Transcription Factors That Govern Development and Disease: An Achilles Heel in Cancer.

Development requires the careful orchestration of several biological events in order to create any structure and, eventually, to build an entire organism. On the other hand, the fate transformation of terminally differentiated cells is a consequence of erroneous development, and ultimately leads to cancer. In this review, we elaborate how development and cancer share several biological processes, including molecular controls. Transcription factors (TF) are at the helm of both these processes, among many others, and are evolutionarily conserved, ranging from yeast to humans. Here, we discuss four families of TFs that play a pivotal role and have been studied extensively in both embryonic development and cancer-high mobility group box (HMG), GATA, paired box (PAX) and basic helix-loop-helix (bHLH) in the context of their role in development, cancer, and their conservation across several species. Finally, we review TFs as possible therapeutic targets for cancer and reflect on the importance of natural resistance against cancer in certain organisms, yielding knowledge regarding TF function and cancer biology.

Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation.

A previously identified enhancer 10 kb upstream of the Aggrecan (Acan) gene (UE) can drive cartilage specific reporter expression in vivo. Here, we report that the paralogous transcription factors PAX1 and PAX9 differentially drive UE, depending on the presence or absence of SOX9-driven transactivation. In the developing vertebral column, PAX1/9 expression was inversely correlated with Acan expression. Moreover, PAX1/9 was co-expressed with SOX9/5/6 in the intervertebral mesenchyme and the inner annulus fibrosus (AF), and with SOX9 in the outer AF. Significant Acan upregulation was observed during chondrification of Pax1-silenced AF cells, while, Acan was significantly downregulated by persistent expression of Pax1 in cartilage. Deletion of UE using CRISPR/Cas9 resulted in ~30% and ~40% reduction of Acan expression in cartilage and the AF, respectively. In the UE, PAX1/9 acts as weak transactivators through a PAX1/9-binding site partially overlapped with a SOX9-binding site. In the presence of SOX9, which otherwise drives robust Acan expression along with SOX5/6, PAX1/9 competes with SOX9 for occupancy of the binding site, resulting in reduced transactivation of Acan. Coimmunoprecipitation revealed the physical interaction of Pax1 with SOX9. Thus, transactivation of the UE is differentially regulated by concerted action of PAX1/9, SOX9, and SOX5/6 in a context-dependent manner.

Nonsynonymous Variants in PAX4 and GLP1R Are Associated With Type 2 Diabetes in an East Asian Population.

We investigated ethnicity-specific exonic variants of type 2 diabetes (T2D) and its related clinical phenotypes in an East Asian population. We performed whole-exome sequencing in 917 T2D case and control subjects, and the findings were validated by exome array genotyping in 3,026 participants. In silico replication was conducted for seven nonsynonymous variants in an additional 13,122 participants. Single-variant and gene-based association tests for T2D were analyzed. A total of 728,838 variants were identified by whole-exome sequencing. Among nonsynonymous variants, PAX4 Arg192His increased risk of T2D and GLP1R Arg131Gln decreased risk of T2D in genome-wide significance (odds ratio [OR] 1.48, P = 4.47 × 10-16 and OR 0.84, P = 3.55 × 10-8, respectively). Another variant at PAX4 192 codon Arg192Ser was nominally associated with T2D (OR 1.62, P = 5.18 × 10-4). In T2D patients, PAX4 Arg192His was associated with earlier age at diagnosis, and GLP1R Arg131Gln was associated with decreased risk of cardiovascular disease. In control subjects without diabetes, the PAX4 Arg192His was associated with higher fasting glucose and GLP1R Arg131Gln was associated with lower fasting glucose and HbA1c level. Gene-based analysis revealed that SLC30A8 was most significantly associated with decreased risk of T2D (P = 1.0 × 10-4). In summary, we have identified nonsynonymous variants associated with risk of T2D and related phenotypes in Koreans.

Impact of G-quadruplex loop conformation in the PITX1 mRNA on protein and small molecule interaction.

Intramolecular G-quadruplexes (G4s) are G-rich nucleic acid structures that fold back on themselves via interrupting loops to create stacked planar G-tetrads, in which four guanine bases associate via Hoogsteen hydrogen bonding. The G4 structure is further stabilized by monovalent cations centered between the stacked tetrads. The G-tetrad face on the top and bottom planes of G4s are often the site of interaction with proteins and small molecules. To investigate the potential impact of interrupting loops on both G4 structure and interaction with proteins/small molecules, we characterized a specific G4 from the 3'-UTR of PITX1 mRNA that contains loops of 6 nucleotides using biophysical approaches. We then introduced mutations to specific loops to determine the impact on G4 structure and the ability to interact with both proteins and a G4-specific ligand. Our results suggest that mutation of a specific loop both affects the global G4 structure and impacts the ability to interact with a G4 binding protein and small molecule ligand.

Ancestral and novel roles of Pax family genes in mollusks.

BACKGROUND: Pax genes are transcription factors with significant roles in cell fate specification and tissue differentiation during animal ontogeny. Most information on their tempo-spatial mode of expression is available from well-studied model organisms where the Pax-subfamilies Pax2/5/8, Pax6, and Paxα/ß are mainly involved in the development of the central nervous system (CNS), the eyes, and other sensory organs. In certain taxa, Pax2/5/8 seems to be additionally involved in the development of excretion organs. Data on expression patterns in lophotrochozoans, and in particular in mollusks, are very scarce for all the above-mentioned Pax-subfamilies, which hampers reconstruction of their putative ancestral roles in bilaterian animals. Thus, we studied the developmental expression of Pax2/5/8, Pax6, and the lophotrochozoan-specific Paxß in the worm-shaped mollusk Wirenia argentea, a member of Aplacophora that together with Polyplacophora forms the Aculifera, the proposed sister taxon to all primarily single-shelled mollusks (Conchifera). RESULTS: All investigated Pax genes are expressed in the developing cerebral ganglia and in the ventral nerve cords, but not in the lateral nerve cords of the tetraneural nervous system. Additionally, Pax2/5/8 is expressed in epidermal spicule-secreting or associated cells of the larval trunk and in the region of the developing protonephridia. We found no indication for an involvement of the investigated Pax genes in the development of larval or adult sensory organs of Wirenia argentea. CONCLUSIONS: Pax2/5/8 seems to have a conserved role in the development of the CNS, whereas expression in the spicule-secreting tissues of aplacophorans and polyplacophorans suggests co-option in aculiferan skeletogenesis. The Pax6 expression pattern in Aculifera largely resembles the common bilaterian expression during CNS development. All data available on Paxß expression argue for a common role in lophotrochozoan neurogenesis.

De Novo Assembly and Characterization of Early Embryonic Transcriptome of the Horseshoe Crab Tachypleus tridentatus.

The horseshoe crab Tachypleus tridentatus is a unique marine species and a potential model for marine invertebrate. Limited genomic and transcriptional data are currently available to understand the molecular mechanisms underlying the embryonic development of T. tridentatus. Here, we reported for the first time the de novo transcriptome assembly for T. tridentatus at embryonic developmental stage using Illumina RNA-seq platform. Approximate 38 million reads were obtained and further assembled into 133,212 unigenes. Sequence homology analysis against public databases revealed that 33,796 unigenes could be annotated with gene descriptions. Of the annotated unigenes, we identified a number of key components of several conserved metazoan signaling pathways (Hedgehog, Wnt, TGF-beta and Notch pathways) and other important regulatory genes involved in embryonic development. Targeted searching of Pax family genes which play critical roles in the formation of tissue and organ during embryonic development identified a complete set of Pax family genes. Moreover, the full length T. tridentatus Pax1/9a (TtPax1/9a) and Pax1/9b (TtPax1/9b) cDNA sequences were determined based on the transcriptome, demonstrating the immediate application of our database. Using quantitative real time PCR, we analyzed the expression patterns of TtPax1/9a and TtPax1/9b in different tissues of horseshoe crab. Taking advantage of Drosophila model, we further found that TtPax1/9b, but not TtPax1/9a, can partly rescue the Drosophila homolog Poxm dysfunction-caused lethality at the larval stage. Our study provides the embryonic transcriptome of T. tridentatus which could be immediately used for gene discovery and characterization, functional genomics studies in T. tridentatus. This transcriptome database will also facilitate the investigations of molecular mechanisms underlying embryonic development of T. tridentatus and other marine arthropods as well.

A rare PAX6 mutation in a Chinese family with congenital aniridia.

Aniridia is an autosomal dominant disorder characterized by the complete or partial loss of the iris and is almost associated with mutations in the paired box gene 6 (PAX6). We examined three generations of a Chinese family with congenital aniridia and observed genetic defects. Exons of PAX6 from 12 family members were amplified by polymerase chain reaction, sequenced, and compared with reference sequences in NCBI reference sequence database (http://www.ncbi.nlm.nih.gov/nuccore/NG_008679.1?from=5001&to=38170&report=genbank). A rare mutation c.2T>A (M1K) in exon 4 of PAX6 was identified in all affected family members but not in unaffected family members. Our results suggest that the c.2T>A (M1K) mutation may be responsible for the pathogenesis of congenital aniridia in this family. To our knowledge, this is the first report of the M1K mutation in PAX6 in a Chinese family with this disease and the second report worldwide.

miR-19b regulates hTERT mRNA expression through targeting PITX1 mRNA in melanoma cells.

Human telomerase reverse transcriptase (hTERT) plays a crucial role in cancer development. We previously identified paired-like homeodomain1 (PITX1) as an hTERT suppressor gene. However, the underlying mechanisms that are involved in the regulation of PITX1 remain unknown. Here, we report that the microRNA-19b (miR-19b) regulates hTERT expression and cell proliferation through inhibition of PITX1. Compared with normal melanocyte cells, miR-19b expression was higher in most melanoma cells and was accompanied by downregulation of PITX1. Moreover, overexpression of miR-19b inhibited PITX1 mRNA translation through a miR-19b binding site within the 3'UTR of the PITX1 mRNA. Our combined findings indicate the participation of miR-19b as a novel upstream effector of hTERT transcription via direct targeting of PITX1.

Wnt1 positively regulates CD36 expression via TCF4 and PPAR-γ in macrophages.

BACKGROUND: Scavenger receptors including CD36 control the phagocytosis of oxidized low-density lipoprotein and play an important role in macrophage physiology, but the underlying molecular mechanism by which CD36 is regulated in macrophages or during macrophage differentiation from monocytes remains to be determined. METHODS: Here, we investigated the relationship between Wnt1 and CD36 during macrophage differentiation. CD36 was suppressed following knockdown of Wnt1 by siRNA, while it was increased by ectopic overexpression of Wnt1 in macrophages. Using a ß-catenin inhibitor, peroxisome proliferator-activated receptor gamma (PPAR-γ) siRNA, and transcription factor 4 (TCF4) siRNA, we demonstrated that Wnt1 regulates the expression of CD36 through TCF4 and PPAR-γ. Co-immunoprecipitation, chromatin immunoprecipitation, and immunofluorescence experiments showed that ß-catenin interacted with PPAR-γ and that PPAR-γ and TCF4 colocalized in the nucleus. Furthermore, Pax3 regulated Wnt1 via binding to the first binding site in the Wnt1 promoter. RESULTS: Our study demonstrated that during macrophage differentiation from monocytes, Wnt1 promotes CD36 expression via activation of PPAR-γ and TCF4. CONCLUSIONS: Our findings suggest that Wnt1 plays an important role in macrophage physiology via activation of the canonical Wnt pathway.

A novel splice site mutation in the PAX6 gene in a Korean family with isolated aniridia.

Aniridia is a rare congenital ocular disorder of complete or partial iris hypoplasia. Frequently associated ocular changes include corneal abnormalities, cataract, glaucoma, and foveal hypoplasia. In most cases, aniridia is caused by decreased dosage of the paired box 6 (PAX6) gene, which is located in chromosome 11p13. We report the case of a Korean family with isolated aniridia inherited in an autosomal dominant manner. The proband was a one-month-old boy. He presented with bilateral complete aniridia and congenital glaucoma. His four-year-old sister had bilateral complete aniridia, glaucoma, and a corneal ulcer. His father had bilateral microcornea and cataract without aniridia. Using PAX6 sequencing analysis, we identified a deletion at the splice donor site of intron 8 in the proband (c.357+1delG). To our knowledge, this variant has not been previously described.

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

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.

Search by proteins for their DNA target site: 2. The effect of DNA conformation on the dynamics of multidomain proteins.

Multidomain transcription factors, which are especially abundant in eukaryotic genomes, are advantageous to accelerate the search kinetics for target site because they can follow the intersegment transfer via the monkey-bar mechanism in which the protein forms a bridged intermediate between two distant DNA regions. Monkey-bar dynamics highly depends on the properties of the multidomain protein (the affinity of each of the constituent domains to the DNA and the length of the linker) and the DNA molecules (their inter-distance and inter-angle). In this study, we investigate using coarse-grained molecular dynamics simulations how the local conformation of the DNA may affect the DNA search performed by a multidomain protein Pax6 in comparison to that of the isolated domains. Our results suggest that in addition to the common rotation-coupled translation along the DNA major groove, for curved DNA the tethered domains may slide in a rotation-decoupled sliding mode. Furthermore, the multidomain proteins move by longer jumps on curved DNA compared with those performed by the single domain protein. The long jumps originate from the DNA curvature bringing two sequentially distant DNA sites into close proximity with each other and they suggest that multidomain proteins may move on highly curved DNA faster than linear DNA.

Crystallization and preliminary X-ray diffraction analysis of the Pax9 paired domain bound to a DC5 enhancer DNA element.

Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based on in vitro optimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6-DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Šresolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 M NaNO3, 0.03 M Na2HPO4, 0.03 M NH2SO4, 0.1 M MES/imidazole pH 6.5. The data set was indexed and merged in space group C2221, with unit-cell parameters a = 75.74, b = 165.59, c = 70.14 Å, α = ß = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit.

PAX6, a novel target of miR-335, inhibits cell proliferation and invasion in glioma cells.

Paired box 6 (PAX6), a highly conserved transcription factor, is important in glioma. However, the molecular mechanisms involved remain unclear. The present study demonstrated that the expression of PAX6 was significantly reduced with the malignancy of glioma and also identified PAX6 as a novel target of microRNA (miR)­335, which was significantly upregulated in glioma. The inhibition of miR­335 increased the protein expression of PAX6, whereas the upregulation of miR­335 suppressed its expression in human glioma U251 and U87 cells. Furthermore, upregulation of miR-335 promoted U251 cell proliferation, colony formation and invasion, which was reversed by the overexpression of PAX6. Furthermore, the present study demonstrated that the effect of miR­335 on U251 cell invasion was via the modulation of matrix metalloproteinase (MMP)­2 and MMP­9 expression by targeting PAX6. In conclusion, the present study demonstrated that PAX6, as a novel target of miR­335, has an anti­oncogenic function in glioma, and thus PAX6 may serve as a therapeutic target for glioma.

Gene regulation by PAX6: structural-functional correlations of missense mutants and transcriptional control of Trpm3/miR-204.

PURPOSE: Pax6 is a key regulatory gene for eye, brain, and pancreas development. It acts as a transcriptional activator and repressor. Loss-of-function of Pax6 results in down- and upregulation of a comparable number of genes, although many are secondary targets. Recently, we found a prototype of a Pax6-binding site that acts as a transcriptional repressor. We also identified the Trpm3 gene as a Pax6-direct target containing the miR-204 gene located in intron 6. Thus, there are multiple Pax6-dependent mechanisms of transcriptional repression in the cell. More than 50 Pax6 missense mutations have been identified in humans and mice. Two of these mutations, N50K (Leca4) and R128C (Leca2), were analyzed in depth resulting in different numbers of regulated genes and different ratios of down- and upregulated targets. Thus, additional studies of these mutants are warranted to better understand the molecular mechanisms of the mutants' action. METHODS: Mutations in PAX6 and PAX6(5a), including G18W, R26G, N50K, G64V, R128C, and R242T, were generated with site-directed mutagenesis. A panel of ten luciferase reporters driven by six copies of Pax6-binding sites representing a spectrum of sites that act as repressors, moderate activators, and strong activators were used. Two additional reporters, including the Pax6-regulated enhancer from mouse Trpm3 and six copies of its individual Pax6-binding site, were also tested in P19 cells. RESULTS: PAX6 (N50K) acted either as a loss-of-function or neutral mutation. In contrast, PAX6 (R128C) and (R242T) acted as loss-, neutral, and gain-of-function mutations. With three distinct reporters, the PAX6 (N50K) mutation broke the pattern of effects produced by substitutions in the surrounding helices of the N-terminal region of the paired domain. All six mutations tested acted as loss-of-function using the Trpm3 Pax6-binding site. CONCLUSIONS: These studies highlight the complexity of Pax6-dependent transcriptional activation and repression mechanisms, and identify the N50K and R128C substitutions as valuable tools for testing interactions between Pax6, Pax6 (N50K), and Pax6 (R128C) with other regulatory proteins, including chromatin remodelers.

Single molecule visualization and characterization of Sox2-Pax6 complex formation on a regulatory DNA element using a DNA origami frame.

We report the use of atomic force microscopy (AFM) to study Sox2-Pax6 complex formation on the regulatory DNA element at a single molecule level. Using an origami DNA scaffold containing two DNA strands with different levels of tensile force, we confirmed that DNA bending is necessary for Sox2 binding. We also demonstrated that two transcription factors bind cooperatively by observing the increased occupancy of Sox2-Pax6 on the DNA element compared to that of Sox2 alone.

Cephalopod eye evolution was modulated by the acquisition of Pax-6 splicing variants.

Previous studies have reported that the developmental processes of vertebrate eyes are controlled by four Pax-6 splicing variants, each modulating different downstream genes, whereas those of insect eyes are controlled by duplicated Pax-6 genes. Cephalopods belong to the Protostomes but possess a camera-type eye similar to those in vertebrates. We examined Pax-6 variations in the squid and found five types of Pax-6 splicing variants but no duplication of the Pax-6 gene. In the five splicing variants, the splicing patterns were produced by the combination of two additional exons to the ortholog and one jettisoned exon containing most of the Homeobox domain (HD). These five variants show spatio-temporal patterns of gene expression during development in the squid. Our study suggests that cephalopods acquired Pax-6 splicing variants independent of those in vertebrates and that these variants were similarly utilized in the development of the squid eye.

Nano-analysis of DNA conformation changes induced by transcription factor complex binding using plasmonic nanodimers.

The plasmon resonant wavelength for a pair of gold nanoparticles, or gold nanodimer, increases inversely with the gap distance between the two nanoparticles. Taking advantage of this property, we performed nanoscale measurements of DNA conformation changes induced by transcription factor binding. Gold nanoparticles were bridged by double-stranded DC5 DNA that included binding sequences for the transcription factors SOX2 and PAX6, which interact on the DC5 enhancer sequence and activate transcription. The gold nanodimers bound by SOX2 shifted the plasmon resonant wavelength from 586.8 to 604.1 nm, indicating that SOX2 binding induces DNA bending. When the SOX2 formed a ternary complex with PAX6 on DC5, the plasmon resonant wavelength showed a further shift to 611.6 nm, indicating additional bending in the DC5 sequence. Furthermore, we investigated DNA conformation changes induced by SOX2 and PAX6 on the DC5-con sequence, which is a consensus sequence of DC5 for the PAX6 binding region that strengthens the PAX6 binding but at the same time disrupts SOX2-PAX6-dependent transcriptional activation. When the PAX6 binding sequence in DC5 was altered to DC5-con, the plasmon resonant wavelength shifted much less to 606.5 nm, which is more comparable to the 603.9 nm by SOX2 alone. These results demonstrate that SOX2-PAX6 cobinding induces a large conformation change in DC5 DNA.

Mutation analysis of paired box 6 gene in inherited aniridia in northern China.

PURPOSE: Aniridia is phenotypically and genetically heterogeneous. This study is to summarize the phenotypes and identify the underlying genetic cause of the paired box 6 (PAX6) gene responsible for aniridia in two three-generation Chinese families in northern China. METHODS: A detailed family history and clinical data were collected from patients during an ophthalmologic examination. All exons and flanking intronic sequences of the PAX6 gene were amplified with PCR and screened for mutation with direct DNA sequencing. Haplotyping was used to confirm the mutation sequence. Real-time PCR was used to determine the PAX6 messenger ribonucleic acid(mRNA) level in patients with aniridia and in unaffected family members. RESULTS: The probands and other patients in the two families were affected with aniridia accompanied with or without congenital cataract. A heterozygous PAX6 mutation in exon 5 (c.112delC, p.Arg38GlyfsX16) was identified in FAMILY-1, which was predicted to generate a frameshift and created a premature termination codon. A heterozygous PAX6 mutation in exon 7 (c.362C>T, p.Ser121Leu) was identified in FAMILY-2. Each mutation cosegregated with the affected individuals in the family and did not exist in unaffected family members and 200 unrelated normal controls. The PAX6 messenger ribonucleic acid level was about 50% lower in patients with aniridia than in unaffected family members in FAMILY-1. CONCLUSIONS: The deletion mutation (c.112delC) in the PAX6 gene was first identified in a Chinese family with aniridia, congenital progressive cataract, developmental delay, or the absence of ulna. The mutation (c.362C>T, p.Ser121Leu) in the PAX6 gene was first identified in a patient with aniridia with congenital ptosis. We summarized the variable phenotypes among the patients, which expanded the phenotypic spectrum of aniridia in a different ethnic background.

Functional dissection of the paired domain of Pax6 reveals molecular mechanisms of coordinating neurogenesis and proliferation.

To achieve adequate organ development and size, cell proliferation and differentiation have to be tightly regulated and coordinated. The transcription factor Pax6 regulates patterning, neurogenesis and proliferation in forebrain development. The molecular basis of this regulation is not well understood. As the bipartite DNA-binding paired domain of Pax6 regulates forebrain development, we examined mice with point mutations in its individual DNA-binding subdomains PAI (Pax6(Leca4), N50K) and RED (Pax6(Leca2), R128C). This revealed distinct roles in regulating proliferation in the developing cerebral cortex, with the PAI and RED subdomain mutations reducing and increasing, respectively, the number of mitoses. Conversely, neurogenesis was affected only by the PAI subdomain mutation, phenocopying the neurogenic defects observed in full Pax6 mutants. Genome-wide expression profiling identified molecularly discrete signatures of Pax6(Leca4) and Pax6(Leca2) mutations. Comparison to Pax6 targets identified by chromatin immunoprecipitation led to the identification and functional characterization of distinct DNA motifs in the promoters of target genes dysregulated in the Pax6(Leca2) or Pax6(Leca4) mutants, further supporting the distinct regulatory functions of the DNA-binding subdomains. Thus, Pax6 achieves its key roles in the developing forebrain by utilizing particular subdomains to coordinate patterning, neurogenesis and proliferation simultaneously.