Limits...
Compensatory interactions between Sir3p and the nucleosomal LRS surface imply their direct interaction.

Norris A, Bianchet MA, Boeke JD - PLoS Genet. (2008)

Bottom Line: We found that all alleles of the SIR3 BAH domain were able to 1) generally suppress the loss of telomeric silencing of LRS alleles, but 2) could not suppress SIN (Swi/Snf Independent) alleles or 3) could not suppress the telomeric silencing defect of H4 tail alleles.Moreover, we noticed a complementary trend in the electrostatic changes resulting from most of the histone mutations that gain or lose silencing and the suppressor alleles isolated in SIR3, and the genes for histones H3 and H4.Our results provide genetic evidence for recent data suggesting that the Sir3p BAH domain directly binds the LRS domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

ABSTRACT
The previously identified LRS (Loss of rDNA Silencing) domain of the nucleosome is critically important for silencing at both ribosomal DNA and telomeres. To understand the function of the LRS surface in silencing, we performed an EMS mutagenesis screen to identify suppressors of the H3 A75V LRS allele. We identified dominant and recessive mutations in histones H3, H4, and dominant mutations in the BAH (Bromo Adjacent Homology) domain of SIR3. We further characterized a surface of Sir3p critical for silencing via the LRS surface. We found that all alleles of the SIR3 BAH domain were able to 1) generally suppress the loss of telomeric silencing of LRS alleles, but 2) could not suppress SIN (Swi/Snf Independent) alleles or 3) could not suppress the telomeric silencing defect of H4 tail alleles. Moreover, we noticed a complementary trend in the electrostatic changes resulting from most of the histone mutations that gain or lose silencing and the suppressor alleles isolated in SIR3, and the genes for histones H3 and H4. Mutations in H3 and H4 genes that lose silencing tend to make the LRS surface more electronegative, whereas mutations that increase silencing make it less electronegative. Conversely, suppressors of LRS alleles in either SIR3, histone H3, or H4 also tend to make their respective surfaces less electronegative. Our results provide genetic evidence for recent data suggesting that the Sir3p BAH domain directly binds the LRS domain. Based on these findings, we propose an electrostatic model for how an extensive surface on the Sir3p BAH domain may regulate docking onto the LRS surface.

Show MeSH

Related in: MedlinePlus

Model for Sir3p BAH domain binding to the LRS surface.The 2Fl7 [35] crystal structure was docked to the 1ID3 nucleosome structure [29]. The helix α8 of Sir3p BAH packs against a group of Histone alpha helices consisting of H2B, H4, and H3. This juxtaposes the LRS surface with the BAH domain found to be important for suppression of LRS alleles. (A) Sighted along the SHL+/−3.5 axis, the DNA was removed to show the details of the docking. (B) A close-up of the docking structure to show the juxtaposition of the LRS surface and the Sir3p BAH domain suppressor residues. (C) An example of potential inhibitory interactions between H3 D77 and Sir3p E178. The introduction of an asparagine at H3 position 77 would ameliorate the inhibitory interactions. (D) A 90° rotation about the X axis from A, showing an overview of the docked structure. The DNA has been removed to show details of the docking. (E) A close-up of the key residues Sir3p D205 and H3 K79. H4 E74 could also be destabilizing, considering its proximity to D205. (F) Showing the interactions between the LRS residues H3 T80, D81, and H4 K79, and key residues W86 and E84. The images were made using PyMOL [62], and the docked structure is available as Datasets S1 and S2.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2587916&req=5

pgen-1000301-g007: Model for Sir3p BAH domain binding to the LRS surface.The 2Fl7 [35] crystal structure was docked to the 1ID3 nucleosome structure [29]. The helix α8 of Sir3p BAH packs against a group of Histone alpha helices consisting of H2B, H4, and H3. This juxtaposes the LRS surface with the BAH domain found to be important for suppression of LRS alleles. (A) Sighted along the SHL+/−3.5 axis, the DNA was removed to show the details of the docking. (B) A close-up of the docking structure to show the juxtaposition of the LRS surface and the Sir3p BAH domain suppressor residues. (C) An example of potential inhibitory interactions between H3 D77 and Sir3p E178. The introduction of an asparagine at H3 position 77 would ameliorate the inhibitory interactions. (D) A 90° rotation about the X axis from A, showing an overview of the docked structure. The DNA has been removed to show details of the docking. (E) A close-up of the key residues Sir3p D205 and H3 K79. H4 E74 could also be destabilizing, considering its proximity to D205. (F) Showing the interactions between the LRS residues H3 T80, D81, and H4 K79, and key residues W86 and E84. The images were made using PyMOL [62], and the docked structure is available as Datasets S1 and S2.

Mentions: Genetic suppressors of a defective nucleosome that has lost the ability to bind Sir3p, should reveal side-chains of Sir3 or the nucleosome that would either normally make inhibitory interactions between Sir3 and the nucleosome, or reveal neutral interactions that could be improved by the introduction of a new side-chain. Substitutions of H3 D77 have been isolated as enhancers of telomeric silencing (Table 2). The histone suppressor allele H3 D77N and D77G are the strongest intragenic suppressors of H3 A75V, suppressing both its telomeric and rDNA silencing phenotypes, and the nature of these negative− > uncharged substitutions are consistent with the simple notion that electrostatics underlie LRS surface function. Similarly, many SIR3 suppressor alleles decrease the negative charge of the BAH domain surface. These results are all consistent with the hypothesis that electronegative regions of the nucleosome and Sir3 normally repel each other in wild-type yeast to some extent and it is the slightly unstable nature of this interaction that underlies silencing. Thus what the mutants do is decrease repulsion, which presumably increases nucleosome binding. Our genetics suggested that two surfaces, one on the nucleosome and one on Sir3p, could directly interact. To this end, we performed studies to attempt to dock the BAH domain on the nucleosome using the mutated “patches” of these surfaces as guides. Considering the large positively charged stretch of amino acids 24 and 34 in SIR3 we surmised that this section was likely to lie in proximity to the DNA. Additionally, there is a deep cleft in the nucleosome neighboring the LRS domain created by the H2B-H4 four helix bundle at the interface between the H2A/B dimer and the H3/4 tetramer (see Figure 7A and 7B). Guided by these constraints, the helix α8 of the BAH domain was readily docked into this cleft, maintaining the packing of the α-helices of H4 and H2B that make up the proposed docking site (see Figure 7B). The area buried in this structure is approximately 2300 Å2, consistent with a strong interaction. Importantly, this configuration directly juxtaposes the LRS domain with the surface of the BAH domain identified here (Figure 7A–7F). We looked deeper into the structure to find evidence of consistent electrostatic interactions between the two proteins. Remarkably, we found that many residues of both surfaces not only line up well, with few clashes, but importantly, we found adjacencies/interactions consistent with the observed silencing-inhibitory and silencing-enhancing mutations isolated (Figure 7). The most frequently isolated SIR3 suppressor mutants were D205N, L138P, and W86R. The importance of these residues in the docking structure are demonstrated in Figure 7E and 7F. SIR3 D205 and L138 are all in close proximity (within 5 Å) to each other and to H3 K79. Given the importance of H3 K79 methylation in Sir3p binding to the nucleosome, we also found in this proposed docking scheme that tri-methylation of H3 K79 is predicted to sterically hinder binding of Sir3p to the LRS surface (Figure 7E). SIR3 W86 is within 5 Å of H3 T80, H3 D81 and H4 K79. A substitution of an arginine (R) at position W86 could enhance an electrostatic interaction between H3 D81 and Sir3p. Also H3 D77 makes potentially inhibitory repulsive interactions with Sir3p-E178 (Figure 7C), which would be ameliorated by introduction of glycine (G) or asparagine (N) residues into either partner. There are of course stabilizing interactions that would not necessarily be revealed by our mutagenesis but were revealed in the docking study. While our genetic data revealed important side-chains in limiting the interaction between Sir3p and the nucleosome, the docking model showed that there are potential attractive forces as well, namely the α7 helix of the Sir3p BAH domain makes favorable contacts with helices αC and α3 of H2B.


Compensatory interactions between Sir3p and the nucleosomal LRS surface imply their direct interaction.

Norris A, Bianchet MA, Boeke JD - PLoS Genet. (2008)

Model for Sir3p BAH domain binding to the LRS surface.The 2Fl7 [35] crystal structure was docked to the 1ID3 nucleosome structure [29]. The helix α8 of Sir3p BAH packs against a group of Histone alpha helices consisting of H2B, H4, and H3. This juxtaposes the LRS surface with the BAH domain found to be important for suppression of LRS alleles. (A) Sighted along the SHL+/−3.5 axis, the DNA was removed to show the details of the docking. (B) A close-up of the docking structure to show the juxtaposition of the LRS surface and the Sir3p BAH domain suppressor residues. (C) An example of potential inhibitory interactions between H3 D77 and Sir3p E178. The introduction of an asparagine at H3 position 77 would ameliorate the inhibitory interactions. (D) A 90° rotation about the X axis from A, showing an overview of the docked structure. The DNA has been removed to show details of the docking. (E) A close-up of the key residues Sir3p D205 and H3 K79. H4 E74 could also be destabilizing, considering its proximity to D205. (F) Showing the interactions between the LRS residues H3 T80, D81, and H4 K79, and key residues W86 and E84. The images were made using PyMOL [62], and the docked structure is available as Datasets S1 and S2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2587916&req=5

pgen-1000301-g007: Model for Sir3p BAH domain binding to the LRS surface.The 2Fl7 [35] crystal structure was docked to the 1ID3 nucleosome structure [29]. The helix α8 of Sir3p BAH packs against a group of Histone alpha helices consisting of H2B, H4, and H3. This juxtaposes the LRS surface with the BAH domain found to be important for suppression of LRS alleles. (A) Sighted along the SHL+/−3.5 axis, the DNA was removed to show the details of the docking. (B) A close-up of the docking structure to show the juxtaposition of the LRS surface and the Sir3p BAH domain suppressor residues. (C) An example of potential inhibitory interactions between H3 D77 and Sir3p E178. The introduction of an asparagine at H3 position 77 would ameliorate the inhibitory interactions. (D) A 90° rotation about the X axis from A, showing an overview of the docked structure. The DNA has been removed to show details of the docking. (E) A close-up of the key residues Sir3p D205 and H3 K79. H4 E74 could also be destabilizing, considering its proximity to D205. (F) Showing the interactions between the LRS residues H3 T80, D81, and H4 K79, and key residues W86 and E84. The images were made using PyMOL [62], and the docked structure is available as Datasets S1 and S2.
Mentions: Genetic suppressors of a defective nucleosome that has lost the ability to bind Sir3p, should reveal side-chains of Sir3 or the nucleosome that would either normally make inhibitory interactions between Sir3 and the nucleosome, or reveal neutral interactions that could be improved by the introduction of a new side-chain. Substitutions of H3 D77 have been isolated as enhancers of telomeric silencing (Table 2). The histone suppressor allele H3 D77N and D77G are the strongest intragenic suppressors of H3 A75V, suppressing both its telomeric and rDNA silencing phenotypes, and the nature of these negative− > uncharged substitutions are consistent with the simple notion that electrostatics underlie LRS surface function. Similarly, many SIR3 suppressor alleles decrease the negative charge of the BAH domain surface. These results are all consistent with the hypothesis that electronegative regions of the nucleosome and Sir3 normally repel each other in wild-type yeast to some extent and it is the slightly unstable nature of this interaction that underlies silencing. Thus what the mutants do is decrease repulsion, which presumably increases nucleosome binding. Our genetics suggested that two surfaces, one on the nucleosome and one on Sir3p, could directly interact. To this end, we performed studies to attempt to dock the BAH domain on the nucleosome using the mutated “patches” of these surfaces as guides. Considering the large positively charged stretch of amino acids 24 and 34 in SIR3 we surmised that this section was likely to lie in proximity to the DNA. Additionally, there is a deep cleft in the nucleosome neighboring the LRS domain created by the H2B-H4 four helix bundle at the interface between the H2A/B dimer and the H3/4 tetramer (see Figure 7A and 7B). Guided by these constraints, the helix α8 of the BAH domain was readily docked into this cleft, maintaining the packing of the α-helices of H4 and H2B that make up the proposed docking site (see Figure 7B). The area buried in this structure is approximately 2300 Å2, consistent with a strong interaction. Importantly, this configuration directly juxtaposes the LRS domain with the surface of the BAH domain identified here (Figure 7A–7F). We looked deeper into the structure to find evidence of consistent electrostatic interactions between the two proteins. Remarkably, we found that many residues of both surfaces not only line up well, with few clashes, but importantly, we found adjacencies/interactions consistent with the observed silencing-inhibitory and silencing-enhancing mutations isolated (Figure 7). The most frequently isolated SIR3 suppressor mutants were D205N, L138P, and W86R. The importance of these residues in the docking structure are demonstrated in Figure 7E and 7F. SIR3 D205 and L138 are all in close proximity (within 5 Å) to each other and to H3 K79. Given the importance of H3 K79 methylation in Sir3p binding to the nucleosome, we also found in this proposed docking scheme that tri-methylation of H3 K79 is predicted to sterically hinder binding of Sir3p to the LRS surface (Figure 7E). SIR3 W86 is within 5 Å of H3 T80, H3 D81 and H4 K79. A substitution of an arginine (R) at position W86 could enhance an electrostatic interaction between H3 D81 and Sir3p. Also H3 D77 makes potentially inhibitory repulsive interactions with Sir3p-E178 (Figure 7C), which would be ameliorated by introduction of glycine (G) or asparagine (N) residues into either partner. There are of course stabilizing interactions that would not necessarily be revealed by our mutagenesis but were revealed in the docking study. While our genetic data revealed important side-chains in limiting the interaction between Sir3p and the nucleosome, the docking model showed that there are potential attractive forces as well, namely the α7 helix of the Sir3p BAH domain makes favorable contacts with helices αC and α3 of H2B.

Bottom Line: We found that all alleles of the SIR3 BAH domain were able to 1) generally suppress the loss of telomeric silencing of LRS alleles, but 2) could not suppress SIN (Swi/Snf Independent) alleles or 3) could not suppress the telomeric silencing defect of H4 tail alleles.Moreover, we noticed a complementary trend in the electrostatic changes resulting from most of the histone mutations that gain or lose silencing and the suppressor alleles isolated in SIR3, and the genes for histones H3 and H4.Our results provide genetic evidence for recent data suggesting that the Sir3p BAH domain directly binds the LRS domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

ABSTRACT
The previously identified LRS (Loss of rDNA Silencing) domain of the nucleosome is critically important for silencing at both ribosomal DNA and telomeres. To understand the function of the LRS surface in silencing, we performed an EMS mutagenesis screen to identify suppressors of the H3 A75V LRS allele. We identified dominant and recessive mutations in histones H3, H4, and dominant mutations in the BAH (Bromo Adjacent Homology) domain of SIR3. We further characterized a surface of Sir3p critical for silencing via the LRS surface. We found that all alleles of the SIR3 BAH domain were able to 1) generally suppress the loss of telomeric silencing of LRS alleles, but 2) could not suppress SIN (Swi/Snf Independent) alleles or 3) could not suppress the telomeric silencing defect of H4 tail alleles. Moreover, we noticed a complementary trend in the electrostatic changes resulting from most of the histone mutations that gain or lose silencing and the suppressor alleles isolated in SIR3, and the genes for histones H3 and H4. Mutations in H3 and H4 genes that lose silencing tend to make the LRS surface more electronegative, whereas mutations that increase silencing make it less electronegative. Conversely, suppressors of LRS alleles in either SIR3, histone H3, or H4 also tend to make their respective surfaces less electronegative. Our results provide genetic evidence for recent data suggesting that the Sir3p BAH domain directly binds the LRS domain. Based on these findings, we propose an electrostatic model for how an extensive surface on the Sir3p BAH domain may regulate docking onto the LRS surface.

Show MeSH
Related in: MedlinePlus