A mobile insulator system to detect and disrupt <i>cis</i>-regulatory landscapes in vertebrates

Bessa, José; Luengo, M.Á. Sánchez; Rivero-Gil, Solangel; Ariza-Cosano, Ana; Maia, António H.F.; Ruiz‐Ruano, Francisco J.; Caballero, Pablo; Naranjo, Silvia; Carvajal, Jaime J.; Gómez-Skármeta, José Luis

doi:10.1101/gr.165654.113

Cited by 13 publications

(10 citation statements)

References 53 publications

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“…nog2 Controls Non-autonomously the Embryonic Endocrine Pancreas Size nog2 encodes a zebrafish Bmp antagonist that starts to be expressed at 10 hpf in the primordium of the notochord (F€ urthauer et al, 1999). To identify possible non-coding genetic modifications affecting pancreas development, we mobilized a transposable element containing a potent enhancer blocking insulator (Bessa et al, 2014). We found a novel integration of this construct (ED301) located 2 kb downstream of nog2 (nog2 ED301 ) (Figure 1A; Figure S1).…”

Section: Resultsmentioning

confidence: 99%

“…Zebrafish transgenesis ED301 line was generated as described by Bessa et al (2014). Enhancer reporter assays were performed by cloning the selected genomic fragments into the ZED vector (Bessa et al, 2009).…”

Section: Methods Detailsmentioning

confidence: 99%

“…In this work, we disrupted the function of nog2 , observed consistent impairment in the number of pancreatic progenitor cells and insulin-expressing cells, and showed nog2 to be an important Bmp antagonist required for pancreatic development. The introduction of an enhancer blocking insulator ( Bessa et al., 2014 ) downstream of nog2 also impaired β cell differentiation and reduced the number of pancreatic progenitor cells, suggesting the existence of an enhancer required for the nog2 pancreatic function. By analyzing histone modifications associated with enhancer activity (histone H3 lysine 27 acetylation [H3K27ac] and histone H3 lysine 4 monomethylation [H3K4me1]) ( Bogdanovic et al., 2012 ; Marlétaz et al., 2018 ; Rada-Iglesias et al., 2011 ) and performing circular chromosome conformation capture coupled with next-generation sequencing (4C-seq) ( Zhao et al., 2006 ), we identified a nog2 notochord enhancer, among others.…”

Section: Introductionmentioning

confidence: 99%

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A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates

et al. 2020

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Summary The notochord is an evolutionary novelty in vertebrates that functions as an important signaling center during development. Notochord ablation in chicken has demonstrated that it is crucial for pancreas development; however, the molecular mechanism has not been fully described. Here, we show that in zebrafish, the loss of function of nog2, a Bmp antagonist expressed in the notochord, impairs β cell differentiation, compatible with the antagonistic role of Bmp in β cell differentiation. In addition, we show that nog2 expression in the notochord is induced by at least one notochord enhancer and its loss of function reduces the number of pancreatic progenitors and impairs β cell differentiation. Tracing Nog2 diffusion, we show that Nog2 emanates from the notochord to the pancreas progenitor domain. Finally, we find a notochord enhancer in human and mice Nog genomic landscapes, suggesting that the acquisition of a Nog notochord enhancer occurred early in the vertebrate phylogeny and contributes to the development of complex organs like the pancreas.

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Section: Resultsmentioning

confidence: 99%

Section: Methods Detailsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates

et al. 2020

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“…In this sense, using CTCF binding sites as an enhancer blocker is a very appealing approach, as it can blunt gene activation as long as the element is inserted between the gene and the enhancers without knowing precisely where the enhancers are. The insulator element identified in the chicken β-globin locus, which harbors a CTCF binding site (Bell et al, 1999), has been utilized in heterologous systems (Bessa et al, 2014). From our view, however, those elements have not been spread widely as tools to control gene expression, probably because the underlying mechanism was not fully understood and convincing explanation for its general utility was missing when they were reported.…”

Section: Introductionmentioning

confidence: 99%

Controlling gene activation by enhancers through a drug-inducible topological insulator

Tsujimura

Takase

Yoshikawa

et al. 2019

Preprint

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AbstractWhile regulation of gene-enhancer interaction is better understood, its application remains limited. Here, we reconstituted arrays of CTCF binding sites and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. We applied this to dissect MYC regulation in human pluripotent stem cells. Insertion of STITCH between MYC and the enhancer down-regulated MYC and affected its target transcriptome. Progressive mutagenesis of STITCH led to preferential escalation of the gene-enhancer interaction, corroborating the strong insulation ability of STITCH. The STITCH insertion altered epigenetic states around MYC. Time-course analysis by drug induction uncovered deposition and removal of H3K27me3 repressive marks follows and reflects, but does not precede and determine, the expression change. Thus the tool provided important insights in gene regulation, demonstrating its potency.

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“…Already there are many works published using medaka to analyse the spatio-temporal activity domain of this kind of elements ( 7 – 10 ). Other model organisms serve similar purpose and there are as well online databases making available these data like REDfly ( 11 ) for the fruit fly, Expression disruption screen ( 12 ) or Enhancer screen ( http://www.upo.es/CABD/EnhancerScreen/ ) for zebrafish and the Vista enhancer browser ( 13 ) for mouse. Nonetheless there is not yet any site that integrates both sets of expression data, genes and regulatory elements, using the same vocabulary and ontology for annotation.…”

Section: Introductionmentioning

confidence: 99%

MEPD: medaka expression pattern database, genes and more

et al. 2015

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The Medaka Expression Pattern Database (MEPD; http://mepd.cos.uni-heidelberg.de/) is designed as a repository of medaka expression data for the scientific community. In this update we present two main improvements. First, we have changed the previous clone-centric view for in situ data to a gene-centric view. This is possible because now we have linked all the data present in MEPD to the medaka gene annotation in ENSEMBL. In addition, we have also connected the medaka genes in MEPD to their corresponding orthologous gene in zebrafish, again using the ENSEMBL database. Based on this, we provide a link to the Zebrafish Model Organism Database (ZFIN) to allow researches to compare expression data between these two fish model organisms. As a second major improvement, we have modified the design of the database to enable it to host regulatory elements, promoters or enhancers, expression patterns in addition to gene expression. The combination of gene expression, by traditional in situ, and regulatory element expression, typically by fluorescence reporter gene, within the same platform assures consistency in terms of annotation. In our opinion, this will allow researchers to uncover new insights between the expression domain of genes and their regulatory landscape.

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A mobile insulator system to detect and disrupt cis-regulatory landscapes in vertebrates

Cited by 13 publications

References 53 publications

A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates

A Conserved Notochord Enhancer Controls Pancreas Development in Vertebrates

Controlling gene activation by enhancers through a drug-inducible topological insulator

MEPD: medaka expression pattern database, genes and more

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