39 results on '"Plajzer-Frick I"'
Search Results
2. Integration of cytogenetic landmarks into the draft sequence of the human genome
- Author
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BAC Resource Consortium, The, Cheung, V. G., Nowak, N., Jang, W., Kirsch, I. R., Zhao, S., Chen, X.-N., Furey, T. S., Kim, U.-J., Kuo, W.-L., Olivier, M., Conroy, J., Kasprzyk, A., Massa, H., Yonescu, R., Sait, S., Thoreen, C., Snijders, A., Lemyre, E., Bailey, J. A., Bruzel, A., Burrill, W. D., Clegg, S. M., Collins, S., Dhami, P., Friedman, C., Han, C. S., Herrick, S., Lee, J., Ligon, A. H., Lowry, S., Morley, M., Narasimhan, S., Osoegawa, K., Peng, Z., Plajzer-Frick, I., Quade, B. J., Scott, D., Sirotkin, K., Thorpe, A. A., Gray, J. W., Hudson, J., Pinkel, D., Ried, T., Rowen, L., Shen-Ong, G. L., Strausberg, R. L., Birney, E., Callen, D. F., Cheng, J.-F., Cox, D. R., Doggett, N. A., Carter, N. P., Eichler, E. E., Haussler, D., Korenberg, J. R., Morton, C. C., Albertson, D., Schuler, G., de Jong, P. J., and Trask, B. J.
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- 2001
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3. 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, Enhancer Elements, Genetic, Gene Expression Regulation, Developmental, Homeodomain Proteins genetics, Homeodomain Proteins metabolism
- Abstract
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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4. 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.
- Published
- 2024
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5. 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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6. 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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7. 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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8. 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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9. 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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10. 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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11. 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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12. 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
- Published
- 2021
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13. 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.
- Published
- 2020
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14. 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
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- 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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15. The changing mouse embryo transcriptome at whole tissue and single-cell resolution.
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He P, Williams BA, Trout D, Marinov GK, Amrhein H, Berghella L, Goh ST, Plajzer-Frick I, Afzal V, Pennacchio LA, Dickel DE, Visel A, Ren B, Hardison RC, Zhang Y, and Wold BJ
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- Animals, Cell Differentiation genetics, Cell Lineage genetics, Chromatin genetics, Embryo, Mammalian metabolism, Enhancer Elements, Genetic, Epigenomics, Extremities embryology, Female, Male, Mice, Poly A genetics, Poly A metabolism, Promoter Regions, Genetic, RNA-Seq, Transcription Factors metabolism, Embryo, Mammalian cytology, Embryo, Mammalian embryology, Embryonic Development genetics, Gene Expression Regulation, Developmental, Single-Cell Analysis, Transcriptome
- Abstract
During mammalian embryogenesis, differential gene expression gradually builds the identity and complexity of each tissue and organ system
1 . Here we systematically quantified mouse polyA-RNA from day 10.5 of embryonic development to birth, sampling 17 tissues and organs. The resulting developmental transcriptome is globally structured by dynamic cytodifferentiation, body-axis and cell-proliferation gene sets that were further characterized by the transcription factor motif codes of their promoters. We decomposed the tissue-level transcriptome using single-cell RNA-seq (sequencing of RNA reverse transcribed into cDNA) and found that neurogenesis and haematopoiesis dominate at both the gene and cellular levels, jointly accounting for one-third of differential gene expression and more than 40% of identified cell types. By integrating promoter sequence motifs with companion ENCODE epigenomic profiles, we identified a prominent promoter de-repression mechanism in neuronal expression clusters that was attributable to known and novel repressors. Focusing on the developing limb, single-cell RNA data identified 25 candidate cell types that included progenitor and differentiating states with computationally inferred lineage relationships. We extracted cell-type transcription factor networks and complementary sets of candidate enhancer elements by using single-cell RNA-seq to decompose integrative cis-element (IDEAS) models that were derived from whole-tissue epigenome chromatin data. These ENCODE reference data, computed network components and IDEAS chromatin segmentations are companion resources to the matching epigenomic developmental matrix, and are available for researchers to further mine and integrate.- Published
- 2020
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16. 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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- 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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17. 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
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- 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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18. 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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19. 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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20. Limb-Enhancer Genie: An accessible resource of accurate enhancer predictions in the developing limb.
- Author
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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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21. Germline Chd8 haploinsufficiency alters brain development in mouse.
- Author
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Gompers AL, Su-Feher L, Ellegood J, Copping NA, Riyadh MA, Stradleigh TW, Pride MC, Schaffler MD, Wade AA, Catta-Preta R, Zdilar I, Louis S, Kaushik G, Mannion BJ, Plajzer-Frick I, Afzal V, Visel A, Pennacchio LA, Dickel DE, Lerch JP, Crawley JN, Zarbalis KS, Silverman JL, and Nord AS
- Subjects
- Animals, Brain metabolism, Cell Cycle Proteins genetics, Chromatin metabolism, Mice, Transgenic, Mutation genetics, Phenotype, Transcription Factors genetics, DNA-Binding Proteins genetics, Gene Expression Regulation, Developmental genetics, Gene Regulatory Networks genetics, Haploinsufficiency genetics
- Abstract
The chromatin remodeling gene CHD8 represents a central node in neurodevelopmental gene networks implicated in autism. We examined the impact of germline heterozygous frameshift Chd8 mutation on neurodevelopment in mice. Chd8
+/del5 mice displayed normal social interactions with no repetitive behaviors but exhibited cognitive impairment correlated with increased regional brain volume, validating that phenotypes of Chd8+/del5 mice overlap pathology reported in humans with CHD8 mutations. We applied network analysis to characterize neurodevelopmental gene expression, revealing widespread transcriptional changes in Chd8+/del5 mice across pathways disrupted in neurodevelopmental disorders, including neurogenesis, synaptic processes and neuroimmune signaling. We identified a co-expression module with peak expression in early brain development featuring dysregulation of RNA processing, chromatin remodeling and cell-cycle genes enriched for promoter binding by Chd8, and we validated increased neuronal proliferation and developmental splicing perturbation in Chd8+/del5 mice. This integrative analysis offers an initial picture of the consequences of Chd8 haploinsufficiency for brain development.- Published
- 2017
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- View/download PDF
22. Progressive Loss of Function in a Limb Enhancer during Snake Evolution.
- Author
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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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23. Genome-wide compendium and functional assessment of in vivo heart enhancers.
- Author
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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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24. Tissue-specific SMARCA4 binding at active and repressed regulatory elements during embryogenesis.
- Author
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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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25. Function-based identification of mammalian enhancers using site-specific integration.
- Author
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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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26. Rapid and pervasive changes in genome-wide enhancer usage during mammalian development.
- Author
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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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27. 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
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- View/download PDF
28. 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
29. 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
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- View/download PDF
30. 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
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31. 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
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32. 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
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- View/download PDF
33. 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
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34. In vivo enhancer analysis of human conserved non-coding sequences.
- Author
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Pennacchio LA, Ahituv N, Moses AM, Prabhakar S, Nobrega MA, Shoukry M, Minovitsky S, Dubchak I, Holt A, Lewis KD, Plajzer-Frick I, Akiyama J, De Val S, Afzal V, Black BL, Couronne O, Eisen MB, Visel A, and Rubin EM
- Subjects
- Animals, Base Sequence, Chromosomes, Human, Pair 16, Conserved Sequence, Embryo, Mammalian metabolism, Embryo, Nonmammalian, Gene Expression, Genomics methods, Humans, Mice, Mice, Transgenic, Nervous System embryology, Nervous System metabolism, Prosencephalon embryology, Prosencephalon metabolism, Takifugu genetics, Transcription Factors genetics, Enhancer Elements, Genetic, Genome, Human
- Abstract
Identifying the sequences that direct the spatial and temporal expression of genes and defining their function in vivo remains a significant challenge in the annotation of vertebrate genomes. One major obstacle is the lack of experimentally validated training sets. In this study, we made use of extreme evolutionary sequence conservation as a filter to identify putative gene regulatory elements, and characterized the in vivo enhancer activity of a large group of non-coding elements in the human genome that are conserved in human-pufferfish, Takifugu (Fugu) rubripes, or ultraconserved in human-mouse-rat. We tested 167 of these extremely conserved sequences in a transgenic mouse enhancer assay. Here we report that 45% of these sequences functioned reproducibly as tissue-specific enhancers of gene expression at embryonic day 11.5. While directing expression in a broad range of anatomical structures in the embryo, the majority of the 75 enhancers directed expression to various regions of the developing nervous system. We identified sequence signatures enriched in a subset of these elements that targeted forebrain expression, and used these features to rank all approximately 3,100 non-coding elements in the human genome that are conserved between human and Fugu. The testing of the top predictions in transgenic mice resulted in a threefold enrichment for sequences with forebrain enhancer activity. These data dramatically expand the catalogue of human gene enhancers that have been characterized in vivo, and illustrate the utility of such training sets for a variety of biological applications, including decoding the regulatory vocabulary of the human genome.
- Published
- 2006
- Full Text
- View/download PDF
35. Genomic deletion of a long-range bone enhancer misregulates sclerostin in Van Buchem disease.
- Author
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Loots GG, Kneissel M, Keller H, Baptist M, Chang J, Collette NM, Ovcharenko D, Plajzer-Frick I, and Rubin EM
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, Bone Density genetics, Genetic Linkage, Humans, Male, Mice, Mice, Transgenic, Phenotype, Rats, Sequence Alignment, Tumor Cells, Cultured, Untranslated Regions, Bone Morphogenetic Proteins genetics, Enhancer Elements, Genetic, Gene Deletion, Gene Expression Regulation, Developmental, Genetic Markers genetics, Hyperostosis genetics
- Abstract
Mutations in distant regulatory elements can have a negative impact on human development and health, yet because of the difficulty of detecting these critical sequences, we predominantly focus on coding sequences for diagnostic purposes. We have undertaken a comparative sequence-based approach to characterize a large noncoding region deleted in patients affected by Van Buchem (VB) disease, a severe sclerosing bone dysplasia. Using BAC recombination and transgenesis, we characterized the expression of human sclerostin (SOST) from normal (SOST(wt)) or Van Buchem (SOST(vbDelta) alleles. Only the SOST(wt) allele faithfully expressed high levels of human SOST in the adult bone and had an impact on bone metabolism, consistent with the model that the VB noncoding deletion removes a SOST-specific regulatory element. By exploiting cross-species sequence comparisons with in vitro and in vivo enhancer assays, we were able to identify a candidate enhancer element that drives human SOST expression in osteoblast-like cell lines in vitro and in the skeletal anlage of the embryonic day 14.5 (E14.5) mouse embryo, and discovered a novel function for sclerostin during limb development. Our approach represents a framework for characterizing distant regulatory elements associated with abnormal human phenotypes.
- Published
- 2005
- Full Text
- View/download PDF
36. In vivo characterization of a vertebrate ultraconserved enhancer.
- Author
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Poulin F, Nobrega MA, Plajzer-Frick I, Holt A, Afzal V, Rubin EM, and Pennacchio LA
- Subjects
- Animals, DNA-Binding Proteins, Humans, Mice, Mice, Transgenic, Transcription Factors, Conserved Sequence genetics, Enhancer Elements, Genetic genetics, Gene Expression Regulation, Developmental, Nuclear Proteins genetics, Takifugu genetics
- Abstract
Genomic sequence comparisons among human, mouse, and pufferfish (Takifugu rubripes (Fugu)) have revealed a set of extremely conserved noncoding sequences. While this high degree of sequence conservation suggests severe evolutionary constraint and predicts a lack of tolerance to change to retain in vivo functionality, such elements have been minimally explored experimentally. In this study, we describe the in-depth characterization of an ancient conserved enhancer, Dc2, located near the dachshund gene, which displays a human-Fugu identity of 84% over 424 basepairs (bp). In addition to this large overall conservation, we find that Dc2 is characterized by the presence of a large block of sequence (144 bp) that is completely identical among human, mouse, chicken, zebrafish, and Fugu. Through the testing of reporter vector constructs in transgenic mice, we observed that the 424-bp Dc2-conserved element is necessary and sufficient for brain tissue enhancer activity. In vivo analyses also revealed that the 144-bp 100% conserved sequence is necessary, but not sufficient, to replicate Dc2 enhancer function. However, the introduction of two separate 16-bp insertions into the highly conserved enhancer core did not cause any detectable modification of its in vivo activity. Our observations indicate that the 144-bp 100% conserved element is tolerant of change at least at the resolution of this transgenic mouse assay and suggest that purifying selection on the Dc2 sequence might not be as strong as we predicted or that some unknown property also constrains this highly conserved enhancer sequence.
- Published
- 2005
- Full Text
- View/download PDF
37. Megabase deletions of gene deserts result in viable mice.
- Author
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Nóbrega MA, Zhu Y, Plajzer-Frick I, Afzal V, and Rubin EM
- Subjects
- Animals, Chromosomes, Mammalian genetics, Conserved Sequence genetics, Female, Gene Expression Profiling, Genes, Essential genetics, Homozygote, Humans, Longevity genetics, Male, Mice, Mice, Mutant Strains, Mice, Transgenic, Phenotype, Reproduction genetics, Chromosome Deletion, Genome, Sequence Deletion genetics
- Abstract
The functional importance of the roughly 98% of mammalian genomes not corresponding to protein coding sequences remains largely undetermined. Here we show that some large-scale deletions of the non-coding DNA referred to as gene deserts can be well tolerated by an organism. We deleted two large non-coding intervals, 1,511 kilobases and 845 kilobases in length, from the mouse genome. Viable mice homozygous for the deletions were generated and were indistinguishable from wild-type littermates with regard to morphology, reproductive fitness, growth, longevity and a variety of parameters assaying general homeostasis. Further detailed analysis of the expression of multiple genes bracketing the deletions revealed only minor expression differences in homozygous deletion and wild-type mice. Together, the two deleted segments harbour 1,243 non-coding sequences conserved between humans and rodents (more than 100 base pairs, 70% identity). Some of the deleted sequences might encode for functions unidentified in our screen; nonetheless, these studies further support the existence of potentially 'disposable DNA' in the genomes of mammals.
- Published
- 2004
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38. Integration of cytogenetic landmarks into the draft sequence of the human genome.
- Author
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Cheung VG, Nowak N, Jang W, Kirsch IR, Zhao S, Chen XN, Furey TS, Kim UJ, Kuo WL, Olivier M, Conroy J, Kasprzyk A, Massa H, Yonescu R, Sait S, Thoreen C, Snijders A, Lemyre E, Bailey JA, Bruzel A, Burrill WD, Clegg SM, Collins S, Dhami P, Friedman C, Han CS, Herrick S, Lee J, Ligon AH, Lowry S, Morley M, Narasimhan S, Osoegawa K, Peng Z, Plajzer-Frick I, Quade BJ, Scott D, Sirotkin K, Thorpe AA, Gray JW, Hudson J, Pinkel D, Ried T, Rowen L, Shen-Ong GL, Strausberg RL, Birney E, Callen DF, Cheng JF, Cox DR, Doggett NA, Carter NP, Eichler EE, Haussler D, Korenberg JR, Morton CC, Albertson D, Schuler G, de Jong PJ, and Trask BJ
- Subjects
- Chromosome Mapping, Chromosomes, Artificial, Bacterial, Cytogenetic Analysis, Human Genome Project, Humans, In Situ Hybridization, Fluorescence, Radiation Hybrid Mapping, Sequence Tagged Sites, Chromosome Aberrations, Genetic Markers, Genome, Human
- Abstract
We have placed 7,600 cytogenetically defined landmarks on the draft sequence of the human genome to help with the characterization of genes altered by gross chromosomal aberrations that cause human disease. The landmarks are large-insert clones mapped to chromosome bands by fluorescence in situ hybridization. Each clone contains a sequence tag that is positioned on the genomic sequence. This genome-wide set of sequence-anchored clones allows structural and functional analyses of the genome. This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome; provides an independent validation of the sequence map and framework for contig order and orientation; surveys the genome for large-scale duplications, which are likely to require special attention during sequence assembly; and allows a stringent assessment of sequence differences between the dark and light bands of chromosomes. It also provides insight into large-scale chromatin structure and the evolution of chromosomes and gene families and will accelerate our understanding of the molecular bases of human disease and cancer.
- Published
- 2001
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39. Characterization of the mouse collectin gene locus.
- Author
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Akiyama J, Volik SV, Plajzer-Frick I, Prince A, Sago H, Weier HU, Vanderbilt JN, Hawgood S, and Poulain FR
- Subjects
- Animals, Base Sequence, Blotting, Southern, Chromosome Mapping, Chromosomes, Bacterial, Cloning, Molecular, Collectins, DNA, Complementary analysis, Exons, Genomic Library, Glycoproteins genetics, In Situ Hybridization, Fluorescence, Introns, Mannose-Binding Lectins, Models, Genetic, Molecular Sequence Data, Proteolipids genetics, Pulmonary Surfactant-Associated Protein A, Pulmonary Surfactant-Associated Protein D, Pulmonary Surfactant-Associated Proteins, Pulmonary Surfactants genetics, Carrier Proteins genetics, Mannose-Binding Lectin analogs & derivatives, Mice genetics
- Abstract
Three of the four known mouse collectin genes have been mapped to chromosome 14. To further characterize the spatial relationship of these genes, a bacterial artificial chromosome (BAC) library of mouse chromosome 14 was screened using mouse surfactant protein (SP)-A and -D complementary DNAs (cDNAs). One large clone hybridized to both SP-A and SP-D cDNAs and was found by polymerase chain reaction (PCR) to contain sequences from one of the mouse mannose-binding lectin genes (Mbl1). We used Southern mapping and subcloning of overlapping restriction fragments to characterize the gene locus. Mapping was confirmed by fluorescent in situ hybridization of fiber-stretched BAC DNA and by Southern hybridization of restriction endonuclease-digested and PCR-amplified genomic DNA. We found that the SP-A, Mbl1, and SP-D genes reside contiguously within a 55-kb region. The SP-A and Mbl1 genes are in the same 5' to 3' orientation and 16 kb apart. The SP-D gene is in the opposite orientation to the two other collectin genes, 13 kb away from the 3' end of the Mbl1 gene. The mouse SP-D gene had not previously been characterized. We found its size (13 kb) and organization to be similar to that of human SP-D. Exon I is untranslated. The second exon is a hybrid exon that contains signal for initiation of translation, signal peptide, N-terminal domain, and the first seven collagen triplets of the collagen-like domain of the protein. Four short exons (III through VI) encode the collagen-like domain of the protein, and exons VII and VIII the linking and the carbohydrate-recognition domains, respectively.
- Published
- 1999
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