed_fulltex

Crystal structure of the dimeric Oct6 (POU3f1) POU domain bound to palindromic MORE DNA

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Structural basis for the SOX-dependent genomic redistribution of OCT4 in stem cell differentiation

In pluripotent cells, OCT4 associates with SOX2 to maintain pluripotency or with SOX17 to induce primitive endoderm commitment. The OCT4-SOX2 and OCT4-SOX17 combinations bind mutually exclusive to two distinct composite DNA elements, known as the "canonical" and "compressed" motifs, respectively. The structural basis for the OCT4-SOX17 cooperativity is unknown. Whereas SOX17 has been engineered to replace SOX2 in the pluripotency circuitry, all generated SOX2 mutants have failed to act like SOX17. From molecular simulations, we revealed the OCT4-SOX17 interaction interface and elucidated the SOX-dependent motif preference of OCT4. Moreover, we designed a SOX2 mutant that we predicted and confirmed experimentally to bind cooperatively with OCT4 to the compressed motif. Ultimately, we found a strong correlation between the experimental and calculated relative cooperative-binding free energies of 12 OCT4-SOX-DNA complexes. Therefore, we validated the OCT4-SOX interfaces and demonstrated that in silico design of DNA-binding cooperativity is suitable for altering transcriptional circuitries. © 2014 Elsevier Ltd.Link_to_subscribed_fulltex

Crystal Structure and DNA Binding of the Homeodomain of the Stem Cell Transcription Factor Nanog

The transcription factor Nanog is an upstream regulator in early mammalian development and a key determinant of pluripotency in embryonic stem cells. Nanog binds to promoter elements of hundreds of target genes and regulates their expression by an as yet unknown mechanism. Here, we report the crystal structure of the murine Nanog homeodomain (HD) and analysis of its interaction with a DNA element derived from the Tcf3 promoter. Two Nanog amino acid pairs, unique among HD sequences, appear to affect the mechanism of nonspecific DNA recognition as well as maintain the integrity of the structural scaffold. To assess selective DNA recognition by Nanog, we performed electrophoretic mobility shift assays using a panel of modified DNA binding sites and found that Nanog HD preferentially binds the TAAT(G/T)(G/T) motif. A series of rational mutagenesis experiments probing the role of six variant residues of Nanog on its DNA binding function establish their role in affecting binding affinity but not binding specificity. Together, the structural and functional evidence establish Nanog as a distant member of a Q50-type HD despite having considerable variation at the sequence level. © 2007 Elsevier Ltd. All rights reserved.Link_to_subscribed_fulltex

Purification, crystallization and preliminary X-ray diffraction analysis of the HMG domain of Sox17 in complex with DNA

Crystals of the Sox17 HMG domain bound to LAMA1 enhancer DNA-element crystals that diffracted to 2.75 Å resolution were obtained

Co-motif discovery identifies an esrrb-Sox2-DNA ternary complex as a mediator of transcriptional differences between mouse embryonic and epiblast stem cells

Transcription factors (TF) often bind in heterodimeric complexes with each TF recognizing a specific neighboring cis element in the regulatory region of the genome. Comprehension of this DNA motif grammar is opaque, yet recent developments have allowed the interrogation of genome- wide TF binding sites. We reasoned that within this data novel motif grammars could be identified that controlled distinct biological programs. For this purpose, we developed a novel motif-discovery tool termed fexcom that systematically interrogates ChIP-seq data to discover spatially constrained TF-TF composite motifs occurring over short DNA distances. We applied this to the extensive ChIP-seq data available from mouse embryonic stem cells (ESCs). In addition to the well-known and most prevalent sox-oct motif, we also discovered a novel constrained spacer motif for Esrrb and Sox2 with a gap of between 2 and 8 bps that Essrb and Sox2 cobind in a selective fashion. Through the use of knockdown experiments, we argue that the Esrrb-Sox2 complex is an arbiter of gene expression differences between ESCs and epiblast stem cells (EpiSC). A number of genes downregulated upon dual Esrrb/Sox2 knockdown (e.g., Klf4, Klf5, Jam2, Pecam1) are similarly downregulated in the ESC to EpiSC transition and contain the esrrb-sox motif. The prototypical Esrrb-Sox2 target gene, containing an esrrbsox element conserved throughout eutherian and metatherian mammals, is Nr0b1. Through positive regulation of this transcriptional repressor, we argue the Esrrb- Sox2 complex promotes the ESC state through inhibition of the EpiSC transcriptional program and the same trio may also function to maintain trophoblast stem cells. © 2012 AlphaMed Press.Link_to_subscribed_fulltex