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. 2000 May 15;19(10):2323-31.
doi: 10.1093/emboj/19.10.2323.

The chromo domain protein chd1p from budding yeast is an ATP-dependent chromatin-modifying factor

H G Tran  1 , D J Steger, V R Iyer, A D Johnson
Affiliations

The chromo domain protein chd1p from budding yeast is an ATP-dependent chromatin-modifying factor

H G Tran et al. EMBO J. .

Abstract

CHD proteins are members of the chromo domain family, a class of proteins involved in transcription, DNA degradation and chromatin structure. In higher eukaryotes, there are two distinct subfamilies of CHD proteins: CHD1 and CHD3/4. Analyses carried out in vitro indicate that the CHD3/4 proteins may regulate transcription via alteration of chromatin structure. However, little is known about the role of CHD proteins in vivo, particularly the CHD1 subfamily. To understand better the cellular function of CHD proteins, we initiated a study on the Chd1p protein from budding yeast. Using genomic DNA arrays, we identified genes whose expression is affected by the absence of Chd1p. A synthetic-lethal screen uncovered genetic interactions between SWI/SNF genes and CHD1. Biochemical experiments using Chd1p purified from yeast showed that it reconfigures the structure of nucleosome core particles in a manner distinct from the SWI-SNF complex. Taken together, these results suggest that Chd1p functions as a nucleosome remodeling factor, and that Chd1p may share overlapping roles with the SWI-SNF complex to regulate transcription.

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Figures

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Fig. 1. Analysis of CHD1’s effect on genome-wide expression. (A) Table of genes whose expression is affected by ≥2-fold in both microarrays. The ‘Fold’ column indicates the average between experiments. Upward-pointing arrows signify that gene expression increased in the deletion strain, while downward-pointing arrows indicate that gene expression decreased in the deletion strain. (B) Northern blot of genes identified from genomic DNA array analysis. Four genes affected by the absence of CHD1, as determined by the array experiment, show similar expression differences when analyzed by RNA Northern blots. (C) PCR analysis of DNA cross-linked to Chd1p. PCRs were carried out using either total DNA from whole cell extracts (WCE) or chromatin immunoprecipitated DNA (Ch-IP), and primers targeted against genes affected by the absence of CHD1. Primers for the ACT1 gene, which is unaffected by the loss of CHD1, were also added to the reactions. The right panel shows a control reaction with DNA from an untagged strain.
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Fig. 2. A chd1 mutation is synthetically sick/lethal with swi/snf mutations. (A) A chd1Δ strain is severely sick when combined with a swc1/2 (swi2) or swc3 (alr1) mutation. (B and C) A chd1Δ swi2Δ double mutant (B) and a chd1Δ swi1Δ double mutant (C) are both unable to survive on 5-FOA when forced to lose a wild-type copy of CHD1 on a plasmid containing the URA3 gene (pAJ741). All strains in (B) and (C) possess the pAJ741 plasmid before being streaked on 5-FOA plates. (D) A swi4Δ chd1Δ strain is viable.
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Fig. 3. Purification of Chd1p. (A) Schematic flowchart of the chromatographic steps used to purify Chd1p. (B) Silver-stained gel of the purest fraction of Chd1p from the SP-Sepharose column. Lane 1 contains 1 µl (∼15 ng) and lane 2 contains 5 µl of protein. (C) Western blot showing the elution profile of Chd1p collected from a Superose 6 sizing column. The elution profile of molecular weight standards is indicated as 670 (kDa), 158, 44 and 17.
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Fig. 4. Chd1p possesses DNA- and nucleosome-stimulated ATPase activity. ATPase assays contained purified Chd1p (2 µl) and 1 pmol of nucleosomes (N) or the equivalent amount of DNA (D) or core histones (H). As competitors, unlabeled ATP and ATP-γ-S were added to the indicated reactions at a 5-fold molar excess over the standard ATP concentration. The arrow points to the signal derived from free phosphate.
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Fig. 5. Chd1p alters nucleosome core particle structure. (A) Purified Chd1p (0.5 µl) or purified SWI–SNF was incubated with mock-reconstituted 5S DNA (DNA) or nucleosome-assembled 5S DNA (Nucl. DNA) and subjected to DNase I protection analysis. Where indicated, ATP and its respective analogs were added to a final concentration of 1 mM. To challenge Chd1p activity, 1 µl of anti-Chd1p polyclonal serum (α-Chd1p) or pre-immune serum (pre-imm) was incubated with Chd1p for 5 min before addition of the 5S nucleosome core particles. Brackets and arrows indicate DNase I-protected regions and DNase I-hypersensitive sites, respectively, which are generated by Chd1p activity on mononucleosomes. (B) Reactions identical to those in (A) were subjected to EMSA.
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