Udit Dalwadi, Elaina Corrado, Kaelin D Fleming, Brandon E Moeller, Sung-Eun Nam, John E Burke, Calvin K Yip
{"title":"NuA4复合物TINTIN模块的生化表征揭示了核小体相互作用的变构调节。","authors":"Udit Dalwadi, Elaina Corrado, Kaelin D Fleming, Brandon E Moeller, Sung-Eun Nam, John E Burke, Calvin K Yip","doi":"10.1128/mcb.00170-22","DOIUrl":null,"url":null,"abstract":"<p><p><u>T</u>rimer <u>I</u>ndependent of <u>N</u>uA4 involved in <u>T</u>ranscription <u>I</u>nteractions with <u>N</u>ucleosomes (TINTIN) is an integral module of the essential yeast lysine acetyltransferase complex NuA4 that plays key roles in transcription regulation and DNA repair. Composed of Eaf3, Eaf5, and Eaf7, TINTIN mediates targeting of NuA4 to chromatin through the chromodomain-containing subunit Eaf3 that is shared with the Rpd3S histone deacetylase complex. How Eaf3 mediates chromatin interaction in the context of TINTIN and how is it different from what has been observed in Rpd3S is unclear. Here, we reconstituted recombinant TINTIN and its subassemblies and characterized their biochemical and structural properties. Our coimmunoprecipitation, AlphaFold2 modeling, and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses revealed that the Eaf3 MRG domain contacts Eaf7 and this binding induces conformational changes throughout Eaf3. Nucleosome-binding assays showed that Eaf3 and TINTIN interact non-specifically with the DNA on nucleosomes. Furthermore, integration into TINTIN enhances the affinity of Eaf3 toward nucleosomes and this improvement is a result of allosteric activation of the Eaf3 chromodomain. Negative stain electron microscopy (EM) analysis revealed that TINTIN binds to the edge of nucleosomes with increased specificity in the presence of H3K36me3. Collectively, our work provides insights into the dynamics of TINTIN and the mechanism by which its interactions with chromatin are regulated.</p>","PeriodicalId":18658,"journal":{"name":"Molecular and Cellular Biology","volume":"42 11","pages":"e0017022"},"PeriodicalIF":3.2000,"publicationDate":"2022-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9670870/pdf/mcb.00170-22.pdf","citationCount":"2","resultStr":"{\"title\":\"Biochemical Characterization of the TINTIN Module of the NuA4 Complex Reveals Allosteric Regulation of Nucleosome Interaction.\",\"authors\":\"Udit Dalwadi, Elaina Corrado, Kaelin D Fleming, Brandon E Moeller, Sung-Eun Nam, John E Burke, Calvin K Yip\",\"doi\":\"10.1128/mcb.00170-22\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p><u>T</u>rimer <u>I</u>ndependent of <u>N</u>uA4 involved in <u>T</u>ranscription <u>I</u>nteractions with <u>N</u>ucleosomes (TINTIN) is an integral module of the essential yeast lysine acetyltransferase complex NuA4 that plays key roles in transcription regulation and DNA repair. Composed of Eaf3, Eaf5, and Eaf7, TINTIN mediates targeting of NuA4 to chromatin through the chromodomain-containing subunit Eaf3 that is shared with the Rpd3S histone deacetylase complex. How Eaf3 mediates chromatin interaction in the context of TINTIN and how is it different from what has been observed in Rpd3S is unclear. Here, we reconstituted recombinant TINTIN and its subassemblies and characterized their biochemical and structural properties. Our coimmunoprecipitation, AlphaFold2 modeling, and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses revealed that the Eaf3 MRG domain contacts Eaf7 and this binding induces conformational changes throughout Eaf3. Nucleosome-binding assays showed that Eaf3 and TINTIN interact non-specifically with the DNA on nucleosomes. Furthermore, integration into TINTIN enhances the affinity of Eaf3 toward nucleosomes and this improvement is a result of allosteric activation of the Eaf3 chromodomain. Negative stain electron microscopy (EM) analysis revealed that TINTIN binds to the edge of nucleosomes with increased specificity in the presence of H3K36me3. Collectively, our work provides insights into the dynamics of TINTIN and the mechanism by which its interactions with chromatin are regulated.</p>\",\"PeriodicalId\":18658,\"journal\":{\"name\":\"Molecular and Cellular Biology\",\"volume\":\"42 11\",\"pages\":\"e0017022\"},\"PeriodicalIF\":3.2000,\"publicationDate\":\"2022-11-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9670870/pdf/mcb.00170-22.pdf\",\"citationCount\":\"2\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular and Cellular Biology\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1128/mcb.00170-22\",\"RegionNum\":2,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular and Cellular Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1128/mcb.00170-22","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Biochemical Characterization of the TINTIN Module of the NuA4 Complex Reveals Allosteric Regulation of Nucleosome Interaction.
Trimer Independent of NuA4 involved in Transcription Interactions with Nucleosomes (TINTIN) is an integral module of the essential yeast lysine acetyltransferase complex NuA4 that plays key roles in transcription regulation and DNA repair. Composed of Eaf3, Eaf5, and Eaf7, TINTIN mediates targeting of NuA4 to chromatin through the chromodomain-containing subunit Eaf3 that is shared with the Rpd3S histone deacetylase complex. How Eaf3 mediates chromatin interaction in the context of TINTIN and how is it different from what has been observed in Rpd3S is unclear. Here, we reconstituted recombinant TINTIN and its subassemblies and characterized their biochemical and structural properties. Our coimmunoprecipitation, AlphaFold2 modeling, and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses revealed that the Eaf3 MRG domain contacts Eaf7 and this binding induces conformational changes throughout Eaf3. Nucleosome-binding assays showed that Eaf3 and TINTIN interact non-specifically with the DNA on nucleosomes. Furthermore, integration into TINTIN enhances the affinity of Eaf3 toward nucleosomes and this improvement is a result of allosteric activation of the Eaf3 chromodomain. Negative stain electron microscopy (EM) analysis revealed that TINTIN binds to the edge of nucleosomes with increased specificity in the presence of H3K36me3. Collectively, our work provides insights into the dynamics of TINTIN and the mechanism by which its interactions with chromatin are regulated.
期刊介绍:
Molecular and Cellular Biology (MCB) showcases significant discoveries in cellular morphology and function, genome organization, regulation of genetic expression, morphogenesis, and somatic cell genetics. The journal also examines viral systems, publishing papers that emphasize their impact on the cell.