High-throughput engineering of a mammalian genome reveals building principles of methylation states at CG rich regions
| Autor: | Dirk Schübeler, Sophie Dessus-Babus, Lukas Burger, Arnaud R Krebs |
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| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Mice
CpG islands chemistry.chemical_compound Genomic library Biology (General) Mammals Genetics Regulation of gene expression Genome DNA methylation histone modifications General Neuroscience High-Throughput Nucleotide Sequencing Cell Differentiation General Medicine high throughput genome editing 3. Good health Genomics and Evolutionary Biology Genes and Chromosomes Medicine Genetic Engineering Insight epigenetic Protein Binding Research Article QH301-705.5 Science Biology General Biochemistry Genetics and Molecular Biology DNA sequencing Recombinases Animals Humans Protein–DNA interaction human Gene Embryonic Stem Cells mouse Gene Library Base Sequence Models Genetic General Immunology and Microbiology epigenetics Epigenetic Markers bivalent chromatin Promoter chemistry gene regulation DNA Transcription Factors |
| Zdroj: | eLife eLife, Vol 3 (2014) |
| ISSN: | 2050-084X |
| Popis: | The majority of mammalian promoters are CpG islands; regions of high CG density that require protection from DNA methylation to be functional. Importantly, how sequence architecture mediates this unmethylated state remains unclear. To address this question in a comprehensive manner, we developed a method to interrogate methylation states of hundreds of sequence variants inserted at the same genomic site in mouse embryonic stem cells. Using this assay, we were able to quantify the contribution of various sequence motifs towards the resulting DNA methylation state. Modeling of this comprehensive dataset revealed that CG density alone is a minor determinant of their unmethylated state. Instead, these data argue for a principal role for transcription factor binding sites, a prediction confirmed by testing synthetic mutant libraries. Taken together, these findings establish the hierarchy between the two cis-encoded mechanisms that define the DNA methylation state and thus the transcriptional competence of CpG islands. DOI: http://dx.doi.org/10.7554/eLife.04094.001 eLife digest Regions of DNA called genes produce the proteins and other molecules that are essential for life. The act of making these molecules is known as gene expression, and being able to switch this process on and off allows cells to adapt to changing conditions. For example, some genes may be turned on in response to injury or may only turn on during waking hours. There are several ways gene expression can be switched on and off. Proteins called transcription factors can bind to DNA and act like a switch that affects nearby genes. Alternatively, special tags called methyl groups can attach to the ‘letters’ that make up the DNA code and turn off gene expression. However, it is not understood how these tags work with transcription factors and other forms of gene regulation. Regions of DNA that boost the expression of a neighboring gene are called promoters. Many promoters in mammals contain repeating patterns of the DNA letters ‘C’ (which is a chemical called cytosine) and ‘G’ (guanine), and these regions are tagged less often than other regions of DNA. This led scientists to wonder whether the DNA sequence itself controls where the tags are placed, but existing experimental techniques made it difficult to establish if DNA sequence alone can prevent tagging. Krebs et al. created a technique that allows thousands of different DNA sequences to be inserted into the same part of the genome of mouse stem cells. Comparing the tagging across these different sequences revealed that the CG pattern is not as closely associated with tagging as was thought. If the CG pattern is repeated many times it does seem to prevent tagging, but sequences with fewer repeats also sometimes escape tagging. Krebs et al. found that a sequence was much less likely to be tagged if the nearby DNA also contains a site that transcription factors can bind to. However, regions with a very high number of CG repeats are able to avoid tagging without help from transcription factors. Krebs et al. found that this behavior is not seen in cancer cells. DNA in cancer cells is heavily tagged, even in CG-rich regions, and transcription factors do not appear to play a major role in directing tagging. The new approach developed by Krebs et al. should benefit researchers working to understand the multiple mechanisms that control gene activity. DOI: http://dx.doi.org/10.7554/eLife.04094.002 |
| Databáze: | OpenAIRE |
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