Juan J. Tena

Obesity-associated variants within FTO form long-range functional connections with IRX3

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although 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. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. 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. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.

Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants

Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.

Genome-wide profiling of p63 DNA-binding sites identifies an element that regulates gene expression during limb development in the 7q21 SHFM1 locus.

Heterozygous mutations in p63 are associated with split hand/foot malformations (SHFM), orofacial clefting, and ectodermal abnormalities. Elucidation of the p63 gene network that includes target genes and regulatory elements may reveal new genes for other malformation disorders. We performed genome-wide DNA–binding profiling by chromatin immunoprecipitation (ChIP), followed by deep sequencing (ChIP–seq) in primary human keratinocytes, and identified potential target genes and regulatory elements controlled by p63. We show that p63 binds to an enhancer element in the SHFM1 locus on chromosome 7q and that this element controls expression of DLX6 and possibly DLX5, both of which are important for limb development. A unique micro-deletion including this enhancer element, but not the DLX5/DLX6 genes, was identified in a patient with SHFM. Our study strongly indicates disruption of a non-coding cis-regulatory element located more than 250 kb from the DLX5/DLX6 genes as a novel disease mechanism in SHFM1. These data provide a proof-of-concept that the catalogue of p63 binding sites identified in this study may be of relevance to the studies of SHFM and other congenital malformations that resemble the p63-associated phenotypes.

Dynamics of enhancer chromatin signatures mark the transition from pluripotency to cell specification during embryogenesis

The generation of distinctive cell types that form different tissues and organs requires precise, temporal and spatial control of gene expression. This depends on specific cis-regulatory elements distributed in the noncoding DNA surrounding their target genes. Studies performed on mammalian embryonic stem cells and Drosophila embryos suggest that active enhancers form part of a defined chromatin landscape marked by histone H3 lysine 4 mono-methylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac). Nevertheless, little is known about the dynamics and the potential roles of these marks during vertebrate embryogenesis. Here, we provide genomic maps of H3K4me1/me3 and H3K27ac at four developmental time-points of zebrafish embryogenesis and analyze embryonic enhancer activity. We find that (1) changes in H3K27ac enrichment at enhancers accompany the shift from pluripotency to tissue-specific gene expression, (2) in early embryos, the peaks of H3K27ac enrichment are bound by pluripotent factors such as Nanog, and (3) the degree of evolutionary conservation is higher for enhancers that become marked by H3K27ac at the end of gastrulation, suggesting their implication in the establishment of the most conserved (phylotypic) transcriptome that is known to occur later at the pharyngula stage.

Zebrafish enhancer detection (ZED) vector: A new tool to facilitate transgenesis and the functional analysis of cis-regulatory regions in zebrafish

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 cis‐regulatory 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. Developmental Dynamics 238:2409–2417, 2009.

Evolutionary comparison reveals that diverging CTCF sites are signatures of ancestral topological associating domains borders

Significance Mammalian chromatin is compartmentalized in topologically associating domains (TADs), genomic regions within which sequences preferentially contact each other. This organization has been proposed to be essential to organize the regulatory information contained in mammalian genomes. We show that Six homeobox genes, essential developmental regulators organized in gene clusters across different animal phyla, share a deeply conserved chromatin organization formed by two abutting TADs that predates the Cambrian explosion. This organization is required to generate separate regulatory landscapes for neighboring genes within the cluster, resulting in very different gene expression patterns. Finally, we show that this extremely conserved 3D architecture is associated with a characteristic arrangement of CCCTC-binding factor (CTCF) binding sites in diverging orientations, revealing a genome-wide conserved signature for TAD borders. 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. Genome-wide 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.

Extensive conservation of ancient microsynteny across metazoans due to cis-regulatory constraints.

The order of genes in eukaryotic genomes has generally been assumed to be neutral, since gene order is largely scrambled over evolutionary time. Only a handful of exceptional examples are known, typically involving deeply conserved clusters of tandemly duplicated genes (e.g., Hox genes and histones). Here we report the first systematic survey of microsynteny conservation across metazoans, utilizing 17 genome sequences. We identified nearly 600 pairs of unrelated genes that have remained tightly physically linked in diverse lineages across over 600 million years of evolution. Integrating sequence conservation, gene expression data, gene function, epigenetic marks, and other genomic features, we provide extensive evidence that many conserved ancient linkages involve (1) the coordinated transcription of neighboring genes, or (2) genomic regulatory blocks (GRBs) in which transcriptional enhancers controlling developmental genes are contained within nearby bystander genes. In addition, we generated ChIP-seq data for key histone modifications in zebrafish embryos, which provided further evidence of putative GRBs in embryonic development. Finally, using chromosome conformation capture (3C) assays and stable transgenic experiments, we demonstrate that enhancers within bystander genes drive the expression of genes such as Otx and Islet, critical regulators of central nervous system development across bilaterians. These results suggest that ancient genomic functional associations are far more common than previously thought-involving ∼12% of the ancestral bilaterian genome-and that cis-regulatory constraints are crucial in determining metazoan genome architecture.

An evolutionarily conserved three-dimensional structure in the vertebrate Irx clusters facilitates enhancer sharing and coregulation

Developmental gene clusters are paradigms for the study of gene regulation; however, the mechanisms that mediate phenomena such as coregulation and enhancer sharing remain largely elusive. Here we address this issue by analysing the vertebrate Irx clusters. We first present a deep enhancer screen of a 2-Mbp span covering the IrxA cluster. Using chromosome conformation capture, we show that enhancer sharing is widespread within the cluster, explaining its evolutionarily conserved organization. We also identify a three-dimensional architecture, probably formed through interactions with CCCTC-binding factor, which is present within both Irx clusters of mouse, Xenopus and zebrafish. This architecture brings the promoters of the first two genes together in the same chromatin landscape. We propose that this unique and evolutionarily conserved genomic architecture of the vertebrate Irx clusters is essential for the coregulation of the first two genes and simultaneously maintains the third gene in a partially independent regulatory landscape.

Deep conservation of wrist and digit enhancers in fish

Significance The fossil record shows that the wrist and digits have an aquatic origin, becoming recognizable in a group of (mostly extinct) fish that contained robust fins. Do the fins of living fishes have the equivalent of these structures? Because comparisons of fin and limb morphology have been inconclusive, we sought to investigate this question using developmental and molecular data. By utilizing a nonmodel fish (the spotted gar), we find that the regulatory networks that control “wrist and digit”-building genes (Hox) are deeply conserved between fish and tetrapods. The genomic architecture described here defines Hox gene activity in fins and limbs as equivalent, in turn suggesting equivalence between the distal bones of fish fins and the wrist and/or digits of tetrapods. There is no obvious morphological counterpart of the autopod (wrist/ankle and digits) in living fishes. Comparative molecular data may provide insight into understanding both the homology of elements and the evolutionary developmental mechanisms behind the fin to limb transition. In mouse limbs the autopod is built by a “late” phase of Hoxd and Hoxa gene expression, orchestrated by a set of enhancers located at the 5′ end of each cluster. Despite a detailed mechanistic understanding of mouse limb development, interpretation of Hox expression patterns and their regulation in fish has spawned multiple hypotheses as to the origin and function of “autopod” enhancers throughout evolution. Using phylogenetic footprinting, epigenetic profiling, and transgenic reporters, we have identified and functionally characterized hoxD and hoxA enhancers in the genomes of zebrafish and the spotted gar, Lepisosteus oculatus, a fish lacking the whole genome duplication of teleosts. Gar and zebrafish “autopod” enhancers drive expression in the distal portion of developing zebrafish pectoral fins, and respond to the same functional cues as their murine orthologs. Moreover, gar enhancers drive reporter gene expression in both the wrist and digits of mouse embryos in patterns that are nearly indistinguishable from their murine counterparts. These functional genomic data support the hypothesis that the distal radials of bony fish are homologous to the wrist and/or digits of tetrapods.

Active DNA demethylation at enhancers during the vertebrate phylotypic period

The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases.