Scanning Human Gene Deserts for Long-Range Enhancers

Nóbrega, Marcelo A.; Ovcharenko, Ivan; Afzal, Veena; Rubin, Edward M.

doi:10.1126/science.1088328

Cited by 545 publications

(508 citation statements)

References 6 publications

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“…The Dc2 enhancer was previously identified by comparing the sequences of the orthologous human and Fugu DACH genes [15]. Briefly, a human DNA fragment of 2,086 bp encompassing the sequence conserved in Fugu was tested in transgenic mice and demonstrated to enhance the activity of a minimal heat shock promoter (HSP68) fused to β-galactosidase (LacZ) [25].…”

Section: Comparative Genomics Delineates Conserved Modulesmentioning

confidence: 99%

“…These characteristics led to the original proposal to sequence the Fugu genome in order to assist with the annotation of human genes based on comparative genomics [10]. Furthermore, the availability of Fugu genomic sequence also revealed that human-Fugu conserved noncoding sequences can be used to delineate gene regulatory sequences [1,[11][12][13][14][15].…”

Section: Introductionmentioning

confidence: 99%

“…In vivo analysis of nine DNA elements conserved from human to Fugu demonstrated that seven displayed enhancer activities recapitulating some aspects of DACH expression [15]. The most surprising characteristic of these enhancers is their extremely high degree of conservation between humans and fish.…”

Section: Introductionmentioning

confidence: 99%

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In vivo characterization of a vertebrate ultraconserved enhancer

et al. 2005

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Genomic sequence comparisons between human, mouse and pufferfish (Takifugu rubripes (Fugu)) have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change in order to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2 located near the dachshund gene, which displays a human-Fugu identity of 84% over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence of a large block of sequence (144 bp) that is completely identical between human, mouse, chicken, zebrafish and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424 bp Dc2 conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144 bp 100% conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16 bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144 bp 100% conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence. Thank you for your letter of February 15 th and for the reviewers' comments on our manuscript entitled "In Vivo Characterization of a Vertebrate Ultra-conserved Enhancer", by Poulin et al. We have addressed the reviewers' concerns in a revised manuscript, which we are resubmitting for your consideration.Our detailed response is as follows:Reviewer #21. Abstract -last sentence should read: "tolerant of change at least AT the resolution…"We have changed this typo. 2.Introduction paragraph four (page 4), first sentence. Statement "which is involved in embryonic development" is too vague. Please state, briefly, the function of DACH.We have added an introduction description of DACHs known function and reference to its Drosophila homolog. 3.Introduction paragraph four (page 4). When first introducing Dc2, please define it as a conserved, functional element at DACH (i.e., since the Dc nomenclature was not used previously in the text).We have made this change. 4.Introduction paragraph four (page 4). Please provide a reference for the statement "known degeneracy of the sequences recognized by transcription factors".We have added a reference. 5.Results paragraph one (page 5), first sentence. Please restate the locus (DACH) at which Dc2 resides.We have made this change. 6.Results "Refinement of the Minimal Necessary Dc2 Enhancer". Authors state that the 424 bp fragment drives expression pattern in transgenics that is "indistingu...

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Section: Comparative Genomics Delineates Conserved Modulesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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In vivo characterization of a vertebrate ultraconserved enhancer

et al. 2005

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“…The commonly used transgenesis vectors consist of a shuttle vector, a minimal promoter and an in vivo reporter gene (Nobrega et al, 2003;Pennacchio et al, 2006;Woolfe et al, 2007;Visel et al, 2008). Yet, this simple design faces two major practical problems.…”

Section: Introductionmentioning

confidence: 99%

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

Bessa

Tena

Calle‐Mustienes

et al. 2009

Developmental Dynamics

152

207

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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.

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“…Several testis-specific promoters have also been reported to be sufficient for the appropriate gene activation in transgenic mice (Reddi et al, 2007). However, the full gene activation by its promoter alone seems to be a rare case in mammals, and most genes require other regulatory elements such as an enhancer and an insulator that are often identified in intronic or intergenic regions (Bagheri-Fam et al, 2006;Grosveld et al, 1987;Jeong et al, 2006;Jones et al, 1995;Kim et al, 2007;Nobrega et al, 2003;Shima et al, 2005Shima et al, , 2008. The enhancer is the sequence to which activator proteins bind to increase the rate of transcription (Bulger and Groudine, 2011), and the insulator is a sequence to protect a transgene from position effect (Gaszner and Felsenfeld, 2006;Yang and Corces, 2011).…”

Section: Introductionmentioning

confidence: 99%

A 914-bp promoter is sufficient to reproduce the endogenous prolyl oligopeptidase gene localization in the mouse placenta if not subject to position effect

Matsubara

Maruyama

Kimura

2013

Gene

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Prolyl oligopeptidase (POP) is a widely distributed multifunctional protein which has an endopeptidase activity to cleave a -Pro-X- peptide bond. In spite of numerous studies about POP, the mechanism by which its transcription is controlled has not been well investigated. Here we generated transgenic mice bearing a transgene which contained a 914-bp POP gene promoter linked to the enhanced green fluorescent protein (EGFP) gene to assess the in vivo promoter activity. We established six transgenic lines with different copy numbers, but no EGFP signal was observed in four lines due to a high level of DNA methylation, which suggested that the transgene was subject to position effect. However, in the other two lines, we detected the EGFP expression in many tissues, and its placental localization showed a similar change to POP. A strong EGFP signal was observed in the junctional and labyrinthine zones of E10.5-E12.5 placentas and in the junctional zone and the maternal decidua after that. This placental gene activation might be attributed to AP-2 gamma because we detected its binding to the POP promoter. In contrast, we did not obtain any evidence that EGFP was expressed in a similar pattern compared with POP in the ovary. The current data demonstrated that the 914-bp promoter had sufficient activity to reproduce the POP localization in the placenta if it was not subject to position effect and suggest that the regulatory mechanism of the POP gene expression differs between tissues.(C) 2013 Elsevier B.V. All rights reserved

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Scanning Human Gene Deserts for Long-Range Enhancers

Cited by 545 publications

References 6 publications

In vivo characterization of a vertebrate ultraconserved enhancer

In vivo characterization of a vertebrate ultraconserved enhancer

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

A 914-bp promoter is sufficient to reproduce the endogenous prolyl oligopeptidase gene localization in the mouse placenta if not subject to position effect

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