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Comprehensive analysis of interacting proteins and genome-wide location studies of the Sas3-dependent NuA3 histone acetyltransferase complex.

Vicente-Muñoz S, Romero P, Magraner-Pardo L, Martinez-Jimenez CP, Tordera V, Pamblanco M - FEBS Open Bio (2014)

Bottom Line: Our results also revealed that the protein complexes associated with Sas3p presented HAT activity even in the absence of Gcn5p and vice versa.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.

View Article: PubMed Central - PubMed

Affiliation: Structural Biochemistry Laboratory, Centro de Investigación Príncipe Felipe (CIPF), Eduardo Primo Yúfera, 3, 46012 València, Spain.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Deletion or TAP-tagging of SAS3, unlike GCN5, does not induce growth defects. Aliquots of 5 μL and 10-fold serial dilutions of the overnight cultures at OD600 ≈ 0.6 were spotted onto YPDA plates. Cells were cultured at 30 °C and 37 °C for 3 days. The temperature-sensitive HAT2-TAP hif1Δ strain, was used as a control.
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f0005: Deletion or TAP-tagging of SAS3, unlike GCN5, does not induce growth defects. Aliquots of 5 μL and 10-fold serial dilutions of the overnight cultures at OD600 ≈ 0.6 were spotted onto YPDA plates. Cells were cultured at 30 °C and 37 °C for 3 days. The temperature-sensitive HAT2-TAP hif1Δ strain, was used as a control.

Mentions: Sas3p is the HAT catalytic subunit of the NuA3 complex. Sas3p, as well as the extensively studied Gcn5p HAT, act on nucleosomal H3, specifically at K9 and K14 [34], as primary sites of acetylation [35]. To study how the absence of one of these two HATs might affect the composition and/or the acetylation activity of the HAT complexes, new strains were constructed. Firstly, we assessed whether the integration of the TAP tag into the genome affects the growth of yeast strains. As shown in Fig. 1, TAP-tagging or SAS3 gene deletion did not produce defective growth at either 30 °C or 37 °C when compared to the corresponding WT cells (BMA64-1A and BY4742, Fig. 1 upper and bottom panels, respectively). On the contrary, the growth defects observed for deleted or TAP-tagged GCN5 (Fig. 1) can be partially explained by previous observations that gcn5Δ mutant cells accumulate in the G2/M phase of the cell cycle [31]. Phenotypic growth defects in the absence of GCN5 depended on the strain background, and no drastic visual defects were observed in the BY4742 background if compared with BMA64-1A. As a control, HAT2-TAP hif1Δ was used as a temperature-sensitive strain (Fig. 1).


Comprehensive analysis of interacting proteins and genome-wide location studies of the Sas3-dependent NuA3 histone acetyltransferase complex.

Vicente-Muñoz S, Romero P, Magraner-Pardo L, Martinez-Jimenez CP, Tordera V, Pamblanco M - FEBS Open Bio (2014)

Deletion or TAP-tagging of SAS3, unlike GCN5, does not induce growth defects. Aliquots of 5 μL and 10-fold serial dilutions of the overnight cultures at OD600 ≈ 0.6 were spotted onto YPDA plates. Cells were cultured at 30 °C and 37 °C for 3 days. The temperature-sensitive HAT2-TAP hif1Δ strain, was used as a control.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4248121&req=5

f0005: Deletion or TAP-tagging of SAS3, unlike GCN5, does not induce growth defects. Aliquots of 5 μL and 10-fold serial dilutions of the overnight cultures at OD600 ≈ 0.6 were spotted onto YPDA plates. Cells were cultured at 30 °C and 37 °C for 3 days. The temperature-sensitive HAT2-TAP hif1Δ strain, was used as a control.
Mentions: Sas3p is the HAT catalytic subunit of the NuA3 complex. Sas3p, as well as the extensively studied Gcn5p HAT, act on nucleosomal H3, specifically at K9 and K14 [34], as primary sites of acetylation [35]. To study how the absence of one of these two HATs might affect the composition and/or the acetylation activity of the HAT complexes, new strains were constructed. Firstly, we assessed whether the integration of the TAP tag into the genome affects the growth of yeast strains. As shown in Fig. 1, TAP-tagging or SAS3 gene deletion did not produce defective growth at either 30 °C or 37 °C when compared to the corresponding WT cells (BMA64-1A and BY4742, Fig. 1 upper and bottom panels, respectively). On the contrary, the growth defects observed for deleted or TAP-tagged GCN5 (Fig. 1) can be partially explained by previous observations that gcn5Δ mutant cells accumulate in the G2/M phase of the cell cycle [31]. Phenotypic growth defects in the absence of GCN5 depended on the strain background, and no drastic visual defects were observed in the BY4742 background if compared with BMA64-1A. As a control, HAT2-TAP hif1Δ was used as a temperature-sensitive strain (Fig. 1).

Bottom Line: Our results also revealed that the protein complexes associated with Sas3p presented HAT activity even in the absence of Gcn5p and vice versa.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.

View Article: PubMed Central - PubMed

Affiliation: Structural Biochemistry Laboratory, Centro de Investigación Príncipe Felipe (CIPF), Eduardo Primo Yúfera, 3, 46012 València, Spain.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus