HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins

Abstract

Chromatin remodelling complexes containing the nucleosome-dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Our purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. We show that the human homologues of two novel putative histone-fold proteins in Drosophila CHRAC are present in HuCHRAC. The two human histone-fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homologue of Acf1, a subunit of Drosophila ACF. The N-terminus of mouse ACF1 was reported as a heterochromatin-targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. We discuss a possible function for HuCHRAC in heterochromatin dynamics. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC.

Fig. 1. Human ISWI co-purifies with human ACF1 and histone-fold proteins p17 and p15. (A) Purification scheme for HuCHRAC. (B) Co-fractionation of hISWI with hACF1, p17 and p15, but separation from topoisomerase IIα and IIβ in Mono S chromatography. Proteins in the input and indicated fractions were visualized by western blot analysis with the indicated antibodies. (C) Co-fractionation of hISWI with hACF1, p17 and p15 in Superose 6 gel filtration chromatography. Proteins in the input and indicated fractions (0.5 ml) were visualized by silver staining (two upper panels) or western blot analysis (four lower panels) with the indicated antibodies. ‘–’ indicates hACF1, identified by mass spectroscopy (see text), ‘+’ indicates hISWI isoform hSNF2H, identified by mass spectroscopy (see text), * indicates protein recognized by anti-p17, and the filled square indicates protein recognized by anti-p15. The running position of a molecular size standard (thyroglobulin) is indicated at the top of the panel. (D) Co-fractionation of hSNF2H, hACF1, p17 and p15 in the final CM-Sepharose chromatography. The input and elution fraction were visualized by silver staining (upper two panels). The lower panel represents a western blot analysis with anti-p17 antibodies.

Fig. 2. Human ACF1 is a member of a family of WAL proteins. (A) Predicted amino acid sequence of hACF1. The WAC and WAKZ domains, PHD finger and bromodomain in hACF1 are indicated by shaded boxes. Underlined amino acids indicate tryptic peptides whose sequences were identified by ion trap tandem mass spectrometry. (B) Known human members of a family of WAL (WSTF/Acf1-like) proteins. The percentage amino acid identity between the indicated domains is shown. The human (h)WALp3 sequence was derived from the AB032254 cDNA. hWALp3 contains the TIP5 (TTF1 interaction peptide 5, EST AF000422; Jansa et al., 1998). The hWALp4 sequence was derived from AB032255 cDNA. The TIP5 peptide in hWALp3 and the N-terminal part of hWALp4 contain a putative methyl-binding domain (pMBD). (C) Phylogeny tree of the WAL proteins. Ce acf-1 is encoded by H20J04.2 cDNA, Ce wal-3 is encoded by ZK783.4 cDNA.

Fig. 3. HuCHRAC-15 and HuCHRAC-17 are the human homologues of Drosophila CHRAC-16 and CHRAC-14, respectively. The predicted amino acid sequences of HuCHRAC-15 and HuCHRAC-17, abbreviated as p15 and p17, were aligned with the sequences of CHRAC-16 and CHRAC-14, respectively. Identical amino acids are assigned; similar amino acids are indicated by +. Putative histone-fold domains are indicated by shaded boxes. Histone-fold domains for p15 and p17 homologues were assigned by homology to the putative histone-fold domains of CBF-C and HAP5 (p15) or CBF-A and HAP3 (p17).

Fig. 4. Human ISWI co-immunoprecipitates with the p15 and p17 histone-fold proteins and hACF1. Immunoprecipitations from HeLa nuclear extracts with anti-hISWI and anti-p17 antibodies. Immuno precipitates with pre-immune (PI) or immune (I) sera and 5% input were western blotted and probed with antibodies against hISWI, p17, ACF1, p15, topo IIα and β, as indicated.

Fig. 5. HuCHRAC has nucleosome spacing activity. The indicated amounts of HuCHRAC or Drosophila CHRAC were added to sarkosyl-stripped chromatin DNA, assembled in Drosophila embryo extracts without ATP. Addition of ATP was as indicated. After the chromatin remodelling reaction, DNA was digested for 30 or 60 s with micrococcal nuclease, separated on a 1.3% agarose gel, Southern blotted and probed with random labelled input DNA.

Fig. 6. Recombinant proteins p15 and p17 form a complex that binds to naked, but not nucleosomal DNA. (A) Coomassie-stained SDS–polyacrylamide gel showing the purification of the p15–p17 complex. Lane 1, elution of the S-p15-HA–p17 complex from glutathione–Sepharose by cleavage with thrombin protease; lane 2, elution of the p15-HA–p17 complex from S-protein–agarose by cleavage with enterokinase-protease; lane 3, control elution from glutathione–Sepharose loaded with lysate from cells expressing only His-S-p15-HA, showing that binding of p15 to this column is only possible via p17–GST. (B) Electrophoretic mobility shift analysis of DNA–p15–p17 complexes with a 150 bp DNA fragment. Lane 1, ³²P-labelled DNA fragment without protein; lane 2, p15-HA–p17 complex (0.2 µg); lane 3, p15-HA–p17 complex (0.2 µg) + excess unrelated plasmid DNA; lane 4, nucleosomal DNA; lane 5, p17 (0.3 µg); lane 6, p15-HA (0.12 µg); lane 7, p15-HA–p17 complex (0.1 µg); lane 8, anti-HA-antibody; lane 9, as lane 2 + anti-HA-antibody (*indicates the supershifted p15-HA–p17–DNA complex); lane 10, as lane 9 +HA-peptide; lane 11, as lane 2 + HA-peptide. The bracket on the left side denotes uncomplexed DNA, the circle indicates single-stranded DNA, a PCR artificial product. (C) Binding of p15–p17 complex to DNA coupled to magnetic beads. p15–p17 complex was incubated with DNA coupled to magnetic beads, or nucleosomal DNA coupled to magnetic beads, or beads alone. Bound proteins were examined by western blot analysis with antibodies against p17, or anti-HA-antibodies (p15-HA).

Fig. 7. Relative amounts of p15 and p17 mRNA in human tissues. Multiple tissue northern blot analysis of p15 and p17 mRNA, and β-actin mRNA as a loading control. The tissue and the position of size markers are indicated.