The WD40-repeat protein Pwp1p associates in vivo with 25S ribosomal chromatin in a histone H4 tail-dependent manner

Abstract

The tails of core histones (H2A, H2B, H3 and H4) are critical for the regulation of chromatin dynamics. Each core histone tail is specifically recognized by various tail binding proteins. Here we screened for budding yeast histone H4-tail binding proteins in a protein differential display approach by two-dimensional gel electrophoresis (2DGE). To obtain highly enriched chromatin proteins, we used a Mg2+-dependent chromatin oligomerization technique. The Mg2+-dependent oligomerized chromatin from H4-tail deleted cells was compared with that from wild-type cells. We used mass spectrometry to identify 22 candidate proteins whose amounts were reduced in the oligomerized chromatin from the H4-tail deleted cells. A Saccharomyces Genome Database search revealed 10 protein complexes, each of which contained more than two candidate proteins. Interestingly, 7 out of the 10 complexes have the potential to associate with the H4-tail. We obtained in vivo evidence, by a chromatin immunoprecipitation assay, that one of the candidate proteins, Pwp1p, associates with the 25S ribosomal DNA (rDNA) chromatin in an H4-tail-dependent manner. We propose that the complex containing Pwp1p regulates the transcription of rDNA. Our results demonstrate that the protein differential display approach by 2DGE, using a histone-tail mutant, is a powerful method to identify histone-tail binding proteins.

Figure 1

Preparation of MNase-eluted chromatin. (A) The biochemical fractionation scheme. (B) DNA from the MNase-eluted chromatin fraction, either with (+) or without (−) the MNase digestion, was electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. M is DNA size markers.

Figure 2

Fractionation of the MNase-eluted chromatin by the Mg²⁺-dependent oligomerization. (A) Titration of the Mg²⁺ concentration. The DNAs fractionated by various concentrations of Mg²⁺, in the supernatant (S) and precipitate (P), were electrophoresed on a 2% agarose gel and were visualized by ethidium bromide staining. (B) The protein compositions of the fractions (S and P) by the Mg²⁺-dependent oligomerization with 12 mM Mg²⁺. The fractionated proteins and purified histones were resolved by SDS–PAGE and were visualized by silver staining. Molecular weight markers are indicated. (C) The distribution of the proteins fractionated as shown in (B) were analyzed by western blot analyses with the antibodies indicated on the left.

Figure 3

Comparison of the oligomerized chromatin fractions between WT and H4-tailΔ. (A) The oligomerized chromatin proteins from the WT and H4-tailΔ strains were resolved by 15% SDS–PAGE and were visualized by silver staining. Purified histones were run and the position of each core histone is indicated on the left. Molecular weight markers are indicated on the right. (B) Western blot analyses of oligomerized chromatin proteins from the WT and H4-tailΔ strains. Histones (H2A and H3) and tail binding proteins (Sir3p and Bdf1p) in each oligomerized chromatin fraction (WT and H4-tailΔ) were analyzed with the antibodies indicated on the left.

Figure 4

The protein differential display approach of the oligomerized chromatin proteins by 2DGE. The oligomerized chromatin fractions from WT (A) and H4-tailΔ (B) cells were resolved by 2DGE and visualized by silver staining. The protein spots with signal intensity that was reduced in the H4 tail-deletion are indicated and were identified by MS. Predicted contaminant proteins are shown in gray.

Figure 5

The amount of Pwp1p is reduced in H4 tail-deleted chromatin. (A) Western blot analyses were performed as in Figure 3B with Myc-tagged Pwp1p strains. (B) The graph shows the quantification of H2A, Bdf1p and Pwp1p-Myc relative to the H3 signal intensity. Data represent the average of five assays ±SD.

Figure 6

Pwp1p associates with the rDNA chromatin in an H4-tail-dependent manner. (A) ChrIP assays were performed with the cells containing the FLAG-tagged Sir3p, the FLAG-tagged Pwp1p and untagged proteins. Serial 2.5-fold dilutions of input DNA and the immunoprecipitated DNA were analyzed by multiplex PCR using primer pairs directed against the CUP1, HMR, and INO1 loci. (B) The bar graphs show the relative fold enrichment of the FLAG-tagged Sir3p, the FLAG-tagged Pwp1p and the untagged protein at the HMR and the INO1 loci. Data represent the average of three assays ±SD (C) ChrIP assays were performed with the FLAG-tagged and the untagged Pwp1ps in the WT and H4-tailΔ cells. Serial 2.5-fold dilutions of input DNA and the immunoprecipitated DNA were analyzed by multiplex PCR using primer pairs directed against the CUP1, the 5S (at 8.1 kb in E) and the 25S (at 3.1 kb in E) loci. (D) The bar graphs show the relative fold enrichment of the Pwp1p binding at the 5S and 25S loci in (C). Data represent the average of three assays ±SD. (E) ChrIP assays were performed as shown in (C) using 16 sets of primers that spanned the rDNA repeat unit. The graph shows the relative fold enrichment of the Pwp1p binding across an rDNA repeat unit, which is schematically indicated under the X-axis. Each repeat yields a Pol I-transcribed 35S precursor rRNA (shown as an arrow with 35S) and a pol III-transcribed 5S rRNA (shown as an arrow with 5S). The repeat contains the genes for 5S, 5.8S 18S and 25S rRNAs (black boxes), as well as three types of spacer regions: internal transcribed spacers (ITS1, ITS2), external transcribed spacers (5′ ETS, 3′ ETS) and non-transcribed spacers (NTS1, NTS2). Data represent the average of three assays ±SD.