DNA-mediated cooperativity facilitates the co-selection of cryptic enhancer sequences by SOX2 and PAX6 transcription factors

© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. Sox2 and Pax6 are transcription factors that direct cell fate decision during neurogenesis, yet the mechanism behind how they cooperate on enhancer DNA elements and regulate gene expression is unclear. By systematically interrogating Sox2 and Pax6 interaction on minimal enhancer elements, we found that cooperative DNA recognition relies on combinatorial nucleotide switches and precisely spaced, but cryptic composite DNA motifs. Surprisingly, all tested Sox and Pax paralogs have the capacity to cooperate on such enhancer elements. NMR and molecular modeling reveal very few direct protein-protein interactions between Sox2 and Pax6, suggesting that cooperative binding is mediated by allosteric interactions propagating through DNA structure. Furthermore, we detected and validated several novel sites in the human genome targeted cooperatively by Sox2 and Pax6. Collectively, we demonstrate that Sox- Pax partnerships have the potential to substantially alter DNA target specificities and likely enable the pleiotropic and context-specific action of these cell-lineage specifiers.Link_to_subscribed_fulltex

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

Directed Evolution of Reprogramming Factors by Cell Selection and Sequencing

Summary: Directed biomolecular evolution is widely used to tailor and enhance enzymes, fluorescent proteins, and antibodies but has hitherto not been applied in the reprogramming of mammalian cells. Here, we describe a method termed directed evolution of reprogramming factors by cell selection and sequencing (DERBY-seq) to identify artificially enhanced and evolved reprogramming transcription factors. DERBY-seq entails pooled screens with libraries of positionally randomised genes, cell selection based on phenotypic readouts, and genotyping by amplicon sequencing for candidate identification. We benchmark this approach using pluripotency reprogramming with libraries based on the reprogramming factor SOX2 and the reprogramming incompetent endodermal factor SOX17. We identified several SOX2 variants outperforming the wild-type protein in three- and four-factor cocktails. The most effective variants were discovered from the SOX17 library, demonstrating that this factor can be converted into a highly potent inducer of pluripotency with a range of diverse modifications. We propose DERBY-seq as a broad-based approach to discover reprogramming factors for any donor/target cell combination applicable to direct lineage reprogramming in vitro and in vivo. : Transcription factor-driven cell-fate conversions are powerful methods to turn one cell type into another but are typically slow and inefficient. In this article Jauch and Veerapandian et al. showed that naturally occurring factors are not optimally adapted for this feat but can be profoundly improved by artificially evolving the way they read and translate regulatory information stored in the genome. Keywords: directed evolution, protein engineering, cellular reprogramming, synthetic biology, deep mutational scanning, synthetic transcription factors, SOX2, SOX17, OCT

Coop-Seq Analysis Demonstrates that Sox2 Evokes Latent Specificities in the DNA Recognition by Pax6

© 2017 Elsevier Ltd Sox2 and Pax6 co-regulate genes in neural lineages and the lens by forming a ternary complex likely facilitated allosterically through DNA. We used the quantitative and scalable cooperativity-by-sequencing (Coop-seq) approach to interrogate Sox2/Pax6 dimerization on a DNA library where five positions of the Pax6 half-site were randomized yielding 1024 cooperativity factors. Consensus positions normally required for the high-affinity DNA binding by Pax6 need to be mutated for effective dimerization with Sox2. Out of the five randomized bases, a 5′ thymidine is present in most of the top ranking elements. However, this thymidine maps to a region outside of the Pax half site and is not expected to directly interact with Pax6 in known binding modes suggesting structural reconfigurations. Re-analysis of ChIP-seq data identified several genomic regions where the cooperativity promoting sequence pattern is co-bound by Sox2 and Pax6. A highly conserved Sox2/Pax6 bound site near the Sprouty2 locus was verified to promote cooperative dimerization designating Sprouty2 as a potential target reliant on Sox2/Pax6 cooperativity in several neural cell types. Collectively, the functional interplay of Sox2 and Pax6 demands the relaxation of high-affinity binding sites and is enabled by alternative DNA sequences. We conclude that this binding mode evolved to warrant that a subset of target genes is only regulated in the presence of suitable partner factors.Link_to_subscribed_fulltex