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.

Functional characteristics of novel pancreatic Pax6 regulatory elements.

Complex diseases, such as diabetes, are influenced by comprehensive transcriptional networks. Genome-wide association studies have revealed that variants located in regulatory elements for pancreatic transcription factors are linked to diabetes, including those functionally linked to the paired box transcription factor Pax6. Pax6 deletions in adult mice cause rapid onset of classic diabetes, but the full spectrum of pancreatic Pax6 regulators is unknown.

Drug-tunable multidimensional synthetic gene control using inducible degron-tagged dCas9 effectors.

The nuclease-deactivated variant of CRISPR-Cas9 proteins (dCas9) fused to heterologous transactivation domains can act as a potent guide RNA sequence-directed inducer or repressor of gene expression in mammalian cells. In such a system the long-term presence of a stable dCas9 effector can be a draw-back precluding the ability to switch rapidly between repressed and activated target gene expression states, imposing a static environment on the synthetic regulatory circuits in the cell. To address this issue we have generated a toolkit of conditionally degradable or stabilisable orthologous dCas9 or Cpf1 effector proteins, thus opening options for multidimensional control of functional activities through combinations of orthogonal, drug-tunable artificial transcription factors.

Remotely acting SMCHD1 gene regulatory elements: in silico prediction and identification of potential regulatory variants in patients with FSHD.

Background: Facioscapulohumeral dystrophy (FSHD) is commonly associated with contraction of the D4Z4 macro-satellite repeat on chromosome 4q35 (FSHD1) or mutations in the SMCHD1 gene (FSHD2). Recent studies have shown that the clinical manifestation of FSHD1 can be modified by mutations in the SMCHD1 gene within a given family. The absence of either D4Z4 contraction or SMCHD1 mutations in a small cohort of patients suggests that the disease could also be due to disruption of gene regulation.

Regulation of SPRY3 by X chromosome and PAR2-linked promoters in an autism susceptibility region.

Sprouty proteins are regulators of cell growth and branching morphogenesis. Unlike mouse Spry3, which is X-linked, human SPRY3 maps to the pseudoautosomal region 2; however, the human Y-linked allele is not expressed due to epigenetic silencing by an unknown mechanism. SPRY3 maps adjacent to X-linked Trimethyllysine hydroxylase epsilon (TMLHE), recently identified as an autism susceptibility gene.

Functional assessment of disease-associated regulatory variants in vivo using a versatile dual colour transgenesis strategy in zebrafish.

Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking.

Identification of novel craniofacial regulatory domains located far upstream of SOX9 and disrupted in Pierre Robin sequence.

Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ∼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ∼1.

Genomic profiling of type-1 adult diabetic and aged normoglycemic mouse liver.

Background: Hyperglycemia induces chromatin remodeling with consequences on differential gene expression in mouse hepatocytes, similar to what occurs during aging. The liver is the central organ for the regulation of glucose homeostasis and xenobiotic and lipid metabolism and is affected by insulin signaling. The precise transcriptional profiling of the type-1 diabetic liver and its comparison to aging have not been elucidated yet.

Disruption of long-range gene regulation in human genetic disease: a kaleidoscope of general principles, diverse mechanisms and unique phenotypic consequences.

The precise control of gene expression programs is crucial for the establishment of the diverse gene activity patterns required for the correct development, patterning and differentiation of the myriad of cell types within an organism. The crucial importance of non-coding regions of the genome in the control of gene regulation is well established and depends on a diverse group of sequence fragments called cis-regulatory elements that reside in these regions. Advances in novel genome-wide techniques have greatly increased the ability to identify potential regulatory elements.

A survey of ancient conserved non-coding elements in the PAX6 locus reveals a landscape of interdigitated cis-regulatory archipelagos.

Biological differences between cell types and developmental processes are characterised by differences in gene expression profiles. Gene-distal enhancers are key components of the regulatory networks that specify the tissue-specific expression patterns driving embryonic development and cell fate decisions, and variations in their sequences are a major contributor to genetic disease and disease susceptibility. Despite advances in the methods for discovery of putative cis-regulatory sequences, characterisation of their spatio-temporal enhancer activities in a mammalian model system remains a major bottle-neck.

Disruption of SATB2 or its long-range cis-regulation by SOX9 causes a syndromic form of Pierre Robin sequence.

Heterozygous loss-of-function (LOF) mutations in the gene encoding the DNA-binding protein, SATB2, result in micrognathia and cleft palate in both humans and mice. In three unrelated individuals, we show that translocation breakpoints (BPs) up to 896 kb 3' of SATB2 polyadenylation site cause a phenotype which is indistinguishable from that caused by SATB2 LOF mutations. This syndrome comprises long nose, small mouth, micrognathia, cleft palate, arachnodactyly and intellectual disability.

Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes aniridia.

The strictly regulated expression of most pleiotropic developmental control genes is critically dependent on the activity of long-range cis-regulatory elements. This was revealed by the identification of individuals with a genetic condition lacking coding-region mutations in the gene commonly associated with the disease but having a variety of nearby chromosomal abnormalities, collectively described as cis-ruption disease cases. The congenital eye malformation aniridia is caused by haploinsufficiency of the developmental regulator PAX6.

Identification of genomic regions regulating Pax6 expression in embryonic forebrain using YAC reporter transgenic mouse lines.

The transcription factor Pax6 is a crucial regulator of eye and central nervous system development. Both the spatiotemporal patterns and the precise levels of Pax6 expression are subject to tight control, mediated by an extensive set of cis-regulatory elements. Previous studies have shown that a YAC reporter transgene containing 420 Kb of genomic DNA spanning the human PAX6 locus drives expression of a tau-tagged GFP reporter in mice in a pattern that closely resembles that of endogenous Pax6.

Sequencing of Pax6 loci from the elephant shark reveals a family of Pax6 genes in vertebrate genomes, forged by ancient duplications and divergences.

Pax6 is a developmental control gene essential for eye development throughout the animal kingdom. In addition, Pax6 plays key roles in other parts of the CNS, olfactory system, and pancreas. In mammals a single Pax6 gene encoding multiple isoforms delivers these pleiotropic functions.

The developmental regulator Pax6 is essential for maintenance of islet cell function in the adult mouse pancreas.

The transcription factor Pax6 is a developmental regulator with a crucial role in development of the eye, brain, and olfactory system. Pax6 is also required for correct development of the endocrine pancreas and specification of hormone producing endocrine cell types. Glucagon-producing cells are almost completely lost in Pax6-null embryos, and insulin-expressing beta and somatostatin-expressing delta cells are reduced.

DNaseI hypersensitivity and ultraconservation reveal novel, interdependent long-range enhancers at the complex Pax6 cis-regulatory region.

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene.

Discovery and assessment of conserved Pax6 target genes and enhancers.

The characterization of transcriptional networks (TNs) is essential for understanding complex biological phenomena such as development, disease, and evolution. In this study, we have designed and implemented a procedure that combines in silico target screens with zebrafish and mouse validation, in order to identify cis-elements and genes directly regulated by Pax6. We chose Pax6 as the paradigm because of its crucial roles in organogenesis and human disease.

Effects of elevated Pax6 expression and genetic background on mouse eye development.

Purpose: To analyze the effects of Pax6 overexpression and its interaction with genetic background on eye development.

Methods: Histologic features of eyes from hemizygous PAX77(+/-) transgenic (high Pax6 gene dose) and wild-type mice were compared on different genetic backgrounds. Experimental PAX77(+/-)<-->wild-type and control wild-type<-->wild-type chimeras were analyzed to investigate the causes of abnormal eye development in PAX77(+/-) mice.

Mosaic analysis of stem cell function and wound healing in the mouse corneal epithelium.

Background: The mouse corneal epithelium is a continuously renewing 5-6 cell thick protective layer covering the corneal surface, which regenerates rapidly when injured. It is maintained by peripherally located limbal stem cells (LSCs) that produce transient amplifying cells (TACs) which proliferate, migrate centripetally, differentiate and are eventually shed from the epithelial surface. LSC activity is required both for normal tissue maintenance and wound healing.

Cited2 is required for the proper formation of the hyaloid vasculature and for lens morphogenesis.

Cited2 is a transcriptional modulator with pivotal roles in different biological processes. Cited2-deficient mouse embryos manifested two major defects in the developing eye. An abnormal corneal-lenticular stalk was characteristic of Cited2(-/-) developing eyes, a feature reminiscent of Peters' anomaly, which can be rescued by increased Pax6 gene dosage in Cited2(-/-) embryonic eyes.

Long-range gene control and genetic disease.

The past two decades have seen great progress in the elucidation of the genetic basis of human genetic disease. Many clinical phenotypes have been linked with mutations or deletions in specific causative genes. However, it is often less recognized that in addition to the integrity of the protein-coding sequences, human health critically also depends on the spatially, temporally, and quantitatively correct expression of those genes.

Subfunctionalization of duplicated zebrafish pax6 genes by cis-regulatory divergence.

Gene duplication is a major driver of evolutionary divergence. In most vertebrates a single PAX6 gene encodes a transcription factor required for eye, brain, olfactory system, and pancreas development. In zebrafish, following a postulated whole-genome duplication event in an ancestral teleost, duplicates pax6a and pax6b jointly fulfill these roles.

Controlled overexpression of Pax6 in vivo negatively autoregulates the Pax6 locus, causing cell-autonomous defects of late cortical progenitor proliferation with little effect on cortical arealization.

Levels of expression of the transcription factor Pax6 vary throughout corticogenesis in a rostro-lateral(high) to caudo-medial(low) gradient across the cortical proliferative zone. Previous loss-of-function studies have indicated that Pax6 is required for normal cortical progenitor proliferation, neuronal differentiation, cortical lamination and cortical arealization, but whether and how its level of expression affects its function is unclear. We studied the developing cortex of PAX77 YAC transgenic mice carrying several copies of the human PAX6 locus with its full complement of regulatory regions.

Long-range downstream enhancers are essential for Pax6 expression.

Pax6 is a developmental control gene with an essential role in development of the eye, brain and pancreas. Pax6, as many other developmental regulators, depends on a substantial number of cis-regulatory elements in addition to its promoters for correct spatiotemporal and quantitative expression. Here we report on our analysis of a set of mice transgenic for a modified yeast artificial chromosome carrying the human PAX6 locus.