Volume 10, Issue 5, Pages 1119-1128 (November 2002) ALL-1 Is a Histone Methyltransferase that Assembles a Supercomplex of Proteins Involved in Transcriptional Regulation  Tatsuya Nakamura, Toshiki Mori, Shinichiro Tada, Wladyslaw Krajewski, Tanya Rozovskaia, Richard Wassell, Garrett Dubois, Alexander Mazo, Carlo M Croce, Eli Canaani  Molecular Cell  Volume 10, Issue 5, Pages 1119-1128 (November 2002) DOI: 10.1016/S1097-2765(02)00740-2

Figure 1 Identification and Characterization of the Processed p300 and p180ALL-1 Polypeptides (A) Nuclear extracts from three cell lines (1–3) were analyzed by Western blotting with six different anti-ALL-1 Ab (their relevant epitopes on the protein are indicated below the blots). Major motifs within the ALL-1 protein are indicated. The approximate extent of the two ALL-1 processed proteins and the region spanning the cleavage point are shown. (B) Cytosolic enzymatic activity splits a peptide spanning ALL-1 cleavage point. The intact peptide, spanning residues 2594–2900, and the cleaved product are identified by Western blotting and reaction with anti-T7 Ab. Sequence of the T7-tagged product was determined by Edman degradation. (C) In vivo pulse labeling experiment. For details, see text. Size of the full-length ALL-1 protein is roughly estimated as 430 kDa, based on the position on the gel of the 200 kDa myosin and of the 300 kDa baculovirus-expressed p300HAT. Molecular Cell 2002 10, 1119-1128DOI: (10.1016/S1097-2765(02)00740-2)

Figure 2 Silver Staining and Western Blotting Pattern of Proteins Eluted from ALL-1 Immunoaffinity Columns (A) The proteins were identified by mass spectrometry and/or Western blotting (right). The gel on the right shows a full blot of eluates from the ALL-1 columns, probed with a mixture of Abs 170 and 173 directed against ALL-1. (B) Association of supercomplex components with ALL-1 is not disrupted by EtBr and therefore is DNA independent (left). The same associations occur in HeLa cells (right). Immunoprecipitations were done with anti-ALL-1 Ab 173. Control Ab used (con) was anti-GST. See text for details. Molecular Cell 2002 10, 1119-1128DOI: (10.1016/S1097-2765(02)00740-2)

Figure 3 ALL-1 and the Proteins Associated with It Are Present within a Very Large Complex which Can Be Affinity Purified with Abs against Its Different Components (A) Superose 6 column fractionation of the enriched ALL-1 complex. Volumes of 0.5 ml and 0.25 ml were collected in fractions 1–14 and 15 and up, respectively. Ten microliter aliquots were used for Western blotting analysis. Analysis of fractions 12–27 is shown. Scanning results for part of the blotting analysis are shown below. In, input. Similar distribution of proteins was obtained when the KCl concentration in the running buffer was increased to 0.5 M (data not shown). (B) Portions of the Superose 6 pools I, II, and III were subjected to immunoaffinity columns (IAC) containing different Abs (con = anti-GST, anti-p180ALL-1, anti-Sin3A, anti-BRM, anti-TAFII80), and the eluted proteins identified by Western blotting. See text for details. Molecular Cell 2002 10, 1119-1128DOI: (10.1016/S1097-2765(02)00740-2)

Figure 4 Chromatin Remodeling (A), Histone Deacetylation (B), and Acetylation (C) by the ALL-1 Complex (A) Increasing amounts of purified ALL-1 and Sin3A+SWI/SNF complexes eluted from immunoaffinity columns are assayed for remodeling of mononucleosomes. + and − indicate increase or decrease, respectively, in signal intensity as a function of the amount of the complex in the reaction. N, naked DNA. (B) The same purified complexes are assayed for histone deacetylation of in vivo labeled core histones. (C) Mononucleosomes are assayed for acetylation with beads linked to anti-ALL-1 Ab and loaded with the enriched ALL-1 complex (lanes 2 and 6), with beads linked to anti-GST Ab (negative control) and similarly loaded (lane 1), with purified TFIID complex (lane 3), or with the purified histone acetylases p300 and PCAF (lanes 4 and 5, respectively). In lane 6, sodium butyrate is not included in the enzymatic reaction. Fluorogram shows acetylation of each of the four histones. Molecular Cell 2002 10, 1119-1128DOI: (10.1016/S1097-2765(02)00740-2)

Figure 5 Histone Methylase Activity of the Purified ALL-1 Complex and of the ALL-1 SET Domain (A) Methylation of H3 by purified ALL-1 complex loaded on beads of immunoaffinity column containing the anti-p180ALL-1 Ab 173 (lanes 2 and 3) or loaded on beads containing anti-GST Ab (lane 1). Also assayed was histone methylation by ALL-1 SET polypeptide linked to GST (lanes 4–6). Substrates were core histones (core), mononucleosomes (mono), or recombinant H3 (rH3). (B) Edman degradation analysis of H3 methylated in vitro by ALL-1 SET. Thirty-seven cycles were performed. (C) Assay of the capacity of ALL-1 SET polypeptide linked to GST or purified ALL-1 complex attached to beads to methylate N-terminal H3 peptides containing unmodified K4 and K9, dimethylated K4, dimethylated K9, or dimethylated K4 and K9. Molecular Cell 2002 10, 1119-1128DOI: (10.1016/S1097-2765(02)00740-2)

Figure 6 ChIP Analysis of the Hox a9 Locus in HeLa Cells (A) HeLa cells transfected with either Scramble siRNA duplex (Scr.) or with ALL-1 siRNA duplex were examined for expression of the ALL-1 and some ALL-1 complex components by Western blotting. Downregulation of Hox a9 and GAPDH RNA was examined in these cells by RT-PCR. For detection of Hox a9 and GAPDH, 31 PCR cycles and 21 PCR cycles were performed, respectively. (B) ChIP analysis to examine the presence of components of the ALL-1 supercomplex and of RNA polymerase II, as well as of histone methylation and acetylation, on different regions of the Hox a9 locus and on the 5′ end of the GAPDH locus. Chromatin was prepared from HeLa cells transfected with Scramble siRNA or with ALL-1 siRNA. The analyzed portion of the Hox a9 locus is depicted on the right. The box shown on the scheme of the Hox a9 locus indicates the first exon, and the dark area corresponds to the coding region. The arrow points to the transcription initiation site. a–c correspond to the sequences amplified by PCR following immunoprecipitation with each of the 11 Abs indicated (con = GST, BRM, Mi2, Sin3A, TBP, TAFII80, ALL-1, dimethylated H3-K4, acetylated H3 and H4, and RNA polymerase II). For input, 1% of the amount of sonicated chromation to be subsequently processed for ChIP was removed and PCR amplified. The marker indicates 310, 281/271, 234, and 194 bp DNA fragments, corresponding to HaeIII digestion products of φX174 phage DNA. Molecular Cell 2002 10, 1119-1128DOI: (10.1016/S1097-2765(02)00740-2)