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Intra- and inter-nucleosomal interactions of the histone H4 tail revealed with a human nucleosome core particle with genetically-incorporated H4 tetra-acetylation.

Wakamori M, Fujii Y, Suka N, Shirouzu M, Sakamoto K, Umehara T, Yokoyama S - Sci Rep (2015)

Bottom Line: However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4.In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP.The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.

ABSTRACT
Post-translational modifications (PTMs) of histones, such as lysine acetylation of the N-terminal tails, play crucial roles in controlling gene expression. Due to the difficulty in reconstituting site-specifically acetylated nucleosomes with crystallization quality, structural analyses of histone acetylation are currently performed using synthesized tail peptides. Through engineering of the genetic code, translation termination, and cell-free protein synthesis, we reconstituted human H4-mono- to tetra-acetylated nucleosome core particles (NCPs), and solved the crystal structures of the H4-K5/K8/K12/K16-tetra-acetylated NCP and unmodified NCP at 2.4 Å and 2.2 Å resolutions, respectively. The structure of the H4-tetra-acetylated NCP resembled that of the unmodified NCP, and the DNA wrapped the histone octamer as precisely as in the unmodified NCP. However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4. In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP. The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs.

No MeSH data available.


Related in: MedlinePlus

Electrophoresis mobility shift assay of the binding between H4-acetylated NCPs and the TAF1 double bromodomain (TAF1-BrD).The indicated ratios of the TAF1-BrD were mixed with the respective NCPs. The bands corresponding to the TAF1-BrD-bound NCP, the TAF1-BrD-unbound NCP, and the free DNA are respectively indicated by the arrows and brackets on the left. The positions of the wells are indicated by arrowheads.
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f6: Electrophoresis mobility shift assay of the binding between H4-acetylated NCPs and the TAF1 double bromodomain (TAF1-BrD).The indicated ratios of the TAF1-BrD were mixed with the respective NCPs. The bands corresponding to the TAF1-BrD-bound NCP, the TAF1-BrD-unbound NCP, and the free DNA are respectively indicated by the arrows and brackets on the left. The positions of the wells are indicated by arrowheads.

Mentions: Lysine acetylation of the histone tail reportedly enhances the binding with a bromodomain, as assessed by biochemical analyses performed mostly with acetylated peptides as binding substrates, but very rarely with modified nucleosomes3751. To validate the utility of the Kac-incorporated NCPs, we examined the stoichiometric association between the prepared Kac-incorporated NCPs and the double bromodomain of TAF1 (TAF1-BrD), which reportedly interacts with acetylated H452, by a native PAGE mobility shift assay. When the unmodified NCP was used as the substrate, partial formation of the NCP•TAF1-BrD complex was detected at a high molar ratio of TAF1-BrD (Fig. 6). When the single Kac-incorporated NCPs at K12 and K16 were used, the band shifts were also modest. When the number of acetylation sites was increased to 2 (both K5 and K8) or 4 (K5/K8/K12/K16), the binding affinity of the TAF1-BrD for the acetylated NCP increased (Fig. 6). The dissociation constants estimated from triplicate electrophoresis mobility shift assays were: 19.3 ± 0.3 μM for the unmodified NCP, 12.3 ± 1.9 μM for the K12-acetylated NCP, 13.3 ± 1.6 μM for the K16-acetylated NCP, 6.1 ± 0.4 μM for the K5/K8-di-acetylated NCP, and 3.9 ± 0.2 μM for the K5/K8/K12/K16-tetra-acetylated NCP. These results indicate that the affinity of the TAF1-BrD for the NCP is enhanced by increased numbers of H4 acetylation sites, as expected. However, the gradient of the affinity is rather modest, as compared with the analysis using acetylated tail peptides52. We also detected the weak affinity of the TAF1-BrD to free nucleosomal DNA at a high molar ratio (Fig. 6, bottom), which may be a reason for the modest affinity gradient for the NCP (see Discussion).


Intra- and inter-nucleosomal interactions of the histone H4 tail revealed with a human nucleosome core particle with genetically-incorporated H4 tetra-acetylation.

Wakamori M, Fujii Y, Suka N, Shirouzu M, Sakamoto K, Umehara T, Yokoyama S - Sci Rep (2015)

Electrophoresis mobility shift assay of the binding between H4-acetylated NCPs and the TAF1 double bromodomain (TAF1-BrD).The indicated ratios of the TAF1-BrD were mixed with the respective NCPs. The bands corresponding to the TAF1-BrD-bound NCP, the TAF1-BrD-unbound NCP, and the free DNA are respectively indicated by the arrows and brackets on the left. The positions of the wells are indicated by arrowheads.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660432&req=5

f6: Electrophoresis mobility shift assay of the binding between H4-acetylated NCPs and the TAF1 double bromodomain (TAF1-BrD).The indicated ratios of the TAF1-BrD were mixed with the respective NCPs. The bands corresponding to the TAF1-BrD-bound NCP, the TAF1-BrD-unbound NCP, and the free DNA are respectively indicated by the arrows and brackets on the left. The positions of the wells are indicated by arrowheads.
Mentions: Lysine acetylation of the histone tail reportedly enhances the binding with a bromodomain, as assessed by biochemical analyses performed mostly with acetylated peptides as binding substrates, but very rarely with modified nucleosomes3751. To validate the utility of the Kac-incorporated NCPs, we examined the stoichiometric association between the prepared Kac-incorporated NCPs and the double bromodomain of TAF1 (TAF1-BrD), which reportedly interacts with acetylated H452, by a native PAGE mobility shift assay. When the unmodified NCP was used as the substrate, partial formation of the NCP•TAF1-BrD complex was detected at a high molar ratio of TAF1-BrD (Fig. 6). When the single Kac-incorporated NCPs at K12 and K16 were used, the band shifts were also modest. When the number of acetylation sites was increased to 2 (both K5 and K8) or 4 (K5/K8/K12/K16), the binding affinity of the TAF1-BrD for the acetylated NCP increased (Fig. 6). The dissociation constants estimated from triplicate electrophoresis mobility shift assays were: 19.3 ± 0.3 μM for the unmodified NCP, 12.3 ± 1.9 μM for the K12-acetylated NCP, 13.3 ± 1.6 μM for the K16-acetylated NCP, 6.1 ± 0.4 μM for the K5/K8-di-acetylated NCP, and 3.9 ± 0.2 μM for the K5/K8/K12/K16-tetra-acetylated NCP. These results indicate that the affinity of the TAF1-BrD for the NCP is enhanced by increased numbers of H4 acetylation sites, as expected. However, the gradient of the affinity is rather modest, as compared with the analysis using acetylated tail peptides52. We also detected the weak affinity of the TAF1-BrD to free nucleosomal DNA at a high molar ratio (Fig. 6, bottom), which may be a reason for the modest affinity gradient for the NCP (see Discussion).

Bottom Line: However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4.In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP.The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs.

View Article: PubMed Central - PubMed

Affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.

ABSTRACT
Post-translational modifications (PTMs) of histones, such as lysine acetylation of the N-terminal tails, play crucial roles in controlling gene expression. Due to the difficulty in reconstituting site-specifically acetylated nucleosomes with crystallization quality, structural analyses of histone acetylation are currently performed using synthesized tail peptides. Through engineering of the genetic code, translation termination, and cell-free protein synthesis, we reconstituted human H4-mono- to tetra-acetylated nucleosome core particles (NCPs), and solved the crystal structures of the H4-K5/K8/K12/K16-tetra-acetylated NCP and unmodified NCP at 2.4 Å and 2.2 Å resolutions, respectively. The structure of the H4-tetra-acetylated NCP resembled that of the unmodified NCP, and the DNA wrapped the histone octamer as precisely as in the unmodified NCP. However, the B-factors were significantly increased for the peripheral DNAs near the N-terminal tail of the intra- or inter-nucleosomal H4. In contrast, the B-factors were negligibly affected by the H4 tetra-acetylation in histone core residues, including those composing the acidic patch, and at H4-R23, which interacts with the acidic patch of the neighboring NCP. The present study revealed that the H4 tetra-acetylation impairs NCP self-association by changing the interactions of the H4 tail with DNA, and is the first demonstration of crystallization quality NCPs reconstituted with genuine PTMs.

No MeSH data available.


Related in: MedlinePlus