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Title: Limb-Enhancer Genie: An accessible resource of accurate enhancer predictions in the developing limb

Abstract

Epigenomic mapping of enhancer-associated chromatin modifications facilitates the genome-wide discovery of tissue-specific enhancers in vivo. However, reliance on single chromatin marks leads to high rates of false-positive predictions. More sophisticated, integrative methods have been described, but commonly suffer from limited accessibility to the resulting predictions and reduced biological interpretability. Here we present the Limb-Enhancer Genie (LEG), a collection of highly accurate, genome-wide predictions of enhancers in the developing limb, available through a user-friendly online interface. We predict limb enhancers using a combination of > 50 published limb-specific datasets and clusters of evolutionarily conserved transcription factor binding sites, taking advantage of the patterns observed at previously in vivo validated elements. By combining different statistical models, our approach outperforms current state-of-the-art methods and provides interpretable measures of feature importance. Our results indicate that including a previously unappreciated score that quantifies tissue-specific nuclease accessibility significantly improves prediction performance. We demonstrate the utility of our approach through in vivo validation of newly predicted elements. Moreover, we describe general features that can guide the type of datasets to include when predicting tissue-specific enhancers genome-wide, while providing an accessible resource to the general biological community and facilitating the functional interpretation of genetic studies of limb malformations.

Authors:
 [1]; ORCiD logo [2]; ORCiD logo [2];  [2];  [2];  [2];  [2];  [2];  [2];  [2]; ORCiD logo [3];  [2];  [4];  [1];  [5]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
  2. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  3. ETH Zurich (Switzerland). Dept. of Biosystems Science and Engineering
  4. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); Univ. of California, Merced, CA (United States). School of Natural Sciences
  5. Ottawa Univ., ON (Canada)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1408453
Grant/Contract Number:
AC02-05CH11231
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
PLoS Computational Biology (Online)
Additional Journal Information:
Journal Name: PLoS Computational Biology (Online); Journal Volume: 13; Journal Issue: 8; Journal ID: ISSN 1553-7358
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES

Citation Formats

Monti, Remo, Barozzi, Iros, Osterwalder, Marco, Lee, Elizabeth, Kato, Momoe, Garvin, Tyler H., Plajzer-Frick, Ingrid, Pickle, Catherine S., Akiyama, Jennifer A., Afzal, Veena, Beerenwinkel, Niko, Dickel, Diane E., Visel, Axel, Pennacchio, Len A., and Ioshikhes, Ilya. Limb-Enhancer Genie: An accessible resource of accurate enhancer predictions in the developing limb. United States: N. p., 2017. Web. doi:10.1371/journal.pcbi.1005720.
Monti, Remo, Barozzi, Iros, Osterwalder, Marco, Lee, Elizabeth, Kato, Momoe, Garvin, Tyler H., Plajzer-Frick, Ingrid, Pickle, Catherine S., Akiyama, Jennifer A., Afzal, Veena, Beerenwinkel, Niko, Dickel, Diane E., Visel, Axel, Pennacchio, Len A., & Ioshikhes, Ilya. Limb-Enhancer Genie: An accessible resource of accurate enhancer predictions in the developing limb. United States. doi:10.1371/journal.pcbi.1005720.
Monti, Remo, Barozzi, Iros, Osterwalder, Marco, Lee, Elizabeth, Kato, Momoe, Garvin, Tyler H., Plajzer-Frick, Ingrid, Pickle, Catherine S., Akiyama, Jennifer A., Afzal, Veena, Beerenwinkel, Niko, Dickel, Diane E., Visel, Axel, Pennacchio, Len A., and Ioshikhes, Ilya. 2017. "Limb-Enhancer Genie: An accessible resource of accurate enhancer predictions in the developing limb". United States. doi:10.1371/journal.pcbi.1005720. https://www.osti.gov/servlets/purl/1408453.
@article{osti_1408453,
title = {Limb-Enhancer Genie: An accessible resource of accurate enhancer predictions in the developing limb},
author = {Monti, Remo and Barozzi, Iros and Osterwalder, Marco and Lee, Elizabeth and Kato, Momoe and Garvin, Tyler H. and Plajzer-Frick, Ingrid and Pickle, Catherine S. and Akiyama, Jennifer A. and Afzal, Veena and Beerenwinkel, Niko and Dickel, Diane E. and Visel, Axel and Pennacchio, Len A. and Ioshikhes, Ilya},
abstractNote = {Epigenomic mapping of enhancer-associated chromatin modifications facilitates the genome-wide discovery of tissue-specific enhancers in vivo. However, reliance on single chromatin marks leads to high rates of false-positive predictions. More sophisticated, integrative methods have been described, but commonly suffer from limited accessibility to the resulting predictions and reduced biological interpretability. Here we present the Limb-Enhancer Genie (LEG), a collection of highly accurate, genome-wide predictions of enhancers in the developing limb, available through a user-friendly online interface. We predict limb enhancers using a combination of > 50 published limb-specific datasets and clusters of evolutionarily conserved transcription factor binding sites, taking advantage of the patterns observed at previously in vivo validated elements. By combining different statistical models, our approach outperforms current state-of-the-art methods and provides interpretable measures of feature importance. Our results indicate that including a previously unappreciated score that quantifies tissue-specific nuclease accessibility significantly improves prediction performance. We demonstrate the utility of our approach through in vivo validation of newly predicted elements. Moreover, we describe general features that can guide the type of datasets to include when predicting tissue-specific enhancers genome-wide, while providing an accessible resource to the general biological community and facilitating the functional interpretation of genetic studies of limb malformations.},
doi = {10.1371/journal.pcbi.1005720},
journal = {PLoS Computational Biology (Online)},
number = 8,
volume = 13,
place = {United States},
year = 2017,
month = 8
}

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  • The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. In this paper, we identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologsmore » caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution.« less
  • We have utilized a modification of the limb bud mesenchyme micromass culture system to screen compounds that might stimulate chondrogenesis. Two compounds in the sulfoxide family (methylphenylsulfoxide and p-chlorophenyl methyl sulfoxide) were stimulatory at 10(-2) M and 10(-3) M, respectively; whereas other sulfoxides and organic solvents were not active at these concentrations. In addition, specific growth factors (basic FGF, IGF-I, IGF-II) were not chondroinductive at concentrations that are active in other cell systems. Both sulfoxide compounds stimulated cartilage nodule formation, ({sup 35}S)sulfate incorporation, and activity of the regulatory sequences of the collagen II gene. In contrast, transforming growth factor beta-1more » (10 ng/ml) stimulated sulfate incorporation but produced only a diffuse deposition of cartilage matrix and reduced the ability of the cells to utilize the regulatory sequences of the collagen II gene. The sulfoxides appear to promote the differentiation of limb bud cells to chondrocytes and thus exhibit chondroinductive activity.« less
  • Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the resultsmore » of these and other studies. Results: TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. Conclusions: Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.« less