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Binding of PHF1 Tudor to H3K36me3 enhances nucleosome accessibility.

Musselman CA, Gibson MD, Hartwick EW, North JA, Gatchalian J, Poirier MG, Kutateladze TG - Nat Commun (2013)

Bottom Line: Here we detail the molecular mechanism of binding of the Tudor domain to the H3KC36me3-nucleosome core particle (H3KC36me3-NCP).Binding of the PHF1 Tudor domain to the H3KC36me3-NCP stabilizes the nucleosome in a conformation in which the nucleosomal DNA is more accessible to DNA-binding regulatory proteins.Our data provide a mechanistic explanation for the consequence of reading of the active mark H3K36me3 by the PHF1 Tudor domain.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado 80045, USA [2].

ABSTRACT
The Tudor domain of human PHF1 recognizes trimethylated lysine 36 of histone H3 (H3K36me3). This interaction modulates the methyltransferase activity of the PRC2 complex and has a role in retention of PHF1 at DNA damage sites. We have previously determined the structural basis for the association of Tudor with a methylated histone peptide. Here we detail the molecular mechanism of binding of the Tudor domain to the H3KC36me3-nucleosome core particle (H3KC36me3-NCP). Using a combination of TROSY NMR and FRET, we show that Tudor concomitantly interacts with H3K36me3 and DNA. Binding of the PHF1 Tudor domain to the H3KC36me3-NCP stabilizes the nucleosome in a conformation in which the nucleosomal DNA is more accessible to DNA-binding regulatory proteins. Our data provide a mechanistic explanation for the consequence of reading of the active mark H3K36me3 by the PHF1 Tudor domain.

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Tudor interacts non-specifically with double stranded DNA(a) 1H, 15N HSQC overlay of Tudor in the presence of increasing concentrations of a 10 bp fragment of double stranded (top) or single stranded (middle) DNA or the double stranded Widom 601 DNA sequence (bottom). (b) A plot of the normalized chemical shift change induced upon titration of the 10 bp DS-DNA fragment as a function of Tudor residue, with changes greater than the average plus 1/3 and average plus 2/3 the standard deviation shown in light blue and dark blue, respectively. (c). Residues showing significant chemical shift changes in (b) are mapped onto a surface representation of Tudor. (d) The Tudor domain binds weaker to DS-DNA and stronger to H3K36me3. Superimposed 1H, 15N HSQC spectra of the Tudor domain collected upon addition of a 10 bp DS-DNA fragment (fast exchange regime), followed by addition of H3K36me3 peptide (intermediate exchange regime). Molar ratios of Tudor to DNA to peptide are shown to the right.
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Figure 3: Tudor interacts non-specifically with double stranded DNA(a) 1H, 15N HSQC overlay of Tudor in the presence of increasing concentrations of a 10 bp fragment of double stranded (top) or single stranded (middle) DNA or the double stranded Widom 601 DNA sequence (bottom). (b) A plot of the normalized chemical shift change induced upon titration of the 10 bp DS-DNA fragment as a function of Tudor residue, with changes greater than the average plus 1/3 and average plus 2/3 the standard deviation shown in light blue and dark blue, respectively. (c). Residues showing significant chemical shift changes in (b) are mapped onto a surface representation of Tudor. (d) The Tudor domain binds weaker to DS-DNA and stronger to H3K36me3. Superimposed 1H, 15N HSQC spectra of the Tudor domain collected upon addition of a 10 bp DS-DNA fragment (fast exchange regime), followed by addition of H3K36me3 peptide (intermediate exchange regime). Molar ratios of Tudor to DNA to peptide are shown to the right.

Mentions: To explore whether the PHF1 Tudor domain makes contacts other than to the histone tail sequence in the nucleosome, we tested its ability to bind DNA. Titration of a 10 bp double-stranded (DS) DNA fragment into 15N-labelled Tudor induced substantial chemical shift changes in the protein (Fig. 3a, first panel). In contrast, titration of a single strand of the same DNA fragment did not lead to resonance perturbations, indicating that this interaction is specific for DS-DNA and likely depends on the presence of a major/minor groove (Fig. 3a, second panel). Analysis of the chemical shift perturbations (CSPs) afforded a Kd of 201 μM for binding of the Tudor domain to the 10 bp DS-DNA. Furthermore, the 601 DNA sequence, which we used to reconstitute the H3KC36me3-NCP, caused large CSPs in the Tudor domain, which were generally similar in direction to those seen upon addition of the 10 bp DS-DNA, however the presence of ~14 major/minor grooves in the 601 DNA construct, and thus non-stoichiometric association, precluded a straightforward quantitative analysis of this interaction (Fig. 3a, third panel). Nevertheless, the pattern of CSPs inferred that the Tudor domain binds to DS-DNA in a non-specific manner. A plot of the resonance perturbations as a function of Tudor residue revealed that the residues involved in the interaction with DNA lay along the β-barrel sides and around the H3K36me3 binding pocket (Fig. 3b, c). Subsequent titration of the H3K36me3 peptide into the 10 bp DS-DNA-bound Tudor domain caused additional CSPs in the protein, and the intermediate exchange regime indicated a stronger interaction with H3K36me3 (Fig. 3d). Together these data suggest that within the intact NCP the Tudor domain can concomitantly interact specifically with the methylated histone H3 tail as well as non-specifically with the nucleosomal DNA.


Binding of PHF1 Tudor to H3K36me3 enhances nucleosome accessibility.

Musselman CA, Gibson MD, Hartwick EW, North JA, Gatchalian J, Poirier MG, Kutateladze TG - Nat Commun (2013)

Tudor interacts non-specifically with double stranded DNA(a) 1H, 15N HSQC overlay of Tudor in the presence of increasing concentrations of a 10 bp fragment of double stranded (top) or single stranded (middle) DNA or the double stranded Widom 601 DNA sequence (bottom). (b) A plot of the normalized chemical shift change induced upon titration of the 10 bp DS-DNA fragment as a function of Tudor residue, with changes greater than the average plus 1/3 and average plus 2/3 the standard deviation shown in light blue and dark blue, respectively. (c). Residues showing significant chemical shift changes in (b) are mapped onto a surface representation of Tudor. (d) The Tudor domain binds weaker to DS-DNA and stronger to H3K36me3. Superimposed 1H, 15N HSQC spectra of the Tudor domain collected upon addition of a 10 bp DS-DNA fragment (fast exchange regime), followed by addition of H3K36me3 peptide (intermediate exchange regime). Molar ratios of Tudor to DNA to peptide are shown to the right.
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Related In: Results  -  Collection

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Figure 3: Tudor interacts non-specifically with double stranded DNA(a) 1H, 15N HSQC overlay of Tudor in the presence of increasing concentrations of a 10 bp fragment of double stranded (top) or single stranded (middle) DNA or the double stranded Widom 601 DNA sequence (bottom). (b) A plot of the normalized chemical shift change induced upon titration of the 10 bp DS-DNA fragment as a function of Tudor residue, with changes greater than the average plus 1/3 and average plus 2/3 the standard deviation shown in light blue and dark blue, respectively. (c). Residues showing significant chemical shift changes in (b) are mapped onto a surface representation of Tudor. (d) The Tudor domain binds weaker to DS-DNA and stronger to H3K36me3. Superimposed 1H, 15N HSQC spectra of the Tudor domain collected upon addition of a 10 bp DS-DNA fragment (fast exchange regime), followed by addition of H3K36me3 peptide (intermediate exchange regime). Molar ratios of Tudor to DNA to peptide are shown to the right.
Mentions: To explore whether the PHF1 Tudor domain makes contacts other than to the histone tail sequence in the nucleosome, we tested its ability to bind DNA. Titration of a 10 bp double-stranded (DS) DNA fragment into 15N-labelled Tudor induced substantial chemical shift changes in the protein (Fig. 3a, first panel). In contrast, titration of a single strand of the same DNA fragment did not lead to resonance perturbations, indicating that this interaction is specific for DS-DNA and likely depends on the presence of a major/minor groove (Fig. 3a, second panel). Analysis of the chemical shift perturbations (CSPs) afforded a Kd of 201 μM for binding of the Tudor domain to the 10 bp DS-DNA. Furthermore, the 601 DNA sequence, which we used to reconstitute the H3KC36me3-NCP, caused large CSPs in the Tudor domain, which were generally similar in direction to those seen upon addition of the 10 bp DS-DNA, however the presence of ~14 major/minor grooves in the 601 DNA construct, and thus non-stoichiometric association, precluded a straightforward quantitative analysis of this interaction (Fig. 3a, third panel). Nevertheless, the pattern of CSPs inferred that the Tudor domain binds to DS-DNA in a non-specific manner. A plot of the resonance perturbations as a function of Tudor residue revealed that the residues involved in the interaction with DNA lay along the β-barrel sides and around the H3K36me3 binding pocket (Fig. 3b, c). Subsequent titration of the H3K36me3 peptide into the 10 bp DS-DNA-bound Tudor domain caused additional CSPs in the protein, and the intermediate exchange regime indicated a stronger interaction with H3K36me3 (Fig. 3d). Together these data suggest that within the intact NCP the Tudor domain can concomitantly interact specifically with the methylated histone H3 tail as well as non-specifically with the nucleosomal DNA.

Bottom Line: Here we detail the molecular mechanism of binding of the Tudor domain to the H3KC36me3-nucleosome core particle (H3KC36me3-NCP).Binding of the PHF1 Tudor domain to the H3KC36me3-NCP stabilizes the nucleosome in a conformation in which the nucleosomal DNA is more accessible to DNA-binding regulatory proteins.Our data provide a mechanistic explanation for the consequence of reading of the active mark H3K36me3 by the PHF1 Tudor domain.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado 80045, USA [2].

ABSTRACT
The Tudor domain of human PHF1 recognizes trimethylated lysine 36 of histone H3 (H3K36me3). This interaction modulates the methyltransferase activity of the PRC2 complex and has a role in retention of PHF1 at DNA damage sites. We have previously determined the structural basis for the association of Tudor with a methylated histone peptide. Here we detail the molecular mechanism of binding of the Tudor domain to the H3KC36me3-nucleosome core particle (H3KC36me3-NCP). Using a combination of TROSY NMR and FRET, we show that Tudor concomitantly interacts with H3K36me3 and DNA. Binding of the PHF1 Tudor domain to the H3KC36me3-NCP stabilizes the nucleosome in a conformation in which the nucleosomal DNA is more accessible to DNA-binding regulatory proteins. Our data provide a mechanistic explanation for the consequence of reading of the active mark H3K36me3 by the PHF1 Tudor domain.

Show MeSH
Related in: MedlinePlus