Examining cooperative binding of Sox2 on DC5 regulatory element upon complex formation with Pax6 through excess electron transfer assay.

Functional cooperativity among transcription factors on regulatory genetic elements is pivotal for milestone decision-making in various cellular processes including mammalian development. However, their molecular interaction during the cooperative binding cannot be precisely understood due to lack of efficient tools for the analyses of protein-DNA interaction in the transcription complex. Here, we demonstrate that photoinduced excess electron transfer assay can be used for analysing cooperativity of proteins in transcription complex using cooperative binding of Pax6 to Sox2 on the regulatory DNA element (DC5 enhancer) as an example. In this assay, (Br)U-labelled DC5 was introduced for the efficient detection of transferred electrons from Sox2 and Pax6 to the DNA, and guanine base in the complementary strand was replaced with hypoxanthine (I) to block intra-strand electron transfer at the Sox2-binding site. By examining DNA cleavage occurred as a result of the electron transfer process, from tryptophan residues of Sox2 and Pax6 to DNA after irradiation at 280 nm, we not only confirmed their binding to DNA but also observed their increased occupancy on DC5 with respect to that of Sox2 and Pax6 alone as a result of their cooperative interaction.

Assessment of PAX6 alleles in 66 families with aniridia.

We report on PAX6 alleles associated with a clinical diagnosis of classical aniridia in 81 affected individuals representing 66 families. Allelic variants expected to affect PAX6 function were identified in 61 families (76 individuals). Ten cases of sporadic aniridia (10 families) had complete (8 cases) or partial (2 cases) deletion of the PAX6 gene. Sequence changes that introduced a premature termination codon into the open reading frame of PAX6 occurred in 47 families (62 individuals). Three individuals with sporadic aniridia (three families) had sequence changes (one deletion, two run-on mutations) expected to result in a C-terminal extension. An intronic deletion of unknown functional significance was detected in one case of sporadic aniridia (one family), but not in unaffected relatives. Within these 61 families, single nucleotide substitutions accounted for 30/61 (49%), indels for 23/61 (38%), and complete deletion of the PAX6 locus for 8/61 (13%). In five cases of sporadic aniridia (five families), no disease-causing mutation in the coding region was detected. In total, 23 unique variants were identified that have not been reported in the Leiden Open Variation Database (LOVD) database. Within the group assessed, 92% had sequence changes expected to reduce PAX6 function, confirming the primacy of PAX6 haploinsufficiency as causal for aniridia.

Impaired DNA-binding affinity of novel PAX6 mutations.

Mutations in human PAX6 gene are associated with various congenital eye malformations including aniridia, foveal hypoplasia, and congenital nystagmus. These various phenotypes may depend on the mutation spectrums that can affect DNA-binding affinity, although this hypothesis is debatable. We screened PAX6 mutations in two unrelated patients with congenital nystagmus, and measured DNA-binding affinity through isothermal titration calorimetry (ITC). To elucidate phenotypic differences according to DNA-binding affinity, we also compared DNA-binding affinity among the previously reported PAX6 missense mutations within the linker region between two subdomains of the paired domain (PD). We identified two novel mutations of PAX6 gene: c.214 G > T (p.Gly72Cys) and c.249_250delinsCGC (p.Val84Alafs*8). Both were located within the linker region between the two subdomains of the PD. ITC measurement revealed that the mutation p.Val84Alafs*8 had no DNA-binding affinity, while the p.Gly72Cys mutation showed a decreased binding affinity (Kd = 0.58 µM) by approximately 1.4 times compared to the wild type-PAX6 (Kd = 0.41 µM). We also found that there was no close relationship between DNA-binding affinity and phenotypic differences. Our results suggest that the DNA-binding affinity alone might be insufficient to determine PAX6-related phenotypes, and that other modifier genes or environmental factors might affect phenotypes of the PAX6 gene.

Lampreys, the jawless vertebrates, contain three Pax6 genes with distinct expression in eye, brain and pancreas.

The transcription factor Pax6 is crucial for the development of the central nervous system, eye, olfactory system and pancreas, and is implicated in human disease. While a single Pax6 gene exists in human and chicken, Pax6 occurs as a gene family in other vertebrates, with two members in elephant shark, Xenopus tropicalis and Anolis lizard and three members in teleost fish such as stickleback and medaka. However, the complement of Pax6 genes in jawless vertebrates (cyclostomes), the sister group of jawed vertebrates (gnathostomes), is unknown. Using a combination of BAC sequencing and genome analysis, we discovered three Pax6 genes in lampreys. Unlike the paired-less Pax6 present in some gnathostomes, all three lamprey Pax6 have a highly conserved full-length paired domain. All three Pax6 genes are expressed in the eye and brain, with variable expression in other tissues. Notably, lamprey Pax6α transcripts are found in the pancreas, a vertebrate-specific organ, indicating the involvement of Pax6 in development of the pancreas in the vertebrate ancestor. Multi-species sequence comparisons revealed only a single conserved non-coding element, in the lamprey Pax6ß locus, with similarity to the PAX6 neuroretina enhancer. Using a transgenic zebrafish enhancer assay we demonstrate functional conservation of this element over 500 million years of vertebrate evolution.

High-Efficiency CRISPR/Cas9-Mediated Gene Editing in Honeybee (Apis mellifera) Embryos.

The honeybee (Apis mellifera) is an important insect pollinator of wild flowers and crops, playing critical roles in the global ecosystem. Additionally, the honeybee serves as an ideal social insect model. Therefore, functional studies on honeybee genes are of great interest. However, until now, effective gene manipulation methods have not been available in honeybees. Here, we reported an improved CRISPR/Cas9 gene-editing method by microinjecting sgRNA and Cas9 protein into the region of zygote formation within 2 hr after queen oviposition, which allows one-step generation of biallelic knockout mutants in honeybee with high efficiency. We first targeted the Mrjp1 gene. Two batches of honeybee embryos were collected and injected with Mrjp1 sgRNA and Cas9 protein at the ventral cephalic side and the dorsal posterior side of the embryos, respectively. The gene-editing rate at the ventral cephalic side was 93.3%, which was much higher than that (11.8%) of the dorsal-posterior-side injection. To validate the high efficiency of our honeybee gene-editing system, we targeted another gene, Pax6, and injected Pax6 sgRNA and Cas9 protein at the ventral cephalic side in the third batch. A 100% editing rate was obtained. Sanger sequencing of the TA clones showed that 73.3% (for Mrjp1) and 76.9% (for Pax6) of the edited current-generation embryos were biallelic knockout mutants. These results suggest that the CRISPR/Cas9 method we established permits one-step biallelic knockout of target genes in honeybee embryos, thereby demonstrating an efficient application to functional studies of honeybee genes. It also provides a useful reference to gene editing in other insects with elongated eggs.

In silico analysis of Pax6 protein glycosylation in vertebrates.

Pax6 is a transcription factor that involves in the formation of the eye, brain, and central nervous system in vertebrates. Due to various roles in the eye morphogenesis, Pax6 interacts with DNA and various transcription factors via post-translational modifications. Post-translational modifications of Pax6 have been studied extensively but there is a paucity of information about the glycosylation. Here, we focused on predicting the glycosylation positions of Pax6 protein in vertebrates. Also, 3D protein and glycoprotein models were generated using I-TASSER and GLYCAM servers in order to understand the effect of glycosylation on the Pax6 protein structure. We predicted N-glycosylation, mucin-type O-glycosylation, O-α-GlcNAcylation, and O-ß-GlcNAcylation positions on Pax6 protein besides O-GlcNAc modification. Moreover, we found ying-yang positions suggesting the presence of O-GlcNAcylation/phosphorylation competition in Pax6. As to 3D glycoprotein models of Pax6, Ser24, Ser65, and Ser74 residues at the PD domain of Pax6 protein was evaluated as a strong candidate for the DNA binding site. We suggest that determination of the glycosylation positions on 3D glycoprotein model will facilitate the understanding of glycosylation role on Pax6 protein interactions in transcription and intracellular activities.

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

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.