Development of Artificial System to Induce Chromatin Loosening in <i>Saccharomyces cerevisiae</i>
In eukaryotic cells, loosening of chromatin causes changes in transcription and DNA replication. The artificial conversion of tightly packed chromatin (heterochromatin) to loosely packed chromatin (euchromatin) enables gene expression and regulates cell differentiation. Although some chemicals conve...
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MDPI AG
2022-08-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/12/8/1138 |
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| author | Ryota Yamamoto Genki Sato Takamitsu Amai Mitsuyoshi Ueda Kouichi Kuroda |
| author_facet | Ryota Yamamoto Genki Sato Takamitsu Amai Mitsuyoshi Ueda Kouichi Kuroda |
| author_sort | Ryota Yamamoto |
| collection | DOAJ |
| description | In eukaryotic cells, loosening of chromatin causes changes in transcription and DNA replication. The artificial conversion of tightly packed chromatin (heterochromatin) to loosely packed chromatin (euchromatin) enables gene expression and regulates cell differentiation. Although some chemicals convert chromatin structures through histone modifications, they lack sequence specificity. This study attempted to establish a novel technology for inducing chromatin loosening in target regions of <i>Saccharomyces cerevisiae</i>. We focused on histone acetylation, which is one of the mechanisms of euchromatin induction. The sequence-recognizing ability of the dead Cas9 (dCas9) and guide RNA (gRNA) complex was used to promote histone acetylation at a targeted genomic locus. We constructed a plasmid to produce a fusion protein consisting of dCas9 and histone acetyltransferase Gcn5 and a plasmid to express gRNA recognizing the upstream region of heterochromatic <i>URA3</i>. Confocal microscopy revealed that the fusion proteins were localized in the nucleus. The yeast strain producing the fusion protein and gRNA grew well in the uracil-deficient medium, while the strain harboring empty plasmids or the strain containing the mutations that cause loss of nucleosomal histone acetylation activity of Gcn5 did not. This suggests that the heterochromatin was loosened as much as euchromatin through nucleosomal histone acetylation. The amount of euchromatic DNA at the target locus increased, indicating that chromatin loosening was induced by our system. Nucleosomal histone acetylation in heterochromatic loci by our developed system is a promising method for inducing euchromatic state in a target locus. |
| first_indexed | 2024-03-09T10:00:25Z |
| format | Article |
| id | doaj.art-62f2431a18084cc59bf146134b634339 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-09T10:00:25Z |
| publishDate | 2022-08-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-62f2431a18084cc59bf146134b6343392023-12-01T23:29:20ZengMDPI AGBiomolecules2218-273X2022-08-01128113810.3390/biom12081138Development of Artificial System to Induce Chromatin Loosening in <i>Saccharomyces cerevisiae</i>Ryota Yamamoto0Genki Sato1Takamitsu Amai2Mitsuyoshi Ueda3Kouichi Kuroda4Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, JapanDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, JapanDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, JapanDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, JapanDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, JapanIn eukaryotic cells, loosening of chromatin causes changes in transcription and DNA replication. The artificial conversion of tightly packed chromatin (heterochromatin) to loosely packed chromatin (euchromatin) enables gene expression and regulates cell differentiation. Although some chemicals convert chromatin structures through histone modifications, they lack sequence specificity. This study attempted to establish a novel technology for inducing chromatin loosening in target regions of <i>Saccharomyces cerevisiae</i>. We focused on histone acetylation, which is one of the mechanisms of euchromatin induction. The sequence-recognizing ability of the dead Cas9 (dCas9) and guide RNA (gRNA) complex was used to promote histone acetylation at a targeted genomic locus. We constructed a plasmid to produce a fusion protein consisting of dCas9 and histone acetyltransferase Gcn5 and a plasmid to express gRNA recognizing the upstream region of heterochromatic <i>URA3</i>. Confocal microscopy revealed that the fusion proteins were localized in the nucleus. The yeast strain producing the fusion protein and gRNA grew well in the uracil-deficient medium, while the strain harboring empty plasmids or the strain containing the mutations that cause loss of nucleosomal histone acetylation activity of Gcn5 did not. This suggests that the heterochromatin was loosened as much as euchromatin through nucleosomal histone acetylation. The amount of euchromatic DNA at the target locus increased, indicating that chromatin loosening was induced by our system. Nucleosomal histone acetylation in heterochromatic loci by our developed system is a promising method for inducing euchromatic state in a target locus.https://www.mdpi.com/2218-273X/12/8/1138chromatinhistone acetyltransferaseepigeneticsCRISPR/dCas9 system<i>Saccharomyces cerevisiae</i> |
| spellingShingle | Ryota Yamamoto Genki Sato Takamitsu Amai Mitsuyoshi Ueda Kouichi Kuroda Development of Artificial System to Induce Chromatin Loosening in <i>Saccharomyces cerevisiae</i> Biomolecules chromatin histone acetyltransferase epigenetics CRISPR/dCas9 system <i>Saccharomyces cerevisiae</i> |
| title | Development of Artificial System to Induce Chromatin Loosening in <i>Saccharomyces cerevisiae</i> |
| title_full | Development of Artificial System to Induce Chromatin Loosening in <i>Saccharomyces cerevisiae</i> |
| title_fullStr | Development of Artificial System to Induce Chromatin Loosening in <i>Saccharomyces cerevisiae</i> |
| title_full_unstemmed | Development of Artificial System to Induce Chromatin Loosening in <i>Saccharomyces cerevisiae</i> |
| title_short | Development of Artificial System to Induce Chromatin Loosening in <i>Saccharomyces cerevisiae</i> |
| title_sort | development of artificial system to induce chromatin loosening in i saccharomyces cerevisiae i |
| topic | chromatin histone acetyltransferase epigenetics CRISPR/dCas9 system <i>Saccharomyces cerevisiae</i> |
| url | https://www.mdpi.com/2218-273X/12/8/1138 |
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