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. We discovered a de novo point mutation in an ultraconserved cis-element located 150 kb downstream from PAX6 in an affected individual with intact coding region and chromosomal locus. The element SIMO acts as a strong enhancer in developing ocular structures. The mutation disrupts an autoregulatory PAX6 binding site, causing loss of enhancer activity, resulting in defective maintenance of PAX6 expression. These findings reveal a distinct regulatory mechanism for genetic disease by disruption of an autoregulatory feedback loop critical for maintenance of gene expression through development.
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. Here we provide evidence that the genomes of many other vertebrate species contain multiple Pax6 loci. We sequenced Pax6-containing BACs from the cartilaginous elephant shark (Callorhinchus milii) and found two distinct Pax6 loci. Pax6.1 is highly similar to mammalian Pax6, while Pax6.2 encodes a paired-less Pax6. Using synteny relationships, we identify homologs of this novel paired-less Pax6.2 gene in lizard and in frog, as well as in zebrafish and in other teleosts. In zebrafish two full-length Pax6 duplicates were known previously, originating from the fish-specific genome duplication (FSGD) and expressed in divergent patterns due to paralog-specific loss of cis-elements. We show that teleosts other than zebrafish also maintain duplicate full-length Pax6 loci, but differences in gene and regulatory domain structure suggest that these Pax6 paralogs originate from a more ancient duplication event and are hence renamed as Pax6.3. Sequence comparisons between mammalian and elephant shark Pax6.1 loci highlight the presence of short- and long-range conserved noncoding elements (CNEs). Functional analysis demonstrates the ancient role of long-range enhancers for Pax6 transcription. We show that the paired-less Pax6.2 ortholog in zebrafish is expressed specifically in the developing retina. Transgenic analysis of elephant shark and zebrafish Pax6.2 CNEs with homology to the mouse NRE/Pα internal promoter revealed highly specific retinal expression. Finally, morpholino depletion of zebrafish Pax6.2 resulted in a “small eye” phenotype, supporting a role in retinal development. In summary, our study reveals that the pleiotropic functions of Pax6 in vertebrates are served by a divergent family of Pax6 genes, forged by ancient duplication events and by independent, lineage-specific gene losses.
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 ~1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harbouring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple non-coding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS.
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. These BPs map to a gene desert between PLCL1 and SATB2. We identified three putative cis-regulatory elements (CRE1–3) using a comparative genomic approach each of which would be placed in trans relative to SATB2 by all three BPs. CRE1–3 each bind p300 and mono-methylated H3K4 consistent with enhancer function. In silico analysis suggested that CRE1–3 contain one or more conserved SOX9-binding sites, and this binding was confirmed using chromatin immunoprecipitation on cells derived from mouse embryonic pharyngeal arch. Interphase bacterial artificial chromosome fluorescence in situ hybridization measurements in embryonic craniofacial tissues showed that the orthologous region in mice exhibits Satb2 expression-dependent chromatin decondensation consistent with Satb2 being a target gene of CRE1–3. To assess their in vivo function, we made multiple stable reporter transgenic lines for each enhancer in zebrafish. CRE2 was shown to drive SATB2-like expression in the embryonic craniofacial region. This expression could be eliminated by mutating the SOX9-binding site of CRE2. These observations suggest that SATB2 and SOX9 may be acting together via complex cis-regulation to coordinate the growth of the developing jaw.
MicroRNAs (miRNAs) are small, endogenous, regulatory RNA molecules that can bind to partially complementary regions on target messenger RNAs and impede their expression or translation. We rationalized that miRNAs, being localized to the cytoplasm, will be maternally inherited during fertilization and may play a role in early development. Although Dicer is known to be essential for the transition from single-celled zygote to two-cell embryo, a direct role for miRNAs has not yet been demonstrated. We identified miRNAs with targets in zygotically expressed transcripts in Drosophila using a combination of transcriptome analysis and miRNA target prediction. We experimentally established that Drosophila miRNA dme-miR-34, the fly homologue of the cancer-related mammalian miRNA miR-34, involved in somatic-cell reprogramming and having critical role in early neuronal differentiation, is present in Drosophila embryos before initiation of zygotic transcription. We also show that the Drosophila miR-34 is dependent on maternal Dicer-1 for its expression in oocytes. Further, we show that miR-34 is also abundant in unfertilized oocytes of zebrafish. Its temporal expression profile during early development showed abundant expression in unfertilized oocytes that gradually decreased by 5 days post-fertilization (dpf). We find that knocking down the maternal, but not the zygotic, miR-34 led to developmental defects in the neuronal system during early embryonic development in zebrafish. Here, we report for the first time, the maternal inheritance of an miRNA involved in development of the neuronal system in a vertebrate model system.
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. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function.
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. We identified over 600 putative Pax6 binding sites and more than 200 predicted direct target genes, conserved in evolution from zebrafish to human and to mouse. This was accomplished using hidden Markov models (HMMs) generated from experimentally validated Pax6 binding sites. A small sample of genes, expressed in the neural lineage, was chosen from the predictions for RNA in situ validation using zebrafish and mouse models. Validation of DNA binding to some predicted cis-elements was also carried out using chromatin immunoprecipitation (ChIP) and zebrafish reporter transgenic studies. The results show that this combined procedure is a highly efficient tool to investigate the architecture of TNs and constitutes a useful complementary resource to ChIP and expression data sets because of its inherent spatiotemporal independence. We have identified several novel direct targets, including some putative disease genes, among them Foxp2; these will allow further dissection of Pax6 function in development and disease.
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. In contrast, their functional characterisation and the determination of their diverse modes of action remain a major bottleneck. Greater knowledge of gene expression control is of major importance for human health as disruption of gene regulation has become recognised as a significant cause of human disease. Appreciation of the role of cis-regulatory polymorphism in natural variation and susceptibility to common disease is also growing. While novel techniques such as GWAS and NGS provide the ability to collect large genomic datasets, the challenge for the twenty-first century will be to extract the relevant sequences and how to investigate the functional consequences of disease-associated changes. Here, we review how studies of transcriptional control at selected paradigm disease gene loci have revealed general principles of cis-regulatory logic and regulatory genome organisation, yet also demonstrate how the variety of mechanisms can combine to result in unique phenotypic outcomes. Integration of these principles with the emerging wealth of genome-wide data will provide enhanced insight into the workings of our regulatory genome.
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