Chromatin position effects assayed by thousands of reporters integrated in parallel
Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here, we describe a multiplexing approach for the parallel monitoring of transcript...
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| Vydáno v: | Cell Ročník 154; číslo 4; s. 914 |
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| Hlavní autoři: | , , , , , , , , , |
| Médium: | Journal Article |
| Jazyk: | angličtina |
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United States
15.08.2013
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| ISSN: | 1097-4172, 1097-4172 |
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| Abstract | Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here, we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two different promoters, show ∼1,000-fold variation in expression levels. Data analysis indicates that lamina-associated domains act as attenuators of transcription, likely by reducing access of transcription factors to binding sites. Furthermore, chromatin compaction is predictive of reporter activity. We also found evidence for crosstalk between neighboring genes and estimate that enhancers can influence gene expression on average over ∼20 kb. The multiplexed reporter assay is highly flexible in design and can be modified to query a wide range of aspects of gene regulation. |
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| AbstractList | Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here, we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two different promoters, show ∼1,000-fold variation in expression levels. Data analysis indicates that lamina-associated domains act as attenuators of transcription, likely by reducing access of transcription factors to binding sites. Furthermore, chromatin compaction is predictive of reporter activity. We also found evidence for crosstalk between neighboring genes and estimate that enhancers can influence gene expression on average over ∼20 kb. The multiplexed reporter assay is highly flexible in design and can be modified to query a wide range of aspects of gene regulation. Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here, we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two different promoters, show ∼1,000-fold variation in expression levels. Data analysis indicates that lamina-associated domains act as attenuators of transcription, likely by reducing access of transcription factors to binding sites. Furthermore, chromatin compaction is predictive of reporter activity. We also found evidence for crosstalk between neighboring genes and estimate that enhancers can influence gene expression on average over ∼20 kb. The multiplexed reporter assay is highly flexible in design and can be modified to query a wide range of aspects of gene regulation.Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here, we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two different promoters, show ∼1,000-fold variation in expression levels. Data analysis indicates that lamina-associated domains act as attenuators of transcription, likely by reducing access of transcription factors to binding sites. Furthermore, chromatin compaction is predictive of reporter activity. We also found evidence for crosstalk between neighboring genes and estimate that enhancers can influence gene expression on average over ∼20 kb. The multiplexed reporter assay is highly flexible in design and can be modified to query a wide range of aspects of gene regulation. |
| Author | Pindyurin, Alexey V Wessels, Lodewyk F A van Lohuizen, Maarten Berns, Anton Akhtar, Waseem Meuleman, Wouter de Ridder, Jeroen Pagie, Ludo van Steensel, Bas de Jong, Johann |
| Author_xml | – sequence: 1 givenname: Waseem surname: Akhtar fullname: Akhtar, Waseem organization: Division of Molecular Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands – sequence: 2 givenname: Johann surname: de Jong fullname: de Jong, Johann – sequence: 3 givenname: Alexey V surname: Pindyurin fullname: Pindyurin, Alexey V – sequence: 4 givenname: Ludo surname: Pagie fullname: Pagie, Ludo – sequence: 5 givenname: Wouter surname: Meuleman fullname: Meuleman, Wouter – sequence: 6 givenname: Jeroen surname: de Ridder fullname: de Ridder, Jeroen – sequence: 7 givenname: Anton surname: Berns fullname: Berns, Anton – sequence: 8 givenname: Lodewyk F A surname: Wessels fullname: Wessels, Lodewyk F A – sequence: 9 givenname: Maarten surname: van Lohuizen fullname: van Lohuizen, Maarten – sequence: 10 givenname: Bas surname: van Steensel fullname: van Steensel, Bas |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/23953119$$D View this record in MEDLINE/PubMed |
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