Comprehensive analysis of interacting proteins and genome-wide location studies of the Sas3-dependent NuA3 histone acetyltransferase complex
| Autor: | Mercè Pamblanco, Lorena Magraner-Pardo, Sara Vicente-Muñoz, Paco Romero, Vicente Tordera, Celia P. Martinez-Jimenez |
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| Rok vydání: | 2014 |
| Předmět: |
nt
nucleotide PTM post-translational modification NuA3 histone acetyltransferase complex ChIP-on-chip chromatin immunoprecipitation with genome-wide location arrays Biology Article General Biochemistry Genetics and Molecular Biology Chromatin remodeling Histones Histone H3 NuA3 nucleosomal acetyltransferase of histone H3 Histone H1 Histone H2A Pdp3 TAP–MS strategy Histone code lcsh:QH301-705.5 TAP tandem affinity purification Genetics RNAPII RNA polymerase II Histone acetyltransferase WCE whole cell extract SAGA Spt-Ada-Gcn acetyltransferase WT wild-type Chromatin Yeast Cell biology ChIP-on-chip lcsh:Biology (General) Histone methyltransferase biology.protein HAT histone acetyltransferase TSS transcription start site |
| Zdroj: | FEBS Open Bio FEBS Open Bio, Vol 4, Iss C, Pp 996-1006 (2014) |
| ISSN: | 2211-5463 |
| Popis: | Highlights • We characterise Sas3p and Gcn5p active HAT complexes in WT and deleted TAP-strains. • We confirm that Pdp3p interacts with NuA3, histones and chromatin regulators. • Pdp3p MS-analysis reveals its phosphorylation, ubiquitination and methylation. • Sas3p can substitute Gcn5p in acetylation of histone H3K14 but not of H3K9. • Genome-wide profiling of Sas3p supports its involvement in transcriptional elongation. Histone acetylation affects several aspects of gene regulation, from chromatin remodelling to gene expression, by modulating the interplay between chromatin and key transcriptional regulators. The exact molecular mechanism underlying acetylation patterns and crosstalk with other epigenetic modifications requires further investigation. In budding yeast, these epigenetic markers are produced partly by histone acetyltransferase enzymes, which act as multi-protein complexes. The Sas3-dependent NuA3 complex has received less attention than other histone acetyltransferases (HAT), such as Gcn5-dependent complexes. Here, we report our analysis of Sas3p-interacting proteins using tandem affinity purification (TAP), coupled with mass spectrometry. This analysis revealed Pdp3p, a recently described component of NuA3, to be one of the most abundant Sas3p-interacting proteins. The PDP3 gene, was TAP-tagged and protein complex purification confirmed that Pdp3p co-purified with the NuA3 protein complex, histones, and several transcription-related and chromatin remodelling proteins. Our results also revealed that the protein complexes associated with Sas3p presented HAT activity even in the absence of Gcn5p and vice versa. We also provide evidence that Sas3p cannot substitute Gcn5p in acetylation of lysine 9 in histone H3 in vivo. Genome-wide occupancy of Sas3p using ChIP-on-chip tiled microarrays showed that Sas3p was located preferentially within the 5′-half of the coding regions of target genes, indicating its probable involvement in the transcriptional elongation process. Hence, this work further characterises the function and regulation of the NuA3 complex by identifying novel post-translational modifications in Pdp3p, additional Pdp3p-co-purifying chromatin regulatory proteins involved in chromatin-modifying complex dynamics and gene regulation, and a subset of genes whose transcriptional elongation is controlled by this complex. |
| Databáze: | OpenAIRE |
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