Structural analysis of Rtt106p reveals a DNA binding role required for heterochromatin silencing

Abstract

Rtt106p is a Saccharomyces cerevisiae histone chaperone with roles in heterochromatin silencing and nucleosome assembly. The molecular mechanism by which Rtt106p engages in chromatin dynamics remains unclear. Here, we report the 2.5 A crystal structure of the core domain of Rtt106p, which adopts an unusual "double pleckstrin homology" domain architecture that represents a novel structural mode for histone chaperones. A histone H3-H4-binding region and a novel double-stranded DNA-binding region have been identified. Mutagenesis studies reveal that the histone and DNA binding activities of Rtt106p are involved in Sir protein-mediated heterochromatin formation. Our results uncover the structural basis of the diverse functions of Rtt106p and provide new insights into its cellular roles.

FIGURE 1.

Structure of Rtt106p-M. A, a stereo view of Rtt106p-M. Disordered residues between two PH domains and in the C terminus are modeled as dashed lines. B, the electronic potential surface of Rtt106p-M. The orientations of B (left) and A are the same. The ellipse highlights the positively charged ridge. C, the conserved residues comprising the positively charged ridge. D, conserved and type-conserved residues at the interface of the two PH domains. C and D have the same orientations as A.

FIGURE 2.

Sequence alignment of the double PH domains among Rtt106p and its homologues. The following sequences were aligned using ClustalW2 (available on the World Wide Web): S. cerevisiae Rtt106p (gi|6324123|NP_014193.1); Vanderwaltozyma polyspora Rtt106 (gi|156837701|XP_001642870.1); Candida glabrata Rtt106 (gi|50289433|XP_447148.1); Ashbya gossypii Rtt106 (gi|45185065|NP_982782.1), and Kluyveromyces lactis Rtt106 (gi|50307819|XP_ 453903.1). The secondary structures and residue numbers of Rtt106p-M are shown at the top. The residues comprising the positively charged ridge for dsDNA binding are designated with a black triangle below; the hydrophobic residues at the interface of the two PH domains are designated with an orange triangle; and residues comprising the crucial loop for H3-H4 binding are designated with a star. The three highly conserved negatively charged residues that form a negative patch near the conserved loop are denoted with a blue triangle.

FIGURE 3.

Structure comparison of Rtt106p-M and Pob3p-M. A, overall structure superposition of Rtt106p-M (red) and Pob3p (cyan). B, superposition of the first PH domains. C, superposition of the second PH domains. D, highlighted are the major structural differences between the first PH domains of Rtt106p-M and Pob3p-M. A–C have the same orientations as in Fig. 1.

FIGURE 4.

Physical interaction between Rtt106p-M and histones. A, Ni-NTA pull-down analysis of Rtt106p-M with histones H3-H4 and H2A-H2B in cell lysates. B, highlighted are the mutated residues in Rtt106p-M loop mutants. C, the CD spectra of Rtt106p-M and its mutants. D, Ni-NTA pull-down analysis of Rtt106p-M mutants with histones H3-H4 in cell lysates. The final samples were resolved by Tricine-SDS-PAGE (15%, w/v) and stained with Coomassie Brilliant Blue (CB).

FIGURE 5.

Rtt106p-M interacts with dsDNA. A, results of an EMSA assessing the interaction of Rtt106p-M and the Rtt106p-site1 and Rtt106p-site2 mutants with a ³²P-labeled 33-bp dsDNA, named ds-001, and used at 0.2 nm. Rtt106p-M concentrations are as follows: 0 μm (lane 1), 72 μm (lane 8). Lanes 2–8, 2-fold dilutions from right to left from 72 μm. Lane 9, 165 μm Rtt106p-site1; lane 10, 168 μm Rtt106p-site2. B, FPAs of Rtt106p-M and mutants with a 5′-FAM-labeled 33-bp dsDNA (with the same sequence as ds-001). The data were fitted according to Equation 2. C, EMSA of Rtt106p-M interaction with AT-rich and GC-rich dsDNA sequences. Lane 2, Rtt106p-M at 144 μm; lanes 4–6, 36, 72, and 144 μm, respectively; lanes 8–10, 36, 72, and 144 μm, respectively. D, FPA of Rtt106p-M interaction with AT-rich and GC-rich dsDNA. Rtt106p-M exhibited similar binding affinity to AT-rich and GC-rich dsDNA. E, FPA of Rtt106p-M interaction with dsDNA oligonucleotides of different lengths.

FIGURE 6.

Rtt106p-M binds dsDNA and histone H3-H4 through unrelated regions. A, EMSA of Rtt106p-loopm interacting with dsDNA with similar affinity as Rtt106p-M. The ds-001 probe was used at 0.2 nm. Rtt106p-loopm concentrations were as follows: 0 μm (lane 1) and 144 μm (lane 10). Lanes 2–10, 2-fold dilutions from right to left from 144 μm. B, pull-down assays of Rtt106p-site1 and -site2 mutants interacting with H3-H4 tetramer in cell lysates. C, pull-down assays of Rtt106p-site1 and -site2 mutants interacting with purified H3-H4 tetramer with similar affinity as wild type Rtt106p-M. CB, Coomassie Brilliant Blue.

FIGURE 7.

Rtt106p-histone and -DNA interactions are involved in telomeric heterochromatin formation. A, wild type, sir2Δ, cac1Δrtt106Δ, and rtt106 mutant cells with a URA3 gene integrated at the subtelomeric regions of chromosome VII (TELVII) were serially diluted (5-fold) and spotted onto YC medium with or without 5-FOA, followed by incubation at 30 °C. Photographs were taken after 20 h. B, a sketch map showing the primers designed according to the sequence near the telomere at chromosome IX. C, 13Myc-Sir2p binding to subtelomeric loci was assayed by chromatin immunoprecipitation using anti-Myc antibody in wild type, cac1Δrtt106Δ, and rtt106 mutant cells. Average relative Sir2p enrichments were shown for each primer set. The quantitative PCR data were normalized to an internal control (ACT1) and the input DNA. The results are the average of three independent chromatin immunoprecipitation assays with error bars shown for the S.E. for three independent experiments.