Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type-2 diabetes (T2D) 1–3. While the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes 4–6. Yet, no direct connection between the obesity-associated variants and FTO expression or function has been made 7–9. Here, we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse, and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Supporting this, obesity-associated SNPs are associated with expression of IRX3, but not FTO, in human brains. Directly linking IRX3 expression with regulation of body mass and composition, Irx3-deficient mice exhibit a 25–30% reduction in body weight, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Furthermore, hypothalamic expression of a dominant negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data posit that IRX3 is a functional long-range target of obesity-associated variants within FTO, and represents a novel determinant of body mass and composition.
Vertebrates have greatly elaborated the basic chordate body plan and evolved highly distinctive genomes that have been sculpted by two whole-genome duplications. Here we sequence the genome of the Mediterranean amphioxus ( Branchiostoma lanceolatum ) and characterize DNA methylation, chromatin accessibility, histone modifications and transcriptomes across multiple developmental stages and adult tissues to investigate the evolution of the regulation of the chordate genome. Comparisons with vertebrates identify an intermediate stage in the evolution of differentially methylated enhancers, and a high conservation of gene expression and its cis -regulatory logic between amphioxus and vertebrates that occurs maximally at an earlier mid-embryonic phylotypic period. We analyse regulatory evolution after whole-genome duplications, and find that—in vertebrates—over 80% of broadly expressed gene families with multiple paralogues derived from whole-genome duplications have members that restricted their ancestral expression, and underwent specialization rather than subfunctionalization. Counter-intuitively, paralogues that restricted their expression increased the complexity of their regulatory landscapes. These data pave the way for a better understanding of the regulatory principles that underlie key vertebrate innovations.
The identification and characterization of the regulatory activity of genomic sequences is crucial for understanding how the information contained in genomes is translated into cellular function. The cisregulatory sequences control when, where, and how much genes are transcribed and can activate (enhancers) or repress (silencers) gene expression. Here, we describe a novel Tol2 transposon-based vector for assessing enhancer activity in the zebrafish (Danio rerio). This Zebrafish Enhancer Detector (ZED) vector harbors several key improvements, among them a sensitive and specific minimal promoter chosen for optimal enhancer activity detection, insulator sequences to shield the minimal promoter from position effects, and a positive control for transgenesis. Additionally, we demonstrate that highly conserved noncoding sequences homologous between humans and zebrafish largely with enhancer activity largely retain their tissue-specific enhancer activity during vertebrate evolution. More strikingly, insulator sequences from mouse and chicken, but not conserved in zebrafish, maintain their insulator capacity when tested in this model.
Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genomewide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution.CTCF | TAD | Six cluster | regulatory landscapes | evolution
Recent genome-wide scans for colorectal cancer (CRC) have revealed the SMAD7 (mothers against decapentaplegic homolog 7) gene as a locus associated with a modest, but highly significant increase in CRC risk. To identify the causal basis of the association between 18q21 variation and CRC, we resequenced the 17-kb region of linkage disequilibrium and evaluated all variants in 2532 CRC cases and 2607 controls. A novel C to G single nucleotide polymorphism (SNP) at 44,703,563 bp was maximally associated with CRC risk (P = 5.98 3 10 À7 ; $1.5-fold more likely to be causal than other variants). Using transgenic assays in Xenopus laevis as a functional model, we demonstrate that the G risk allele leads to reduced reporter gene expression in the colorectum (P = 5.4 3 10 À3 ). Electrophoretic mobility shift assays provided evidence for the role of Novel 1 in transcription factor binding. We propose that the novel SNP we have identified is the functional change leading to CRC predisposition through differential SMAD7 expression and, hence, aberrant TGF-beta signaling.
Despite their evolutionary, developmental, and functional importance the origin of vertebrate paired appendages remains uncertain. In mice, a single enhancer termed ZRS is solely responsible for Shh expression in limbs. Here, zebrafish and mouse transgenic assays trace the functional equivalence of ZRS across the gnathostome phylogeny. CRISPR/Cas9-mediated deletion of the medaka-ZRS and enhancer assays reveal the existence of ZRS shadow enhancers in both teleost and human genomes. Deletion of both ZRS and shadow ZRS abolish shh expression and completely truncate pectoral fin formation. Strikingly, deletion of ZRS results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and median fins, and that paired fins likely emerged by the co‐option of developmental programs established in the median fins of stem gnathostomes. Shh function was later reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness.
Common genetic variation at human 8q23.3 is significantly associated with colorectal cancer (CRC) risk. To elucidate the basis of this association we compared the frequency of common variants at 8q23.3 in 1,964 CRC cases and 2,081 healthy controls. Reporter gene studies showed that the single nucleotide polymorphism rs16888589 acts as an allele-specific transcriptional repressor. Chromosome conformation capture (3C) analysis demonstrated that the genomic region harboring rs16888589 interacts with the promoter of gene for eukaryotic translation initiation factor 3, subunit H (EIF3H). We show that increased expression of EIF3H gene increases CRC growth and invasiveness thereby providing a biological mechanism for the 8q23.3 association. These data provide evidence for a functional basis for the non-coding risk variant rs16888589 at 8q23.3 and provides novel insight into the etiological basis of CRC.
POU3F4 encodes a POU-domain transcription factor required for inner ear development. Defects in POU3F4 function are associated with X-linked deafness type 3 (DFN3). Multiple deletions affecting up to ~900-kb upstream of POU3F4 are found in DFN3 patients, suggesting the presence of essential POU3F4 enhancers in this region. Recently, an inner ear enhancer was reported that is absent in most DFN3 patients with upstream deletions. However, two indications suggest that additional enhancers in the POU3F4 upstream region are required for POU3F4 function during inner ear development. First, there is at least one DFN3 deletion that does not eliminate the reported enhancer. Second, the expression pattern driven by this enhancer does not fully recapitulate Pou3f4 expression in the inner ear. Here, we screened a 1-Mb region upstream of the POU3F4 gene for additional cis-regulatory elements and searched for novel DFN3 mutations in the identified POU3F4 enhancers. We found several novel enhancers for otic vesicle expression. Some of these also drive expression in kidney, pancreas and brain, tissues that are known to express Pou3f4. In addition, we report a new and smallest deletion identified so far in a DFN3 family which eliminates 3.9 kb, comprising almost exclusively the previous reported inner ear enhancer. We suggest that multiple enhancers control the expression of Pou3f4 in the inner ear and these may contribute to the phenotype observed in DFN3 patients. In addition, the novel deletion demonstrates that the previous reported enhancer, although not sufficient, is essential for POU3F4 function during inner ear development.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-010-0864-x) contains supplementary material, which is available to authorized users.
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