The HIR corepressor complex binds to nucleosomes generating a distinct protein/DNA complex resistant to remodeling by SWI/SNF

Abstract

The histone regulatory (HIR) and histone promoter control (HPC) repressor proteins regulate three of the four histone gene loci during the Saccharomyces cerevisiae cell cycle. Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 proteins form a stable HIR repressor complex. The HIR complex promotes histone deposition onto DNA in vitro and constitutes a novel nucleosome assembly complex. The HIR complex stably binds to DNA and nucleosomes. Furthermore, HIR complex binding to nucleosomes forms a distinct protein/DNA complex resistant to remodeling by SWI/SNF. Thus, the HIR complex is a novel nucleosome assembly complex which functions with SWI/SNF to regulate transcription.

Figure 1.

Identification and characterization of the HIR complex. (A) HIR complex purified from Hir1-TAP-tagged strain analyzed by SDS-PAGE and silver stain. The subunits are labeled with their locations on the gel. (B) Proteins from the different TAP purifications were identified by MudPIT. The proteins identified are listed with the number of spectrum counts followed by the percentage coverage of each protein obtained from the MudPIT analysis. (C) The number of MS/MS spectra-matching peptides from HIR subunits was divided by each subunit's molecular weight, scaled up by an arbitrary factor of 500,000 (SAF), and normalized to the total spectral count for the entire complex (NSAF). Average values and standard deviations were calculated for NSAFs obtained from seven trypsin and two elastase digests independently analyzed by MudPIT. (D) Immunoblotting of the 10%-40% glycerol gradient sedimentation of the HIR complex. The fractions were assayed for the presence of HIR1-, HIR2-, HIR3-TAP-, and HPC2-Myc-tagged proteins by Western blotting. The peak fractions of HIR complex are indicated by a solid line. The 670-kDa marker indicates the position of thyroglobin fractionated on a 10%-40% glycerol gradient run in parallel. BSA (67-kDa) and catalase (232-kDa) markers fractionate in fractions 5 and 15, respectively. The presence of a dimer of thyroglobin (1338 kDa) could be detected in fraction 34.

Figure 2.

Nucleosome assembly activity of the HIR complex. (A) Nucleosome assembly reactions were carried out with HeLa core histones incubated with 1 μg of NAP1 (lanes 3-5), with 8.65 nM and 17.3 nM of HIR complex (lanes 6-11), or with 8.2 nM and 16.3 nM of HIR B (lanes 9-11). HIR B corresponds to the HIR complex purified directly from the calmodulin step of the TAP protocol. The formation of mononucleosome was monitored on a 6% (29:1) PAGE gel, dried, and exposed on a PhosphorImager screen. (B) Analysis of the nucleosome assembly activity of the HIR complex by supercoiling assay. Mock, 0.2-1 μg of NAP1, or 3.4 nM of TAP-purified HIR complex was combined with topoisomerase I and 500 ng of relaxed plasmid in the presence or absence of HeLa core histones and incubated for 3 h or as indicated. Different amounts of core histone relative to plasmid were used, indicated by the ratio CH/DNA. Purified plasmid products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide staining. Migration positions of DNA plasmid form I (supercoiled), form II (nicked circular), and form Ir (closed circular) are indicated.

Figure 3.

HIR complex binds to naked DNA or to nucleosomes. (Lanes 1-5) Purified HIR complex (0.15, 0.3, 0.6, and 1.2 nM) was incubated with a naked 183-bp DNA probe. (Lanes 6-13) HIR complex (0.56, 1.12, or 2.24 nM) was incubated with reconstituted nucleosome cores using a 183-bp (lanes 6-9) or 147-bp (lanes 10-13) DNA probe. Samples were run on a 4% acrylamide gel (37.5:1, acrylamide/bis) in a 0.5× TBE buffer system. Migration positions of DNA, nucleosomes, and HIR-bound complexes are indicated.

Figure 4.

HIR complex prevents SWI/SNF remodeling activity. In this DNase I digestion assay, a 5′-end-labeled 183-bp (lanes 1-8) or 147-bp (lanes 9-16) template was mock reconstituted (DNA) or reconstituted into mononucleosome cores (nucleosome), and preincubated with 2.9 nM of HIR complex for 1 h at 30°C prior to addition of 2.1 nM of SWI/SNF complex for 1 h at 30°C, as indicated. The remodeling reactions were treated with 0.4 or 0.04 U of DNase I for 1 min at room temperature for nucleosome template or naked DNA respectively, except in lanes 1, 4, 9, and 12 (no DNase I treatment). The DNA product of the reactions was resolved on an 8% acrylamide-8 M urea sequencing gel.

Figure 5.

HIR binding to nucleosomes does not prevent SWI/SNF from binding. Purified HIR complex (lanes 2,3) or SWI/SNF complex (lanes 8,9) was incubated with a reconstituted nucleosome core using a 183-bp DNA template. In lanes 4-7, after preincubation of 0.9 and 1.8 nM of HIR complex with nucleosomes, 2.4 and 4.8 nM of SWI/SNF were added to the reactions. Samples were run on a 4% acrylamide gel (77.5:1, acrylamide/bis) in a 0.5× TBE buffer system. Migration positions of nucleosomes, HIR-bound complexes, and SWI/SNF-HIR-bound complexes are indicated.