43 results on '"Akiyama JA"'
Search Results
2. Author Correction: Perspectives on ENCODE (Nature, (2020), 583, 7818, (693-698), 10.1038/s41586-020-2449-8)
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Abascal, FBC, Acosta, R, Addleman, NJ, Adrian, J, Afzal, V, Aken, B, Ai, R, Akiyama, JA, Jammal, OA, Amrhein, H, Anderson, SM, Dileep, V, Ding, B, Djebali, S, Dobin, A, Dominguez, D, Donaldson, S, Drenkow, J, Dreszer, TR, Snyder, MP, Drier, Y, Duff, MO, Dunn, D, Sisu, C, Eastman, C, Ecker, JR, Edwards, MD, El-Ali, N, Andrews, GR, Antoshechkin, I, Ardlie, KG, Armstrong, J, Astley, M, Banerjee, B, Barkal, AA, Barnes, IHA, Barozzi, I, Barrell, D, Barson, G, Bates, D, Baymuradov, UK, Bazile, C, Beer, MA, Beik, S, Bender, MA, Bennett, R, Bouvrette, LPB, Bernstein, BE, Berry, A, Bhaskar, A, Bignell, A, Blue, SM, Bodine, DM, Boix, C, Boley, N, Borrman, T, Borsari, B, Boyle, AP, Brandsmeier, LA, Breschi, A, Bresnick, EH, Brooks, JA, Buckley, M, Burge, CB, Byron, R, Cahill, E, Cai, L, Cao, L, Carty, M, Castanon, RG, Castillo, A, Chaib, H, Chan, ET, Chee, DR, Chee, S, Chen, H, Chen, JY, Chen, S, Cherry, JM, Chhetri, SB, Choudhary, JS, Chrast, J, Chung, D, Clarke, D, Cody, NAL, Coppola, CJ, Coursen, J, D’Ippolito, AM, Dalton, S, Danyko, C, Davidson, C, Davila-Velderrain, J, Davis, CA, Dekker, J, Deran, A, DeSalvo, G, Despacio-Reyes, G, Dewey, CN, Dickel, DE, Diegel, M, Diekhans, M, and The ENCODE Project Consortium
- Abstract
The Original Article (https://doi.org/10.1038/s41586-020-2449-8) was published on 29 July 2020. Copyright © The Authors 2022. In this Article, the authors Rizi Ai (Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA) and Shantao Li (Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA) were mistakenly omitted from the ENCODE Project Consortium author list. The original Article has been corrected online.
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- 2022
3. Author Correction: Expanded encyclopaedias of DNA elements in the human and mouse genomes
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Moore, JE, Abascal, F, Acosta, R, Addleman, NJ, Adrian, J, Afzal, V, Aken, B, Akiyama, JA, Jammal, OA, Amrhein, H, Anderson, SM, Edwards, MD, El-Ali, N, Elhajjajy, SI, Andrews, GR, Antoshechkin, I, Ardlie, KG, Armstrong, J, Astley, M, Banerjee, B, Barkal, AA, Barnes, IHA, Barozzi, I, Barrell, D, Barson, G, Bates, D, Baymuradov, UK, Bazile, C, Beer, MA, Beik, S, Bender, MA, Bennett, R, Bouvrette, LPB, Bernstein, BE, Berry, A, Bhaskar, A, Bignell, A, Blue, SM, Bodine, DM, Boix, C, Boley, N, Borrman, T, Borsari, B, Boyle, AP, Brandsmeier, LA, Breschi, A, Bresnick, EH, Brooks, JA, Buckley, M, Burge, CB, Byron, R, Cahill, E, Cai, L, Cao, L, Carty, M, Castanon, RG, Castillo, A, Chaib, H, Chan, ET, Chee, DR, Chee, S, Chen, H, Chen, JY, Chen, S, Cherry, JM, Chhetri, SB, Choudhary, JS, Chrast, J, Chung, D, Clarke, D, Cody, NAL, Coppola, CJ, Coursen, J, D’Ippolito, AM, Dalton, S, Danyko, C, Davidson, C, Davila-Velderrain, J, Davis, CA, Dekker, J, Deran, A, DeSalvo, G, Despacio-Reyes, G, Dewey, CN, Dickel, DE, Diegel, M, Diekhans, M, Dileep, V, Ding, B, Djebali, S, Dobin, A, Dominguez, D, Donaldson, S, Drenkow, J, Dreszer, TR, Drier, Y, Duff, MO, Dunn, D, Eastman, C, Ecker, JR, and The ENCODE Project Consortium
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Multidisciplinary ,epigenomics ,data integration ,functional genomics - Abstract
Online Correction for: https://doi.org/10.1038/s41586-020-2493-4 | Erratum for https://bura.brunel.ac.uk/handle/2438/21299 In the version of this article initially published, two members of the ENCODE Project Consortium were missing from the author list. Rizi Ai (Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA) and Shantao Li (Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT, USA) are now included in the author list. These errors have been corrected in the online version of the article : 'Expanded encyclopaedias of DNA elements in the human and mouse genomes'. https://www.nature.com/articles/s41586-021-04226-3 https://www.nature.com/articles/s41586-021-04226-3
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- 2022
4. Perspectives on ENCODE
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Snyder, MP, Gingeras, TR, Abascal, F, Acosta, R, Addleman, NJ, Adrian, J, Afzal, V, Aken, B, Akiyama, JA, Jammal, OA, Amrhein, H, Dileep, V, Ding, B, Djebali, S, Dobin, A, Dominguez, D, Sisu, C, Donaldson, S, Drenkow, J, Dreszer, TR, Drier, Y, Duff, MO, Dunn, D, Anderson, SM, Andrews, GR, Eastman, C, Ecker, JR, Edwards, MD, El-Ali, N, Elhajjajy, SI, Antoshechkin, I, Ardlie, KG, Armstrong, J, Astley, M, Banerjee, B, Barkal, AA, Barnes, IHA, Barozzi, I, Barrell, D, Barson, G, Bates, D, Baymuradov, UK, Bazile, C, Beer, MA, Beik, S, Bender, MA, Bennett, R, Bouvrette, LPB, Bernstein, BE, Berry, A, Bhaskar, A, Bignell, A, Blue, SM, Bodine, DM, Boix, C, Boley, N, Borrman, T, Borsari, B, Boyle, AP, Brandsmeier, LA, Breschi, A, Bresnick, EH, Brooks, JA, Buckley, M, Burge, CB, Byron, R, Cahill, E, Cai, L, Cao, L, Carty, M, Castanon, RG, Castillo, A, Chaib, H, Chan, ET, Chee, DR, Chee, S, Chen, H, Chen, JY, Chen, S, Cherry, JM, Chhetri, SB, Choudhary, JS, Chrast, J, Chung, D, Clarke, D, Cody, NAL, Coppola, CJ, Coursen, J, D’Ippolito, AM, Dalton, S, Danyko, C, Davidson, C, Davila-Velderrain, J, Davis, CA, Dekker, J, Deran, A, DeSalvo, G, Despacio-Reyes, G, Dewey, CN, Dickel, DE, Diegel, M, Diekhans, M, and The ENCODE Project Consortium
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Epigenomics ,Quality Control ,610 Medicine & health ,Computational biology ,Biology ,Regulatory Sequences, Nucleic Acid ,ENCODE ,Genome ,Histones ,03 medical and health sciences ,transcriptomics ,Mice ,0302 clinical medicine ,Databases, Genetic ,Animals ,Humans ,Transcriptomics ,Gene ,genome ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,Multidisciplinary ,Binding Sites ,Genome, Human ,Molecular Sequence Annotation ,Genomics ,DNA Methylation ,Chromatin ,DNA binding site ,Gene Expression Regulation ,030220 oncology & carcinogenesis ,Perspective ,epigenomics ,Human genome ,Epigenetics ,epigenetic ,Transcription Factors - Abstract
The Encylopedia of DNA Elements (ENCODE) Project launched in 2003 with the long-term goal of developing a comprehensive map of functional elements in the human genome. These included genes, biochemical regions associated with gene regulation (for example, transcription factor binding sites, open chromatin, and histone marks) and transcript isoforms. The marks serve as sites for candidate cis-regulatory elements (cCREs) that may serve functional roles in regulating gene expression1. The project has been extended to model organisms, particularly the mouse. In the third phase of ENCODE, nearly a million and more than 300,000 cCRE annotations have been generated for human and mouse, respectively, and these have provided a valuable resource for the scientific community., The authors summarize the history of the ENCODE Project, the achievements of ENCODE 1 and ENCODE 2, and how the new data generated and analysed in ENCODE 3 complement the previous phases.
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- 2019
5. A distal 594 bp ECR specifies Hmx1 expression in pinna and lateral facial morphogenesis and is regulated by the Hox-Pbx-Meis complex
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Rosin, JM, Li, W, Cox, LL, Rolfe, SM, Latorre, V, Akiyama, JA, Visel, A, Kuramoto, T, Bobola, N, Turner, EE, and Cox, TC
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© 2016. Published by The Company of Biologists Ltd. Hmx1 encodes a homeodomain transcription factor expressed in the developing lateral craniofacial mesenchyme, retina and sensory ganglia. Mutation or mis-regulation of Hmx1 underlies malformations of the eye and external ear in multiple species. Deletion or insertional duplication of an evolutionarily conserved region (ECR) downstream of Hmx1 has recently been described in rat and cow, respectively. Here, we demonstrate that the impact of Hmx1 loss is greater than previously appreciated, with a variety of lateral cranioskeletal defects, auriculofacial nerve deficits, and duplication of the caudal region of the external ear. Using a transgenic approach, we demonstrate that a 594 bp sequence encompassing the ECR recapitulates specific aspects of the endogenous Hmx1 lateral facial expression pattern. Moreover, we show that Hoxa2, Meis and Pbx proteins act cooperatively on the ECR, via a core 32 bp sequence, to regulate Hmx1 expression. These studies highlight the conserved role for Hmx1 in BA2-derived tissues and provide an entry point for improved understanding of the causes of the frequent lateral facial birth defects in humans.
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- 2016
6. A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival.
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Abassah-Oppong S, Zoia M, Mannion BJ, Rouco R, Tissières V, Spurrell CH, Roland V, Darbellay F, Itum A, Gamart J, Festa-Daroux TA, Sullivan CS, Kosicki M, Rodríguez-Carballo E, Fukuda-Yuzawa Y, Hunter RD, Novak CS, Plajzer-Frick I, Tran S, Akiyama JA, Dickel DE, Lopez-Rios J, Barozzi I, Andrey G, Visel A, Pennacchio LA, Cobb J, and Osterwalder M
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- Animals, Humans, Mice, Morphogenesis genetics, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Gene Expression Regulation, Developmental, Enhancer Elements, Genetic
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Approximately a quarter of the human genome consists of gene deserts, large regions devoid of genes often located adjacent to developmental genes and thought to contribute to their regulation. However, defining the regulatory functions embedded within these deserts is challenging due to their large size. Here, we explore the cis-regulatory architecture of a gene desert flanking the Shox2 gene, which encodes a transcription factor indispensable for proximal limb, craniofacial, and cardiac pacemaker development. We identify the gene desert as a regulatory hub containing more than 15 distinct enhancers recapitulating anatomical subdomains of Shox2 expression. Ablation of the gene desert leads to embryonic lethality due to Shox2 depletion in the cardiac sinus venosus, caused in part by the loss of a specific distal enhancer. The gene desert is also required for stylopod morphogenesis, mediated via distributed proximal limb enhancers. In summary, our study establishes a multi-layered role of the Shox2 gene desert in orchestrating pleiotropic developmental expression through modular arrangement and coordinated dynamics of tissue-specific enhancers., (© 2024. The Author(s).)
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- 2024
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7. Mutagenesis Sensitivity Mapping of Human Enhancers In Vivo .
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Kosicki M, Zhang B, Pampari A, Akiyama JA, Plajzer-Frick I, Novak CS, Tran S, Zhu Y, Kato M, Hunter RD, von Maydell K, Barton S, Beckman E, Kundaje A, Dickel DE, Visel A, and Pennacchio LA
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Distant-acting enhancers are central to human development. However, our limited understanding of their functional sequence features prevents the interpretation of enhancer mutations in disease. Here, we determined the functional sensitivity to mutagenesis of human developmental enhancers in vivo . Focusing on seven enhancers active in the developing brain, heart, limb and face, we created over 1700 transgenic mice for over 260 mutagenized enhancer alleles. Systematic mutation of 12-basepair blocks collectively altered each sequence feature in each enhancer at least once. We show that 69% of all blocks are required for normal in vivo activity, with mutations more commonly resulting in loss (60%) than in gain (9%) of function. Using predictive modeling, we annotated critical nucleotides at base-pair resolution. The vast majority of motifs predicted by these machine learning models (88%) coincided with changes to in vivo function, and the models showed considerable sensitivity, identifying 59% of all functional blocks. Taken together, our results reveal that human enhancers contain a high density of sequence features required for their normal in vivo function and provide a rich resource for further exploration of human enhancer logic., Competing Interests: Conflicts of Interest A.K. is on the scientific advisory board of SerImmune, AINovo, TensorBio and OpenTargets. A.K. was a scientific co-founder of RavelBio, a paid consultant with Illumina, was on the SAB of PatchBio and owns shares in DeepGenomics, Immunai, Freenome, and Illumina.
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- 2024
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8. Massively parallel reporter assays and mouse transgenic assays provide complementary information about neuronal enhancer activity.
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Kosicki M, Cintrón DL, Page NF, Georgakopoulos-Soares I, Akiyama JA, Plajzer-Frick I, Novak CS, Kato M, Hunter RD, von Maydell K, Barton S, Godfrey P, Beckman E, Sanders SJ, Pennacchio LA, and Ahituv N
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Genetic studies find hundreds of thousands of noncoding variants associated with psychiatric disorders. Massively parallel reporter assays (MPRAs) and in vivo transgenic mouse assays can be used to assay the impact of these variants. However, the relevance of MPRAs to in vivo function is unknown and transgenic assays suffer from low throughput. Here, we studied the utility of combining the two assays to study the impact of non-coding variants. We carried out an MPRA on over 50,000 sequences derived from enhancers validated in transgenic mouse assays and from multiple fetal neuronal ATAC-seq datasets. We also tested over 20,000 variants, including synthetic mutations in highly active neuronal enhancers and 177 common variants associated with psychiatric disorders. Variants with a high impact on MPRA activity were further tested in mice. We found a strong and specific correlation between MPRA and mouse neuronal enhancer activity including changes in neuronal enhancer activity in mouse embryos for variants with strong MPRA effects. Mouse assays also revealed pleiotropic variant effects that could not be observed in MPRA. Our work provides a large catalog of functional neuronal enhancers and variant effects and highlights the effectiveness of combining MPRAs and mouse transgenic assays., Competing Interests: Competing interests N.A. is a cofounder and on the scientific advisory board of Regel Therapeutics. N.A. receives funding from BioMarin Pharmaceutical Incorporate.
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- 2024
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9. Dynamic enhancer landscapes in human craniofacial development.
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Rajderkar SS, Paraiso K, Amaral ML, Kosicki M, Cook LE, Darbellay F, Spurrell CH, Osterwalder M, Zhu Y, Wu H, Afzal SY, Blow MJ, Kelman G, Barozzi I, Fukuda-Yuzawa Y, Akiyama JA, Afzal V, Tran S, Plajzer-Frick I, Novak CS, Kato M, Hunter RD, von Maydell K, Wang A, Lin L, Preissl S, Lisgo S, Ren B, Dickel DE, Pennacchio LA, and Visel A
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- Humans, Animals, Mice, Gene Expression Profiling, Genomics, Protein Processing, Post-Translational, Regulatory Sequences, Nucleic Acid, Chromatin genetics
- Abstract
The genetic basis of human facial variation and craniofacial birth defects remains poorly understood. Distant-acting transcriptional enhancers control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development. However, a lack of accurate maps of the genomic locations and cell type-resolved activities of craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combine histone modification, chromatin accessibility, and gene expression profiling of human craniofacial development with single-cell analyses of the developing mouse face to define the regulatory landscape of facial development at tissue- and single cell-resolution. We provide temporal activity profiles for 14,000 human developmental craniofacial enhancers. We find that 56% of human craniofacial enhancers share chromatin accessibility in the mouse and we provide cell population- and embryonic stage-resolved predictions of their in vivo activity. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development., (© 2024. The Author(s).)
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- 2024
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10. Combinatorial transcription factor binding encodes cis-regulatory wiring of forebrain GABAergic neurogenesis.
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Catta-Preta R, Lindtner S, Ypsilanti A, Price J, Abnousi A, Su-Feher L, Wang Y, Juric I, Jones IR, Akiyama JA, Hu M, Shen Y, Visel A, Pennacchio LA, Dickel D, Rubenstein JLR, and Nord AS
- Abstract
Transcription factors (TFs) bind combinatorially to genomic cis-regulatory elements (cREs), orchestrating transcription programs. While studies of chromatin state and chromosomal interactions have revealed dynamic neurodevelopmental cRE landscapes, parallel understanding of the underlying TF binding lags. To elucidate the combinatorial TF-cRE interactions driving mouse basal ganglia development, we integrated ChIP-seq for twelve TFs, H3K4me3-associated enhancer-promoter interactions, chromatin and transcriptional state, and transgenic enhancer assays. We identified TF-cREs modules with distinct chromatin features and enhancer activity that have complementary roles driving GABAergic neurogenesis and suppressing other developmental fates. While the majority of distal cREs were bound by one or two TFs, a small proportion were extensively bound, and these enhancers also exhibited exceptional evolutionary conservation, motif density, and complex chromosomal interactions. Our results provide new insights into how modules of combinatorial TF-cRE interactions activate and repress developmental expression programs and demonstrate the value of TF binding data in modeling gene regulatory wiring., Competing Interests: STATEMENT OF COMPETING INTERESTS J.L.R.R. is co-founder, stockholder, and currently on the scientific board of Neurona, a company studying the potential therapeutic use of interneuron transplantation. The other authors declare no competing interests.
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- 2023
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11. Cell Type- and Tissue-specific Enhancers in Craniofacial Development.
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Rajderkar SS, Paraiso K, Amaral ML, Kosicki M, Cook LE, Darbellay F, Spurrell CH, Osterwalder M, Zhu Y, Wu H, Afzal SY, Blow MJ, Kelman G, Barozzi I, Fukuda-Yuzawa Y, Akiyama JA, Afzal V, Tran S, Plajzer-Frick I, Novak CS, Kato M, Hunter RD, von Maydell K, Wang A, Lin L, Preissl S, Lisgo S, Ren B, Dickel DE, Pennacchio LA, and Visel A
- Abstract
The genetic basis of craniofacial birth defects and general variation in human facial shape remains poorly understood. Distant-acting transcriptional enhancers are a major category of non-coding genome function and have been shown to control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development
1-3 . However, a lack of accurate maps of the genomic location and cell type-specific in vivo activities of all craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combined histone modification and chromatin accessibility profiling from different stages of human craniofacial development with single-cell analyses of the developing mouse face to create a comprehensive catalogue of the regulatory landscape of facial development at tissue- and single cell-resolution. In total, we identified approximately 14,000 enhancers across seven developmental stages from weeks 4 through 8 of human embryonic face development. We used transgenic mouse reporter assays to determine the in vivo activity patterns of human face enhancers predicted from these data. Across 16 in vivo validated human enhancers, we observed a rich diversity of craniofacial subregions in which these enhancers are active in vivo . To annotate the cell type specificities of human-mouse conserved enhancers, we performed single-cell RNA-seq and single-nucleus ATAC-seq of mouse craniofacial tissues from embryonic days e11.5 to e15.5. By integrating these data across species, we find that the majority (56%) of human craniofacial enhancers are functionally conserved in mice, providing cell type- and embryonic stage-resolved predictions of their in vivo activity profiles. Using retrospective analysis of known craniofacial enhancers in combination with single cell-resolved transgenic reporter assays, we demonstrate the utility of these data for predicting the in vivo cell type specificity of enhancers. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development., Competing Interests: Declaration of Interests Bing Ren is a co-founder of Arima Genomics, Inc, and Epigenome Technologies, Inc.- Published
- 2023
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12. Topologically associating domain boundaries are required for normal genome function.
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Rajderkar S, Barozzi I, Zhu Y, Hu R, Zhang Y, Li B, Alcaina Caro A, Fukuda-Yuzawa Y, Kelman G, Akeza A, Blow MJ, Pham Q, Harrington AN, Godoy J, Meky EM, von Maydell K, Hunter RD, Akiyama JA, Novak CS, Plajzer-Frick I, Afzal V, Tran S, Lopez-Rios J, Talkowski ME, Lloyd KCK, Ren B, Dickel DE, Visel A, and Pennacchio LA
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- Animals, Mice, Phenotype, Chromatin genetics, Genome
- Abstract
Topologically associating domain (TAD) boundaries partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotypes
1-3 , but the overall extent to which this occurs remains unknown. Here we demonstrate that targeted deletions of TAD boundaries cause a range of disruptions to normal in vivo genome function and organismal development. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11-80 kb in size) from the genome. All deletions examined resulted in detectable molecular or organismal phenotypes, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. We observed changes in local 3D chromatin architecture in 7 of 8 (88%) cases, including the merging of TADs and altered contact frequencies within TADs adjacent to the deleted boundary. For 5 of 8 (63%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and reinforce the critical need to carefully consider the potential pathogenicity of noncoding deletions affecting TAD boundaries in clinical genetics screening., (© 2023. The Author(s).)- Published
- 2023
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13. Genome-wide fetalization of enhancer architecture in heart disease.
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Spurrell CH, Barozzi I, Kosicki M, Mannion BJ, Blow MJ, Fukuda-Yuzawa Y, Slaven N, Afzal SY, Akiyama JA, Afzal V, Tran S, Plajzer-Frick I, Novak CS, Kato M, Lee EA, Garvin TH, Pham QT, Kronshage AN, Lisgo S, Bristow J, Cappola TP, Morley MP, Margulies KB, Pennacchio LA, Dickel DE, and Visel A
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- Adult, Epigenome, Epigenomics, Humans, Transcription Factors, Cardiomyopathy, Dilated, Enhancer Elements, Genetic genetics
- Abstract
Heart disease is associated with re-expression of key transcription factors normally active only during prenatal development of the heart. However, the impact of this reactivation on the regulatory landscape in heart disease is unclear. Here, we use RNA-seq and ChIP-seq targeting a histone modification associated with active transcriptional enhancers to generate genome-wide enhancer maps from left ventricle tissue from up to 26 healthy controls, 18 individuals with idiopathic dilated cardiomyopathy (DCM), and five fetal hearts. Healthy individuals have a highly reproducible epigenomic landscape, consisting of more than 33,000 predicted heart enhancers. In contrast, we observe reproducible disease-associated changes in activity at 6,850 predicted heart enhancers. Combined analysis of adult and fetal samples reveals that the heart disease epigenome and transcriptome both acquire fetal-like characteristics, with 3,400 individual enhancers sharing fetal regulatory properties. We also provide a comprehensive data resource (http://heart.lbl.gov) for the mechanistic exploration of DCM etiology., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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14. Characterization of Mammalian In Vivo Enhancers Using Mouse Transgenesis and CRISPR Genome Editing.
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Osterwalder M, Tran S, Hunter RD, Meky EM, von Maydell K, Harrington AN, Godoy J, Novak CS, Plajzer-Frick I, Zhu Y, Akiyama JA, Afzal V, Kvon EZ, Pennacchio LA, Dickel DE, and Visel A
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- Animals, Enhancer Elements, Genetic, Gene Transfer Techniques, Genomics, Mice, Clustered Regularly Interspaced Short Palindromic Repeats, Gene Editing
- Abstract
Embryonic morphogenesis is strictly dependent on tight spatiotemporal control of developmental gene expression, which is typically achieved through the concerted activity of multiple enhancers driving cell type-specific expression of a target gene. Mammalian genomes are organized in topologically associated domains, providing a preferred environment and framework for interactions between transcriptional enhancers and gene promoters. While epigenomic profiling and three-dimensional chromatin conformation capture have significantly increased the accuracy of identifying enhancers, assessment of subregional enhancer activities via transgenic reporter assays in mice remains the gold standard for assigning enhancer activity in vivo. Once this activity is defined, the ideal method to explore the functional necessity of a transcriptional enhancer and its contribution to target gene dosage and morphological or physiological processes is deletion of the enhancer sequence from the mouse genome. Here we present detailed protocols for efficient introduction of enhancer-reporter transgenes and CRISPR-mediated genomic deletions into the mouse genome, including a step-by-step guide for pronuclear microinjection of fertilized mouse eggs. We provide instructions for the assembly and genomic integration of enhancer-reporter cassettes that have been used for validation of thousands of putative enhancer sequences accessible through the VISTA enhancer browser, including a recently published method for robust site-directed transgenesis at the H11 safe-harbor locus. Together, these methods enable rapid and large-scale assessment of enhancer activities and sequence variants in mice, which is essential to understand mammalian genome function and genetic diseases., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
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- 2022
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15. Ultraconserved enhancer function does not require perfect sequence conservation.
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Snetkova V, Ypsilanti AR, Akiyama JA, Mannion BJ, Plajzer-Frick I, Novak CS, Harrington AN, Pham QT, Kato M, Zhu Y, Godoy J, Meky E, Hunter RD, Shi M, Kvon EZ, Afzal V, Tran S, Rubenstein JLR, Visel A, Pennacchio LA, and Dickel DE
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- Alleles, Animals, Base Sequence, Conserved Sequence, Embryo, Mammalian, Humans, Mice, Mutagenesis, Site-Directed, Rats, Transcription Factors metabolism, Enhancer Elements, Genetic, Gene Expression Regulation, Developmental, Mutation, Transcription Factors genetics
- Abstract
Ultraconserved enhancer sequences show perfect conservation between human and rodent genomes, suggesting that their functions are highly sensitive to mutation. However, current models of enhancer function do not sufficiently explain this extreme evolutionary constraint. We subjected 23 ultraconserved enhancers to different levels of mutagenesis, collectively introducing 1,547 mutations, and examined their activities in transgenic mouse reporter assays. Overall, we find that the regulatory properties of ultraconserved enhancers are robust to mutation. Upon mutagenesis, nearly all (19/23, 83%) still functioned as enhancers at one developmental stage, as did most of those tested again later in development (5/9, 56%). Replacement of endogenous enhancers with mutated alleles in mice corroborated results of transgenic assays, including the functional resilience of ultraconserved enhancers to mutation. Our findings show that the currently known activities of ultraconserved enhancers do not necessarily require the perfect conservation observed in evolution and suggest that additional regulatory or other functions contribute to their sequence constraint.
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- 2021
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16. Author Correction: An atlas of dynamic chromatin landscapes in mouse fetal development.
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Gorkin DU, Barozzi I, Zhao Y, Zhang Y, Huang H, Lee AY, Li B, Chiou J, Wildberg A, Ding B, Zhang B, Wang M, Strattan JS, Davidson JM, Qiu Y, Afzal V, Akiyama JA, Plajzer-Frick I, Novak CS, Kato M, Garvin TH, Pham QT, Harrington AN, Mannion BJ, Lee EA, Fukuda-Yuzawa Y, He Y, Preissl S, Chee S, Han JY, Williams BA, Trout D, Amrhein H, Yang H, Cherry JM, Wang W, Gaulton K, Ecker JR, Shen Y, Dickel DE, Visel A, Pennacchio LA, and Ren B
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- 2021
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17. Author Correction: An atlas of dynamic chromatin landscapes in mouse fetal development.
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Gorkin DU, Barozzi I, Zhao Y, Zhang Y, Huang H, Lee AY, Li B, Chiou J, Wildberg A, Ding B, Zhang B, Wang M, Strattan JS, Davidson JM, Qiu Y, Afzal V, Akiyama JA, Plajzer-Frick I, Novak CS, Kato M, Garvin TH, Pham QT, Harrington AN, Mannion BJ, Lee EA, Fukuda-Yuzawa Y, He Y, Preissl S, Chee S, Han JY, Williams BA, Trout D, Amrhein H, Yang H, Cherry JM, Wang W, Gaulton K, Ecker JR, Shen Y, Dickel DE, Visel A, Pennacchio LA, and Ren B
- Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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- 2020
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18. Supervised enhancer prediction with epigenetic pattern recognition and targeted validation.
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Sethi A, Gu M, Gumusgoz E, Chan L, Yan KK, Rozowsky J, Barozzi I, Afzal V, Akiyama JA, Plajzer-Frick I, Yan C, Novak CS, Kato M, Garvin TH, Pham Q, Harrington A, Mannion BJ, Lee EA, Fukuda-Yuzawa Y, Visel A, Dickel DE, Yip KY, Sutton R, Pennacchio LA, and Gerstein M
- Subjects
- Animals, Cell Line, Drosophila, Histones genetics, Histones metabolism, Humans, Mice, Mice, Transgenic, Reproducibility of Results, Epigenesis, Genetic physiology, Pattern Recognition, Automated methods
- Abstract
Enhancers are important non-coding elements, but they have traditionally been hard to characterize experimentally. The development of massively parallel assays allows the characterization of large numbers of enhancers for the first time. Here, we developed a framework using Drosophila STARR-seq to create shape-matching filters based on meta-profiles of epigenetic features. We integrated these features with supervised machine-learning algorithms to predict enhancers. We further demonstrated that our model could be transferred to predict enhancers in mammals. We comprehensively validated the predictions using a combination of in vivo and in vitro approaches, involving transgenic assays in mice and transduction-based reporter assays in human cell lines (153 enhancers in total). The results confirmed that our model can accurately predict enhancers in different species without re-parameterization. Finally, we examined the transcription factor binding patterns at predicted enhancers versus promoters. We demonstrated that these patterns enable the construction of a secondary model that effectively distinguishes enhancers and promoters.
- Published
- 2020
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19. An atlas of dynamic chromatin landscapes in mouse fetal development.
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Gorkin DU, Barozzi I, Zhao Y, Zhang Y, Huang H, Lee AY, Li B, Chiou J, Wildberg A, Ding B, Zhang B, Wang M, Strattan JS, Davidson JM, Qiu Y, Afzal V, Akiyama JA, Plajzer-Frick I, Novak CS, Kato M, Garvin TH, Pham QT, Harrington AN, Mannion BJ, Lee EA, Fukuda-Yuzawa Y, He Y, Preissl S, Chee S, Han JY, Williams BA, Trout D, Amrhein H, Yang H, Cherry JM, Wang W, Gaulton K, Ecker JR, Shen Y, Dickel DE, Visel A, Pennacchio LA, and Ren B
- Subjects
- Animals, Chromatin chemistry, Chromatin Immunoprecipitation Sequencing, Disease genetics, Enhancer Elements, Genetic genetics, Female, Gene Expression Regulation, Developmental genetics, Genetic Variation, Histones chemistry, Humans, Male, Mice, Mice, Inbred C57BL, Organ Specificity genetics, Reproducibility of Results, Transposases metabolism, Chromatin genetics, Chromatin metabolism, Datasets as Topic, Fetal Development genetics, Histones metabolism, Molecular Sequence Annotation, Regulatory Sequences, Nucleic Acid genetics
- Abstract
The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP-seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC-seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.
- Published
- 2020
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20. Comprehensive In Vivo Interrogation Reveals Phenotypic Impact of Human Enhancer Variants.
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Kvon EZ, Zhu Y, Kelman G, Novak CS, Plajzer-Frick I, Kato M, Garvin TH, Pham Q, Harrington AN, Hunter RD, Godoy J, Meky EM, Akiyama JA, Afzal V, Tran S, Escande F, Gilbert-Dussardier B, Jean-Marçais N, Hudaiberdiev S, Ovcharenko I, Dobbs MB, Gurnett CA, Manouvrier-Hanu S, Petit F, Visel A, Dickel DE, and Pennacchio LA
- Subjects
- Animals, Enhancer Elements, Genetic physiology, Gene Expression Regulation, Developmental genetics, Gene Knock-In Techniques methods, Hedgehog Proteins genetics, Hedgehog Proteins metabolism, Humans, Mice, Mutation, Phenotype, Polydactyly metabolism, RNA, Untranslated genetics, Enhancer Elements, Genetic genetics, High-Throughput Screening Assays methods, Polydactyly genetics
- Abstract
Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)
- Published
- 2020
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21. Parkinson-Associated SNCA Enhancer Variants Revealed by Open Chromatin in Mouse Dopamine Neurons.
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McClymont SA, Hook PW, Soto AI, Reed X, Law WD, Kerans SJ, Waite EL, Briceno NJ, Thole JF, Heckman MG, Diehl NN, Wszolek ZK, Moore CD, Zhu H, Akiyama JA, Dickel DE, Visel A, Pennacchio LA, Ross OA, Beer MA, and McCallion AS
- Subjects
- Adult, Aged, Aged, 80 and over, Alleles, Animals, Disease Models, Animal, Female, Genotype, Humans, Introns genetics, Male, Mice, Mice, Transgenic, Middle Aged, Pregnancy, Zebrafish, Chromatin genetics, Dopaminergic Neurons pathology, Enhancer Elements, Genetic genetics, Genetic Predisposition to Disease genetics, Parkinson Disease genetics, alpha-Synuclein genetics
- Abstract
The progressive loss of midbrain (MB) dopaminergic (DA) neurons defines the motor features of Parkinson disease (PD), and modulation of risk by common variants in PD has been well established through genome-wide association studies (GWASs). We acquired open chromatin signatures of purified embryonic mouse MB DA neurons because we anticipated that a fraction of PD-associated genetic variation might mediate the variants' effects within this neuronal population. Correlation with >2,300 putative enhancers assayed in mice revealed enrichment for MB cis-regulatory elements (CREs), and these data were reinforced by transgenic analyses of six additional sequences in zebrafish and mice. One CRE, within intron 4 of the familial PD gene SNCA, directed reporter expression in catecholaminergic neurons from transgenic mice and zebrafish. Sequencing of this CRE in 986 individuals with PD and 992 controls revealed two common variants associated with elevated PD risk. To assess potential mechanisms of action, we screened >16,000 proteins for DNA binding capacity and identified a subset whose binding is impacted by these enhancer variants. Additional genotyping across the SNCA locus identified a single PD-associated haplotype, containing the minor alleles of both of the aforementioned PD-risk variants. Our work posits a model for how common variation at SNCA might modulate PD risk and highlights the value of cell-context-dependent guided searches for functional non-coding variation., (Copyright © 2018 American Society of Human Genetics. All rights reserved.)
- Published
- 2018
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22. Enhancer redundancy provides phenotypic robustness in mammalian development.
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Osterwalder M, Barozzi I, Tissières V, Fukuda-Yuzawa Y, Mannion BJ, Afzal SY, Lee EA, Zhu Y, Plajzer-Frick I, Pickle CS, Kato M, Garvin TH, Pham QT, Harrington AN, Akiyama JA, Afzal V, Lopez-Rios J, Dickel DE, Visel A, and Pennacchio LA
- Subjects
- Animals, Brain embryology, Female, Genome, Heart embryology, Limb Deformities, Congenital embryology, Limb Deformities, Congenital genetics, Male, Mice, Sequence Deletion, Spatio-Temporal Analysis, Enhancer Elements, Genetic genetics, Extremities embryology, Gene Expression Regulation, Developmental genetics, Phenotype
- Abstract
Distant-acting tissue-specific enhancers, which regulate gene expression, vastly outnumber protein-coding genes in mammalian genomes, but the functional importance of this regulatory complexity remains unclear. Here we show that the pervasive presence of multiple enhancers with similar activities near the same gene confers phenotypic robustness to loss-of-function mutations in individual enhancers. We used genome editing to create 23 mouse deletion lines and inter-crosses, including both single and combinatorial enhancer deletions at seven distinct loci required for limb development. Unexpectedly, none of the ten deletions of individual enhancers caused noticeable changes in limb morphology. By contrast, the removal of pairs of limb enhancers near the same gene resulted in discernible phenotypes, indicating that enhancers function redundantly in establishing normal morphology. In a genetic background sensitized by reduced baseline expression of the target gene, even single enhancer deletions caused limb abnormalities, suggesting that functional redundancy is conferred by additive effects of enhancers on gene expression levels. A genome-wide analysis integrating epigenomic and transcriptomic data from 29 developmental mouse tissues revealed that mammalian genes are very commonly associated with multiple enhancers that have similar spatiotemporal activity. Systematic exploration of three representative developmental structures (limb, brain and heart) uncovered more than one thousand cases in which five or more enhancers with redundant activity patterns were found near the same gene. Together, our data indicate that enhancer redundancy is a remarkably widespread feature of mammalian genomes that provides an effective regulatory buffer to prevent deleterious phenotypic consequences upon the loss of individual enhancers.
- Published
- 2018
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23. Ultraconserved Enhancers Are Required for Normal Development.
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Dickel DE, Ypsilanti AR, Pla R, Zhu Y, Barozzi I, Mannion BJ, Khin YS, Fukuda-Yuzawa Y, Plajzer-Frick I, Pickle CS, Lee EA, Harrington AN, Pham QT, Garvin TH, Kato M, Osterwalder M, Akiyama JA, Afzal V, Rubenstein JLR, Pennacchio LA, and Visel A
- Subjects
- Animals, Brain abnormalities, Brain embryology, Brain metabolism, Female, Gene Deletion, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Male, Mice, Transcription Factors genetics, Transcription Factors metabolism, Conserved Sequence, Embryonic Development genetics, Enhancer Elements, Genetic
- Abstract
Non-coding "ultraconserved" regions containing hundreds of consecutive bases of perfect sequence conservation across mammalian genomes can function as distant-acting enhancers. However, initial deletion studies in mice revealed that loss of such extraordinarily constrained sequences had no immediate impact on viability. Here, we show that ultraconserved enhancers are required for normal development. Focusing on some of the longest ultraconserved sites genome wide, located near the essential neuronal transcription factor Arx, we used genome editing to create an expanded series of knockout mice lacking individual or combinations of ultraconserved enhancers. Mice with single or pairwise deletions of ultraconserved enhancers were viable and fertile but in nearly all cases showed neurological or growth abnormalities, including substantial alterations of neuron populations and structural brain defects. Our results demonstrate the functional importance of ultraconserved enhancers and indicate that remarkably strong sequence conservation likely results from fitness deficits that appear subtle in a laboratory setting., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2018
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24. Limb-Enhancer Genie: An accessible resource of accurate enhancer predictions in the developing limb.
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Monti R, Barozzi I, Osterwalder M, Lee E, Kato M, Garvin TH, Plajzer-Frick I, Pickle CS, Akiyama JA, Afzal V, Beerenwinkel N, Dickel DE, Visel A, and Pennacchio LA
- Subjects
- Animals, Genome genetics, Machine Learning, Mice, Enhancer Elements, Genetic genetics, Extremities growth & development, Genomics methods, Growth and Development genetics, Software
- 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.
- Published
- 2017
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25. HAND2 Target Gene Regulatory Networks Control Atrioventricular Canal and Cardiac Valve Development.
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Laurent F, Girdziusaite A, Gamart J, Barozzi I, Osterwalder M, Akiyama JA, Lincoln J, Lopez-Rios J, Visel A, Zuniga A, and Zeller R
- Subjects
- Animals, Base Sequence, Cell Movement genetics, Chromatin metabolism, Epithelial-Mesenchymal Transition genetics, Gene Expression Regulation, Developmental, Genome, Mesoderm cytology, Mesoderm metabolism, Mice, Snail Family Transcription Factors genetics, Snail Family Transcription Factors metabolism, Transcription, Genetic, Basic Helix-Loop-Helix Transcription Factors metabolism, Endocardial Cushions embryology, Endocardial Cushions metabolism, Gene Regulatory Networks, Heart Valves embryology, Heart Valves metabolism
- Abstract
The HAND2 transcriptional regulator controls cardiac development, and we uncover additional essential functions in the endothelial to mesenchymal transition (EMT) underlying cardiac cushion development in the atrioventricular canal (AVC). In Hand2-deficient mouse embryos, the EMT underlying AVC cardiac cushion formation is disrupted, and we combined ChIP-seq of embryonic hearts with transcriptome analysis of wild-type and mutants AVCs to identify the functionally relevant HAND2 target genes. The HAND2 target gene regulatory network (GRN) includes most genes with known functions in EMT processes and AVC cardiac cushion formation. One of these is Snai1, an EMT master regulator whose expression is lost from Hand2-deficient AVCs. Re-expression of Snai1 in mutant AVC explants partially restores this EMT and mesenchymal cell migration. Furthermore, the HAND2-interacting enhancers in the Snai1 genomic landscape are active in embryonic hearts and other Snai1-expressing tissues. These results show that HAND2 directly regulates the molecular cascades initiating AVC cardiac valve development., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
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26. Progressive Loss of Function in a Limb Enhancer during Snake Evolution.
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Kvon EZ, Kamneva OK, Melo US, Barozzi I, Osterwalder M, Mannion BJ, Tissières V, Pickle CS, Plajzer-Frick I, Lee EA, Kato M, Garvin TH, Akiyama JA, Afzal V, Lopez-Rios J, Rubin EM, Dickel DE, Pennacchio LA, and Visel A
- Subjects
- Animals, Base Sequence, Evolution, Molecular, Gene Knock-In Techniques, Mice, Mice, Transgenic, Mutation, Phylogeny, Snakes classification, Biological Evolution, Enhancer Elements, Genetic, Extremities growth & development, Hedgehog Proteins genetics, Snakes genetics
- Abstract
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. 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 orthologs 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. PAPERCLIP., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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27. Enhancer Variants Synergistically Drive Dysfunction of a Gene Regulatory Network In Hirschsprung Disease.
- Author
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Chatterjee S, Kapoor A, Akiyama JA, Auer DR, Lee D, Gabriel S, Berrios C, Pennacchio LA, and Chakravarti A
- Subjects
- Alleles, Animals, Binding Sites, Disease Models, Animal, GATA2 Transcription Factor genetics, GATA2 Transcription Factor metabolism, Gastrointestinal Tract embryology, Humans, Mice, Mice, Transgenic, RNA, Untranslated genetics, Receptors, Retinoic Acid genetics, Receptors, Retinoic Acid metabolism, SOXE Transcription Factors genetics, SOXE Transcription Factors metabolism, Enhancer Elements, Genetic, Gene Expression Regulation, Gene Regulatory Networks, Hirschsprung Disease genetics, Proto-Oncogene Proteins c-ret genetics
- Abstract
Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidence that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2016
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28. Genome-wide compendium and functional assessment of in vivo heart enhancers.
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Dickel DE, Barozzi I, Zhu Y, Fukuda-Yuzawa Y, Osterwalder M, Mannion BJ, May D, Spurrell CH, Plajzer-Frick I, Pickle CS, Lee E, Garvin TH, Kato M, Akiyama JA, Afzal V, Lee AY, Gorkin DU, Ren B, Rubin EM, Visel A, and Pennacchio LA
- Subjects
- Animals, Echocardiography, Epigenomics, Female, Gene Expression Profiling, Gene Expression Regulation, Gene Expression Regulation, Developmental, Genome, Human, Histones metabolism, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mutation, Phenotype, Enhancer Elements, Genetic, Heart physiology
- Abstract
Whole-genome sequencing is identifying growing numbers of non-coding variants in human disease studies, but the lack of accurate functional annotations prevents their interpretation. We describe the genome-wide landscape of distant-acting enhancers active in the developing and adult human heart, an organ whose impairment is a predominant cause of mortality and morbidity. Using integrative analysis of >35 epigenomic data sets from mouse and human pre- and postnatal hearts we created a comprehensive reference of >80,000 putative human heart enhancers. To illustrate the importance of enhancers in the regulation of genes involved in heart disease, we deleted the mouse orthologs of two human enhancers near cardiac myosin genes. In both cases, we observe in vivo expression changes and cardiac phenotypes consistent with human heart disease. Our study provides a comprehensive catalogue of human heart enhancers for use in clinical whole-genome sequencing studies and highlights the importance of enhancers for cardiac function.
- Published
- 2016
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29. A distal 594 bp ECR specifies Hmx1 expression in pinna and lateral facial morphogenesis and is regulated by the Hox-Pbx-Meis complex.
- Author
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Rosin JM, Li W, Cox LL, Rolfe SM, Latorre V, Akiyama JA, Visel A, Kuramoto T, Bobola N, Turner EE, and Cox TC
- Subjects
- Animals, Base Sequence, Conserved Sequence genetics, Craniofacial Abnormalities genetics, Ear Auricle abnormalities, Ear Auricle pathology, Enhancer Elements, Genetic genetics, Face embryology, Gene Expression Regulation, Developmental, Genes, Reporter, Mice, Mutant Strains, Mice, Transgenic, Organ Specificity genetics, Physical Stimulation, Pre-B-Cell Leukemia Transcription Factor 1, Protein Binding genetics, Sensory Receptor Cells pathology, Base Pairing genetics, Ear Auricle metabolism, Evolution, Molecular, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Morphogenesis genetics, Transcription Factors genetics, Transcription Factors metabolism
- Abstract
Hmx1 encodes a homeodomain transcription factor expressed in the developing lateral craniofacial mesenchyme, retina and sensory ganglia. Mutation or mis-regulation of Hmx1 underlies malformations of the eye and external ear in multiple species. Deletion or insertional duplication of an evolutionarily conserved region (ECR) downstream of Hmx1 has recently been described in rat and cow, respectively. Here, we demonstrate that the impact of Hmx1 loss is greater than previously appreciated, with a variety of lateral cranioskeletal defects, auriculofacial nerve deficits, and duplication of the caudal region of the external ear. Using a transgenic approach, we demonstrate that a 594 bp sequence encompassing the ECR recapitulates specific aspects of the endogenous Hmx1 lateral facial expression pattern. Moreover, we show that Hoxa2, Meis and Pbx proteins act cooperatively on the ECR, via a core 32 bp sequence, to regulate Hmx1 expression. These studies highlight the conserved role for Hmx1 in BA2-derived tissues and provide an entry point for improved understanding of the causes of the frequent lateral facial birth defects in humans., (© 2016. Published by The Company of Biologists Ltd.)
- Published
- 2016
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30. Dynamic GATA4 enhancers shape the chromatin landscape central to heart development and disease.
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He A, Gu F, Hu Y, Ma Q, Ye LY, Akiyama JA, Visel A, Pennacchio LA, and Pu WT
- Subjects
- Animals, Blotting, Western, Chromatin Immunoprecipitation, Enhancer Elements, Genetic genetics, High-Throughput Nucleotide Sequencing, Histones metabolism, Luciferases, Mice, Mice, Transgenic, Real-Time Polymerase Chain Reaction, Statistics, Nonparametric, Cardiomegaly metabolism, Chromatin metabolism, GATA4 Transcription Factor metabolism, Gene Expression Regulation, Developmental physiology, Heart embryology, Morphogenesis physiology
- Abstract
How stage-specific enhancer dynamics modulate gene expression patterns essential for organ development, homeostasis and disease is not well understood. Here, we addressed this question by mapping chromatin occupancy of GATA4--a master cardiac transcription factor--in heart development and disease. We find that GATA4 binds and participates in establishing active chromatin regions by stimulating H3K27ac deposition, which facilitates GATA4-driven gene expression. GATA4 chromatin occupancy changes markedly between fetal and adult heart, with a limited binding sites overlap. Cardiac stress restored GATA4 occupancy to a subset of fetal sites, but many stress-associated GATA4 binding sites localized to loci not occupied by GATA4 during normal heart development. Collectively, our data show that dynamic, context-specific transcription factors occupancy underlies stage-specific events in development, homeostasis and disease.
- Published
- 2014
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31. Tissue-specific RNA expression marks distant-acting developmental enhancers.
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Wu H, Nord AS, Akiyama JA, Shoukry M, Afzal V, Rubin EM, Pennacchio LA, and Visel A
- Subjects
- Animals, Gene Expression Profiling, Genomics methods, Mice, Mice, Transgenic, Organ Specificity genetics, Reproducibility of Results, Transcription, Genetic, Enhancer Elements, Genetic, Gene Expression Regulation, Developmental, RNA, Untranslated genetics
- Abstract
Short non-coding transcripts can be transcribed from distant-acting transcriptional enhancer loci, but the prevalence of such enhancer RNAs (eRNAs) within the transcriptome, and the association of eRNA expression with tissue-specific enhancer activity in vivo remain poorly understood. Here, we investigated the expression dynamics of tissue-specific non-coding RNAs in embryonic mouse tissues via deep RNA sequencing. Overall, approximately 80% of validated in vivo enhancers show tissue-specific RNA expression that correlates with tissue-specific enhancer activity. Globally, we identified thousands of tissue-specifically transcribed non-coding regions (TSTRs) displaying various genomic hallmarks of bona fide enhancers. In transgenic mouse reporter assays, over half of tested TSTRs functioned as enhancers with reproducible activity in the predicted tissue. Together, our results demonstrate that tissue-specific eRNA expression is a common feature of in vivo enhancers, as well as a major source of extragenic transcription, and that eRNA expression signatures can be used to predict tissue-specific enhancers independent of known epigenomic enhancer marks.
- Published
- 2014
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32. Tissue-specific SMARCA4 binding at active and repressed regulatory elements during embryogenesis.
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Attanasio C, Nord AS, Zhu Y, Blow MJ, Biddie SC, Mendenhall EM, Dixon J, Wright C, Hosseini R, Akiyama JA, Holt A, Plajzer-Frick I, Shoukry M, Afzal V, Ren B, Bernstein BE, Rubin EM, Visel A, and Pennacchio LA
- Subjects
- Animals, Brain embryology, Brain metabolism, Chromatin genetics, Chromatin metabolism, DNA Helicases genetics, Extremities embryology, Genome, Heart embryology, Histones genetics, Histones metabolism, Mice, Myocardium metabolism, Nuclear Proteins genetics, Organ Specificity, Protein Binding, Transcription Factors genetics, DNA Helicases metabolism, Gene Expression Regulation, Developmental, Nuclear Proteins metabolism, Regulatory Elements, Transcriptional, Transcription Factors metabolism
- Abstract
The SMARCA4 (also known as BRG1 in humans) chromatin remodeling factor is critical for establishing lineage-specific chromatin states during early mammalian development. However, the role of SMARCA4 in tissue-specific gene regulation during embryogenesis remains poorly defined. To investigate the genome-wide binding landscape of SMARCA4 in differentiating tissues, we engineered a Smarca4(FLAG) knock-in mouse line. Using ChIP-seq, we identified ∼51,000 SMARCA4-associated regions across six embryonic mouse tissues (forebrain, hindbrain, neural tube, heart, limb, and face) at mid-gestation (E11.5). The majority of these regions was distal from promoters and showed dynamic occupancy, with most distal SMARCA4 sites (73%) confined to a single or limited subset of tissues. To further characterize these regions, we profiled active and repressive histone marks in the same tissues and examined the intersection of informative chromatin states and SMARCA4 binding. This revealed distinct classes of distal SMARCA4-associated elements characterized by activating and repressive chromatin signatures that were associated with tissue-specific up- or down-regulation of gene expression and relevant active/repressed biological pathways. We further demonstrate the predicted active regulatory properties of SMARCA4-associated elements by retrospective analysis of tissue-specific enhancers and direct testing of SMARCA4-bound regions in transgenic mouse assays. Our results indicate a dual active/repressive function of SMARCA4 at distal regulatory sequences in vivo and support its role in tissue-specific gene regulation during embryonic development., (© 2014 Attanasio et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2014
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33. Function-based identification of mammalian enhancers using site-specific integration.
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Dickel DE, Zhu Y, Nord AS, Wylie JN, Akiyama JA, Afzal V, Plajzer-Frick I, Kirkpatrick A, Göttgens B, Bruneau BG, Visel A, and Pennacchio LA
- Subjects
- Animals, Cell Differentiation, Cell Separation, Chromosomes, Artificial, Bacterial genetics, Flow Cytometry, Gene Expression Regulation, Gene Library, Genes, Reporter, Genetic Vectors, Genomics, High-Throughput Nucleotide Sequencing, Humans, Mice, Mice, Transgenic, Plasmids metabolism, Sequence Analysis, DNA, Embryonic Stem Cells cytology, Enhancer Elements, Genetic, Myocytes, Cardiac cytology, Neural Stem Cells cytology
- Abstract
The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Targeted single-copy genomic integration into pluripotent cells, reporter assays and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. By functionally interrogating >500 kilobases (kb) of mouse and human sequence in mouse embryonic stem cells for enhancer activity we identified enhancers at pluripotency loci including NANOG. In in vitro-differentiated cardiomyocytes and neural progenitor cells, we identified cardiac enhancers and neuronal enhancers, respectively. SIF-seq is a powerful and flexible method for de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.
- Published
- 2014
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34. Rapid and pervasive changes in genome-wide enhancer usage during mammalian development.
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Nord AS, Blow MJ, Attanasio C, Akiyama JA, Holt A, Hosseini R, Phouanenavong S, Plajzer-Frick I, Shoukry M, Afzal V, Rubenstein JL, Rubin EM, Pennacchio LA, and Visel A
- Subjects
- Acetylation, Animals, Epigenesis, Genetic, Evolution, Molecular, Histones metabolism, Mice, Mice, Transgenic, Organ Specificity, Enhancer Elements, Genetic, Gene Expression Regulation, Developmental, Genome-Wide Association Study
- Abstract
Enhancers are distal regulatory elements that can activate tissue-specific gene expression and are abundant throughout mammalian genomes. Although substantial progress has been made toward genome-wide annotation of mammalian enhancers, their temporal activity patterns and global contributions in the context of developmental in vivo processes remain poorly explored. Here we used epigenomic profiling for H3K27ac, a mark of active enhancers, coupled to transgenic mouse assays to examine the genome-wide utilization of enhancers in three different mouse tissues across seven developmental stages. The majority of the ∼90,000 enhancers identified exhibited tightly temporally restricted predicted activity windows and were associated with stage-specific biological functions and regulatory pathways in individual tissues. Comparative genomic analysis revealed that evolutionary conservation of enhancers decreases following midgestation across all tissues examined. The dynamic enhancer activities uncovered in this study illuminate rapid and pervasive temporal in vivo changes in enhancer usage that underlie processes central to development and disease., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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35. Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants.
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Parker SC, Stitzel ML, Taylor DL, Orozco JM, Erdos MR, Akiyama JA, van Bueren KL, Chines PS, Narisu N, Black BL, Visel A, Pennacchio LA, and Collins FS
- Subjects
- Animals, Chromatin Immunoprecipitation, Diabetes Mellitus, Type 2 genetics, Enhancer Elements, Genetic physiology, Gene Expression Profiling, Gene Expression Regulation genetics, Genome-Wide Association Study, High-Throughput Nucleotide Sequencing, Humans, Insulin-Secreting Cells physiology, Luciferases, Mice, Mice, Transgenic, Cell Differentiation physiology, Chromatin physiology, Diabetes Mellitus, Type 2 physiopathology, Enhancer Elements, Genetic genetics, Epigenomics methods, Gene Expression Regulation physiology, Insulin-Secreting Cells metabolism
- Abstract
Chromatin-based functional genomic analyses and genomewide association studies (GWASs) together implicate enhancers as critical elements influencing gene expression and risk for common diseases. Here, we performed systematic chromatin and transcriptome profiling in human pancreatic islets. Integrated analysis of islet data with those from nine cell types identified specific and significant enrichment of type 2 diabetes and related quantitative trait GWAS variants in islet enhancers. Our integrated chromatin maps reveal that most enhancers are short (median = 0.8 kb). Each cell type also contains a substantial number of more extended (≥ 3 kb) enhancers. Interestingly, these stretch enhancers are often tissue-specific and overlap locus control regions, suggesting that they are important chromatin regulatory beacons. Indeed, we show that (i) tissue specificity of enhancers and nearby gene expression increase with enhancer length; (ii) neighborhoods containing stretch enhancers are enriched for important cell type-specific genes; and (iii) GWAS variants associated with traits relevant to a particular cell type are more enriched in stretch enhancers compared with short enhancers. Reporter constructs containing stretch enhancer sequences exhibited tissue-specific activity in cell culture experiments and in transgenic mice. These results suggest that stretch enhancers are critical chromatin elements for coordinating cell type-specific regulatory programs and that sequence variation in stretch enhancers affects risk of major common human diseases.
- Published
- 2013
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- View/download PDF
36. Fine tuning of craniofacial morphology by distant-acting enhancers.
- Author
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Attanasio C, Nord AS, Zhu Y, Blow MJ, Li Z, Liberton DK, Morrison H, Plajzer-Frick I, Holt A, Hosseini R, Phouanenavong S, Akiyama JA, Shoukry M, Afzal V, Rubin EM, FitzPatrick DR, Ren B, Hallgrímsson B, Pennacchio LA, and Visel A
- Subjects
- Animals, Craniofacial Abnormalities genetics, Craniofacial Abnormalities pathology, Enhancer Elements, Genetic genetics, Epigenesis, Genetic, Face abnormalities, Gene Expression Profiling, Gene Targeting, Mice, Mice, Transgenic, Sequence Deletion, Skull abnormalities, Skull anatomy & histology, Enhancer Elements, Genetic physiology, Face anatomy & histology, Gene Expression Regulation, Developmental, Maxillofacial Development genetics, Skull growth & development
- Abstract
The shape of the human face and skull is largely genetically determined. However, the genomic basis of craniofacial morphology is incompletely understood and hypothesized to involve protein-coding genes, as well as gene regulatory sequences. We used a combination of epigenomic profiling, in vivo characterization of candidate enhancer sequences in transgenic mice, and targeted deletion experiments to examine the role of distant-acting enhancers in craniofacial development. We identified complex regulatory landscapes consisting of enhancers that drive spatially complex developmental expression patterns. Analysis of mouse lines in which individual craniofacial enhancers had been deleted revealed significant alterations of craniofacial shape, demonstrating the functional importance of enhancers in defining face and skull morphology. These results demonstrate that enhancers are involved in craniofacial development and suggest that enhancer sequence variation contributes to the diversity of human facial morphology.
- Published
- 2013
- Full Text
- View/download PDF
37. A high-resolution enhancer atlas of the developing telencephalon.
- Author
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Visel A, Taher L, Girgis H, May D, Golonzhka O, Hoch RV, McKinsey GL, Pattabiraman K, Silberberg SN, Blow MJ, Hansen DV, Nord AS, Akiyama JA, Holt A, Hosseini R, Phouanenavong S, Plajzer-Frick I, Shoukry M, Afzal V, Kaplan T, Kriegstein AR, Rubin EM, Ovcharenko I, Pennacchio LA, and Rubenstein JL
- Subjects
- Animals, Embryo, Mammalian metabolism, Fetus metabolism, Genome-Wide Association Study, Humans, Mice, Telencephalon embryology, Transcriptome, p300-CBP Transcription Factors metabolism, Enhancer Elements, Genetic, Telencephalon metabolism
- Abstract
The mammalian telencephalon plays critical roles in cognition, motor function, and emotion. Though many of the genes required for its development have been identified, the distant-acting regulatory sequences orchestrating their in vivo expression are mostly unknown. Here, we describe a digital atlas of in vivo enhancers active in subregions of the developing telencephalon. We identified more than 4,600 candidate embryonic forebrain enhancers and studied the in vivo activity of 329 of these sequences in transgenic mouse embryos. We generated serial sets of histological brain sections for 145 reproducible forebrain enhancers, resulting in a publicly accessible web-based data collection comprising more than 32,000 sections. We also used epigenomic analysis of human and mouse cortex tissue to directly compare the genome-wide enhancer architecture in these species. These data provide a primary resource for investigating gene regulatory mechanisms of telencephalon development and enable studies of the role of distant-acting enhancers in neurodevelopmental disorders., (Copyright © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
38. Large-scale discovery of enhancers from human heart tissue.
- Author
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May D, Blow MJ, Kaplan T, McCulley DJ, Jensen BC, Akiyama JA, Holt A, Plajzer-Frick I, Shoukry M, Wright C, Afzal V, Simpson PC, Rubin EM, Black BL, Bristow J, Pennacchio LA, and Visel A
- Subjects
- Adult, Animals, Chromosome Mapping, Gene Expression Regulation, Developmental, Heart embryology, Humans, Mice, Mice, Transgenic, p300-CBP Transcription Factors, Enhancer Elements, Genetic, Heart physiology
- Abstract
Development and function of the human heart depend on the dynamic control of tissue-specific gene expression by distant-acting transcriptional enhancers. To generate an accurate genome-wide map of human heart enhancers, we used an epigenomic enhancer discovery approach and identified ∼6,200 candidate enhancer sequences directly from fetal and adult human heart tissue. Consistent with their predicted function, these elements were markedly enriched near genes implicated in heart development, function and disease. To further validate their in vivo enhancer activity, we tested 65 of these human sequences in a transgenic mouse enhancer assay and observed that 43 (66%) drove reproducible reporter gene expression in the heart. These results support the discovery of a genome-wide set of noncoding sequences highly enriched in human heart enhancers that is likely to facilitate downstream studies of the role of enhancers in development and pathological conditions of the heart.
- Published
- 2011
- Full Text
- View/download PDF
39. ChIP-Seq identification of weakly conserved heart enhancers.
- Author
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Blow MJ, McCulley DJ, Li Z, Zhang T, Akiyama JA, Holt A, Plajzer-Frick I, Shoukry M, Wright C, Chen F, Afzal V, Bristow J, Ren B, Black BL, Rubin EM, Visel A, and Pennacchio LA
- Subjects
- Animals, Base Sequence, Conserved Sequence genetics, Embryo, Mammalian, Evolution, Molecular, Gene Expression Regulation, Developmental, Heart embryology, Humans, Mice, Mice, Transgenic, Models, Biological, Organ Specificity genetics, Phylogeny, Vertebrates genetics, Vertebrates metabolism, Chromatin Immunoprecipitation methods, Enhancer Elements, Genetic genetics, Myocardium metabolism, Sequence Analysis, DNA methods
- Abstract
Accurate control of tissue-specific gene expression plays a pivotal role in heart development, but few cardiac transcriptional enhancers have thus far been identified. Extreme noncoding-sequence conservation has successfully predicted enhancers that are active in many tissues but has failed to identify substantial numbers of heart-specific enhancers. Here, we used ChIP-Seq with the enhancer-associated protein p300 from mouse embryonic day 11.5 heart tissue to identify over 3,000 candidate heart enhancers genome wide. Compared to enhancers active in other tissues we studied at this time point, most candidate heart enhancers were less deeply conserved in vertebrate evolution. Nevertheless, transgenic mouse assays of 130 candidate regions revealed that most function reproducibly as enhancers active in the heart, irrespective of their degree of evolutionary constraint. These results provide evidence for a large population of poorly conserved heart enhancers and suggest that the evolutionary conservation of embryonic enhancers can vary depending on tissue type.
- Published
- 2010
- Full Text
- View/download PDF
40. Functional autonomy of distant-acting human enhancers.
- Author
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Visel A, Akiyama JA, Shoukry M, Afzal V, Rubin EM, and Pennacchio LA
- Subjects
- Animals, Evolution, Molecular, Genes, Reporter genetics, Humans, Mice, Mice, Transgenic, Enhancer Elements, Genetic genetics, Gene Expression Regulation, Developmental genetics
- Abstract
Many human genes are associated with dispersed arrays of transcriptional enhancers that regulate their expression in time and space. Studies in invertebrate model systems have suggested that these elements could function as discrete and independent regulatory units, but the in vivo combinatorial properties of vertebrate enhancers remain poorly understood. To explore the modularity and regulatory autonomy of human developmental enhancers, we experimentally concatenated up to four enhancers from different genes and used a transgenic mouse assay to compare the in vivo activity of these compound elements with that of the single modules. In all of the six different combinations of elements tested, the reporter gene activity patterns were additive without signs of interference between the individual modules, indicating that regulatory specificity was maintained despite the presence of closely-positioned heterologous enhancers. Even in cases where two elements drove expression in close anatomical proximity, such as within neighboring subregions of the developing limb bud, the compound patterns did not show signs of cross-inhibition between individual elements or novel expression sites. These data indicate that human developmental enhancers are highly modular and functionally autonomous and suggest that genomic enhancer shuffling may have contributed to the evolution of complex gene expression patterns in vertebrates.
- Published
- 2009
- Full Text
- View/download PDF
41. ChIP-seq accurately predicts tissue-specific activity of enhancers.
- Author
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Visel A, Blow MJ, Li Z, Zhang T, Akiyama JA, Holt A, Plajzer-Frick I, Shoukry M, Wright C, Chen F, Afzal V, Ren B, Rubin EM, and Pennacchio LA
- Subjects
- Animals, Conserved Sequence, Embryo, Mammalian embryology, Mice, Chromatin Immunoprecipitation methods, Chromosome Mapping methods, Extremities embryology, Gene Expression Regulation, Developmental, Mesencephalon embryology, Prosencephalon embryology, p300-CBP Transcription Factors metabolism
- Abstract
A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover because they are scattered among the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here we present the results of chromatin immunoprecipitation with the enhancer-associated protein p300 followed by massively parallel sequencing, and map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain and limb tissue. We tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases demonstrated reproducible enhancer activity in the tissues that were predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities, and suggest that such data sets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.
- Published
- 2009
- Full Text
- View/download PDF
42. Human-specific gain of function in a developmental enhancer.
- Author
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Prabhakar S, Visel A, Akiyama JA, Shoukry M, Lewis KD, Holt A, Plajzer-Frick I, Morrison H, Fitzpatrick DR, Afzal V, Pennacchio LA, Rubin EM, and Noonan JP
- Subjects
- Animals, Base Sequence, Binding Sites, Conserved Sequence, Embryonic Development, Evolution, Molecular, Gene Expression Profiling, Humans, Limb Buds embryology, Limb Buds metabolism, Macaca mulatta genetics, Mice, Mice, Transgenic, Molecular Sequence Data, Mutation, PAX9 Transcription Factor metabolism, Pan troglodytes genetics, Selection, Genetic, Transcription Factors metabolism, Body Patterning genetics, Enhancer Elements, Genetic, Extremities embryology, Gene Expression Regulation, Developmental
- Abstract
Changes in gene regulation are thought to have contributed to the evolution of human development. However, in vivo evidence for uniquely human developmental regulatory function has remained elusive. In transgenic mice, a conserved noncoding sequence (HACNS1) that evolved extremely rapidly in humans acted as an enhancer of gene expression that has gained a strong limb expression domain relative to the orthologous elements from chimpanzee and rhesus macaque. This gain of function was consistent across two developmental stages in the mouse and included the presumptive anterior wrist and proximal thumb. In vivo analyses with synthetic enhancers, in which human-specific substitutions were introduced into the chimpanzee enhancer sequence or reverted in the human enhancer to the ancestral state, indicated that 13 substitutions clustered in an 81-base pair module otherwise highly constrained among terrestrial vertebrates were sufficient to confer the human-specific limb expression domain.
- Published
- 2008
- Full Text
- View/download PDF
43. Ultraconservation identifies a small subset of extremely constrained developmental enhancers.
- Author
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Visel A, Prabhakar S, Akiyama JA, Shoukry M, Lewis KD, Holt A, Plajzer-Frick I, Afzal V, Rubin EM, and Pennacchio LA
- Subjects
- Animals, Base Pairing, Base Sequence, Embryo, Mammalian, Evolution, Molecular, Gene Expression Regulation, Developmental, Genes, Reporter, Genomics methods, Humans, Mice, Mice, Transgenic, Molecular Sequence Data, Nervous System embryology, Nervous System metabolism, Regulatory Sequences, Nucleic Acid, Selection, Genetic, Species Specificity, Transcription, Genetic, Conserved Sequence genetics, Enhancer Elements, Genetic, Genome, Human
- Abstract
Extended perfect human-rodent sequence identity of at least 200 base pairs (ultraconservation) is potentially indicative of evolutionary or functional uniqueness. We used a transgenic mouse assay to compare the embryonic enhancer activity of 231 noncoding ultraconserved human genome regions with that of 206 extremely conserved regions lacking ultraconservation. Developmental enhancers were equally prevalent in both populations, suggesting instead that ultraconservation identifies a small, functionally indistinct subset of similarly constrained cis-regulatory elements.
- Published
- 2008
- Full Text
- View/download PDF
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