Genome-wide profiling of DNA methylation and histone modifications answered many questions as to how the genes are regulated on a global scale and what their epigenetic makeup is. Yet, little is ...known about the function of these marks during early vertebrate embryogenesis. Here we provide detailed protocols for ChIP-seq and MethylCap-seq procedures applied to zebrafish (Danio rerio) embryonic material at four developmental stages. As a proof of principle, we have profiled on a global scale a number of post-translational histone modifications including H3K4me1, H3K4me3 and H3K27ac. We demonstrate that these marks are dynamic during early development and that such developmental transitions can be detected by ChIP-seq. In addition, we applied MethylCap-seq to show that developmentally-regulated DNA methylation remodeling can be detected by such a procedure. Our MethylCap-seq data concur with previous DNA methylation studies of early zebrafish development rendering this method highly suitable for the global assessment of DNA methylation in early vertebrate embryos.
New frontiers in the evolution of fin development Freitas, Renata; Gómez-Skarmeta, José Luis; Rodrigues, Pedro Nuno
Journal of experimental zoology. Part B, Molecular and developmental evolution,
November 2014, Letnik:
322, Številka:
7
Journal Article
The organization of animal genomes into topologically associating domains (TADs) provides a structural scaffold in which cis-regulatory elements (CREs) operate on their target genes. Determining the ...position of CREs and genes relative to TADs has become instrumental to trace gene expression changes during evolution and in diseases. Here we will review recent studies and discuss TADs as structural units with respect to their conservation and stability during genome reorganization. Furthermore, we describe how TAD restructuring contributed to morphological novelties during evolution but also their deleterious effects associated with disease. Despite considering TADs as structural units, the nested and dynamic scaffold within TADs contributes to tissue-specific gene expression, implying that such changes can also account for gene expression differences during evolution.
Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that ...acts as an enhancer-blocking element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that the asthma-associated single nucleotide polymorphism (SNP) rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a mechanism through which a regulatory SNP contributes to genetic risk of asthma.
Planarians regenerate a whole animal from a small body piece within a few days 1, 2. Recent studies have shown that the bone morphogenetic protein (BMP) pathway is required to reestablish the ...dorsoventral (DV) axis 3–5. In vertebrates, the specification of the DV axis depends on the coordinated action of a dual organizer defined by BMP and antidorsalizing morphogenetic protein (ADMP) under the control of several factors, including the inhibitors chordin and noggin 6. Planarians have an expanded noggin family (up to ten members), which have been classified as canonical noggin (nog) and noggin-like (nlg) genes, the latter carrying an insertion within the noggin domain 7. Here we show that a BMP/ADMP organizer governs DV axis reestablishment during planarian regeneration, highlighting a greater-than-thought conservation of the mechanisms that establish this axis in protostomes and deuterostomes. Also, we report that whereas noggin genes function as canonical BMP inhibitors, the silencing of planarian nlg8 induces ectopic neurogenesis and enhances ventralizing bmp(RNAi) phenotypes. Finally, we show that noggin-like genes are conserved from cnidarian to vertebrates and that both planarian nlg8 and Xenopus nlg ventralize Xenopus embryos when overexpressed. Remarkably, this ventralization is not associated with an increase in SMAD1/5/8 phosphorylation.
► A BMP/ADMP organizer controls DV patterning outside deuterostomes ► NOGGIN and NOGGIN-LIKE exert opposite functions in both planarians and Xenopus ► Noggin-like genes constitute a new evolutionary conserved family ► NOG, NLG, BMP, and ADMP may form a conserved tool kit to pattern the DV axis
Vertebrate odd-skipped related genes (Osr) have an essential function during the formation of the intermediate mesoderm (IM) and the kidney structures derived from it. Here, we show that these genes ...are also crucial for limb bud formation in the adjacent lateral plate mesoderm (LPM). Reduction of zebrafish Osr function impairs fin development by the failure of tbx5a maintenance in the developing pectoral fin bud. Osr morphant embryos show reduced wnt2b expression, and increasing Wnt signaling in Osr morphant embryos partially rescues tbx5a expression. Thus, Osr genes control limb bud development in a non-cell-autonomous manner, probably through the activation of Wnt2b. Finally, we demonstrate that Osr genes are downstream targets of retinoic acid (RA) signaling. Therefore, Osr genes act as a relay within the genetic cascade of fin bud formation: by controlling the expression of the signaling molecule Wnt2ba in the IM they play an essential function transmitting the RA signaling originated in the somites to the LPM.
The causes and consequences of genome reduction in animals are unclear because our understanding of this process mostly relies on lineages with often exceptionally high rates of evolution. Here, we ...decode the compact 73.8-megabase genome of Dimorphilus gyrociliatus, a meiobenthic segmented worm. The D. gyrociliatus genome retains traits classically associated with larger and slower-evolving genomes, such as an ordered, intact Hox cluster, a generally conserved developmental toolkit and traces of ancestral bilaterian linkage. Unlike some other animals with small genomes, the analysis of the D. gyrociliatus epigenome revealed canonical features of genome regulation, excluding the presence of operons and trans-splicing. Instead, the gene-dense D. gyrociliatus genome presents a divergent Myc pathway, a key physiological regulator of growth, proliferation and genome stability in animals. Altogether, our results uncover a conservative route to genome compaction in annelids, reminiscent of that observed in the vertebrate Takifugu rubripes.
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.
Control of gene expression is dictated by cell-type specific regulatory sequences that physically organize the structure of chromatin, including promoters, enhancers and insulators. While promoters ...and enhancers convey cell-type specific activating signals, insulators prevent the cross-talk of regulatory elements within adjacent loci and safeguard the specificity of action of promoters and enhancers towards their targets in a tissue specific manner. Using the mouse tyrosinase (Tyr) locus as an experimental model, a gene whose mutations are associated with albinism, we described the chromatin structure in cells at two distinct transcriptional states. Guided by chromatin structure, through the use of Chromosome Conformation Capture (3C), we identified sequences at the 5' and 3' boundaries of this mammalian gene that function as enhancers and insulators. By CRISPR/Cas9-mediated chromosomal deletion, we dissected the functions of these two regulatory elements in vivo in the mouse, at the endogenous chromosomal context, and proved their mechanistic role as genomic insulators, shielding the Tyr locus from the expression patterns of adjacent genes.
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