Figure 1 - from "SATB1 targets chromatin remodelling to regulate genes over long distances"

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Received 5 June; accepted 13 August 2002; doi:10.1038/nature01084

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Figure 1 Chromatin remodelling factors co-purify with SATB1 in DNA affinity chromatography. a, Proteins in SATB1 (+/+) thymic extract (SM), mutant BUR (base unpairing region) column flow-through (Mut), wild-type BUR column flow-through (WT) and proteins eluted with increasing concentrations of NaC! were identified by western analysis. Mi-2, ACF1, mSin3A, ISWI, MTA-2, HDAC1 and HDAC2 signals are shown in the top panel. SATB1 signal is shown in the middle panel and PARP, another BUR binding protein, is shown in the lower panel. b, When BUR affinity chromatography was performed with SATB1 (—/—) extract, only PARP was eluted (lower panel). “ “ We next examined whether SATB] directs chromatin remodelling complexes to the SATB1 binding loci in vivo. We first studied HDACI1, a component of the NURD complex. This was done by chromatin immunoprecipitation (ChIP) assays. Briefly, thymocyte chromatin crosslinked with formaldehyde, purified with a urea

Figure 2 SATB1 directly interacts with several chromatin remodelling factors. Proteins immunoprecipitated (IP) with a, anti-SATB1 antibody or b, anti-HDAC1 from SATB1 (+/+) (lane 1) and SATB1 (—/—) thymi (lane 2) were identified by western blot with antibodies as indicated. In both a and b, lane 3 shows control antibody (anti-p27 antibody) immunoprecipitation of SATB1 (-++/-++) extract, and lane 4 shows proteins in starting material. ¢, Protein immunoprecipitated with anti-ISWI, anti-ACF1, anti-SATB1, and anti- PARP antibodies from mixtures of purified FLAG-fused recombinant ISWI (nSNF2H), ACF1 (nACF1), his-tag-fused SATB1 (amino acids 90-763), truncated SATB1 (amino acids 365-763), and full-length PARP were identified by western blot with anti-FLAG, anti- SATB1 and anti-PARP antibodies, Pl, preimmune serum; IP, immunoprecipitation; |B, immunoblot. gradient, and digested with restriction enzymes, was immunopre- cipitated with either anti-SATB1 or anti- HDACI antibody**”’. We focused on the IL-2Ra locus because the IL-2Ra gene is ectopically transcribed in SATB1 null double positive thymocytes*. The double positive subset represents >84% of the total thymocyte population. Within the IL-2Ra locus, we identified a 700-bp fragment that exhibits very high affinity (Ka = 0.1-0.5nM, data not shown), to SATB1 in vitro (SATB1-bound sequence 700; SBS700) which mapped to the first intron, 6.8—7.5 kb downstream of the transcrip- tional start site (Fig. 3c). There is no additional SATB1-bound site

Figure 3 SATB1 targets HDAC1, ISWI and ACF1 to the /L-2Ra locus in vivo. PCR amplification of DNA isolated from immunoprecipitated SATB1 wild-type (+/+) chromatir (upper row) and SATB1 (—/—) chromatin (lower row) using antibodies to a, SATB1, HDAC1, acetylated histone H4 (H4ace), control non-immune serum and b, to ACF1, ISWI or genomic DNA. Each DNA sample was amplified with four primer pairs; /L-2Re promote: (lanes 1), /L-2Re intron (lanes 2), /L-2Ra SBS700 (lanes 3) or SBS336 (lanes 4). DNA size markers are shown to the left of the panel. ¢, A map of /L-2Aa with positions of the primers used for (1) promoter, (2) intron, and (3) SBS700 sequences.

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Figure 4 Alteration of nucleosomal positioning in the /L-2Ra locus in SATB1 null thymocytes. a, MNase cleavage sites downstream of the first exon region in wild type (WT), SATB1 null (KO) thymocyte nuclei and naked DNA determined by Southern hybridization of Pstt-digested DNA with probe 1. b, MNase cleavage sites at the intronic SBS700 determined by Southern hybridization of Pstt-digested DNA with probe 2. c, Summary map of the /L-2Aa gene locus. Location of SBS700 region (hatched boxes) and ATC sequences that binds SATB1 (wavy lines) are indicated. For ¢, MNase cleavage sites restricted to WT (solid arrowheads) or KO nuclei (open arrowheads), and enhanced cleavages for WT (solid circle) or KO nuclei (open circles) are shown.

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Dag Yasui,

Shutao Cai

SATB1 targets chromatin remodelling to regulate genes over long distances

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