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. 2003 Jul 1;17(13):1605-16.
doi: 10.1101/gad.1086803. Epub 2003 Jun 18.

Drosophila FACT contributes to Hox gene expression through physical and functional interactions with GAGA factor

Tsukasa Shimojima  1 , Masahiro Okada, Takahiro Nakayama, Hitoshi Ueda, Katsuya Okawa, Akihiro Iwamatsu, Hiroshi Handa, Susumu Hirose
Affiliations

Drosophila FACT contributes to Hox gene expression through physical and functional interactions with GAGA factor

Tsukasa Shimojima et al. Genes Dev. .

Abstract

Chromatin structure plays a critical role in the regulation of transcription. Drosophila GAGA factor directs chromatin remodeling to its binding sites. We show here that Drosophila FACT (facilitates chromatin transcription), a heterodimer of dSPT16 and dSSRP1, is associated with GAGA factor through its dSSRP1 subunit, binds to a nucleosome, and facilitates GAGA factor-directed chromatin remodeling. Moreover, genetic interactions between Trithorax-like encoding GAGA factor and spt16 implicate the GAGA factor-FACT complex in expression of Hox genes Ultrabithorax, Sex combs reduced, and Abdominal-B. Chromatin immunoprecipitation experiments indicated the presence of the GAGA factor-FACT complex in the regulatory regions of Ultrabithorax and Abdominal-B. These data illustrate a crucial role of FACT in the modulation of chromatin structure for the regulation of gene expression.

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Figures

Figure 1.
Figure 1.
Flag-GAGA factor-associated proteins. (A) Identification of Flag-GAGA factor-associated proteins. Proteins were purified by an anti-Flag M2 affinity column from a nuclear extract of a host line yw (lane 1) or a transgenic line expressing Flag-GAGA factor (lane 2), separated by 7.5% SDS-PAGE, transferred to a membrane, and stained with Coomassie Brilliant Blue. (Lanes 3–5) Proteins were blotted onto a membrane as in lane 2 and probed with the indicated antibodies. Positions of molecular weight markers are shown at the left of lane 1. (B) Peptide sequences from a protein in the middle or upper band. “X” represents an unidentified amino acid. Numbers in parentheses denote amino acid positions. (C) Deduced amino acid sequence of SPT16 from Drosophila embryos. Lines represent the stretches of acidic amino acids.
Figure 2.
Figure 2.
GST pull-down assays. (A) His-GAGA was tested for binding to GST, GST-dSSRP1, or GST-dSPT16. (B) Indicated deletion derivatives of His-GAGA were examined for binding to GST or GST-dSSRP1. (C,D) His-GAGA factor was assayed for binding to GST or indicated deletion derivatives of GST-dSSRP1. (E) His-dSSRP1 was analyzed for binding to GST or indicated deletion derivatives of GST-dSPT16. (F) Indicated deletion derivatives of His-dSSRP1 were tested for binding to GST-dSPT16. (G) Summary of interactions among GAGA factor, dSSRP1, and dSPT16. In B–F, numbers in each deletion represent the remaining amino acid residues. In B and F, positions of molecular weight markers are shown at the left.
Figure 3.
Figure 3.
EMSA for binding of dFACT to nucleosome. (A) Binding of dFACT to nucleosome and naked DNA. Four femtomoles of 32P-labeled naked DNA fragments (lanes 8–14) or mononucleosomes assembled on them (lanes 1–7) were incubated with buffer (lanes 1,8) or 0.24fmoles (lanes 2,9), 0.73 fmoles (lanes 3,10), 2.2 fmoles (lanes 4,11), 6.6 fmoles (lanes 5,12), 20 fmoles (lanes 6,13), and 60 fmoles (lanes 7,14) each of His-dSPT16 and His-dSSRP1. The samples were then analyzed by agarose gel electrophoresis. (B) Supershift by antibodies. Mononucleosomes were incubated with buffer (lane 1) or 12 fmoles each of His-dSPT16 and His-dSSRP1 as in A. In lanes 3–6, the mixtures also contained 1.2 μg of indicated serum. The preparation of mononucleosomes contained naked DNA in A but not in B.
Figure 4.
Figure 4.
Effect of dFACT on GAGA factor-directed chromatin remodeling. (Top) Nucleosome ladders derived from bulk chromatin. Chromatin was reconstituted on a plasmid DNA carrying the ftz promoter in S150 extract and then treated with indicated amounts of His-GAGA and/or a mixture of His-dSPT16 and His-dSSRP1. After incubation, chromatin was digested with micrococcal nuclease, and nucleosome ladders were visualized by staining with SYBR green I dye. (Bottom) Chromatin structure around GAGA factor-binding sites. DNA was then transferred to a nylon membrane and detected by Southern hybridization with 32P-labeled oligonucleotide GAWT harboring two tandemly repeated GAGA factor-binding sites in the ftz promoter (Okada and Hirose 1998).
Figure 5.
Figure 5.
Genetic interactions between Trl and spt16. (A–D) Haltere phenotypes. (A) Ubx130/+. (B) Trl13C+/+ Ubx130. (C) Δspt16+/+ Ubx130. (D) Δspt16 Trl13C+/++ Ubx130. These were photographed at the same magnification so that the size of haltere in each panel can be compared directly. (E–I) Sex comb phenotypes. (E) +/+. (F) Trl13C/+. (G) Δspt16/+. (H) Δspt16 Trl13C/+. (I) P[SPT16+];Δspt16 Trl13C/+. Average number of teeth per sex comb with standard deviation is shown at the bottom of each figure. Numerals in parentheses represent total numbers of combs examined. (J–N) A6-to-A5 transformation. (J) +/+. (K) Trl13C/+. (L) Δspt16/+. (M) Δspt16 Trl13C/+. (N) P[SPT16+]; Δspt16 Trl13C/+. Arrows indicate bristles on A6. Also shown are percentages of males with bristles on A6. Numerals in parentheses represent total numbers of males observed.
Figure 6.
Figure 6.
Binding of GAGA factor, dSSRP1, and dSPT16 to regulatory regions of Ubx and Abd-B in vivo. (A,B) Schematic presentation of the regions in Ubx (A) and Abd-B (B) used for ChIP assays. Positions are relative to the transcription start site. Thick bars represent the PCR amplified regions. (C) PCR amplification of DNA isolated from immunoprecipitated GAGA factor (lanes 3,8), dSSRP1 (lanes 4,9), and dSPT16 (lanes 5,10) chromatin in wild-type or the mutant embryos. Lanes 1 and 6 represent amplification of 0.1% of input DNA. Lanes 2 and 7 show amplification of DNA from mock immunoprecipitated chromatin. (D) Western analyses of GAGA factor, dSSRP1, and dSPT16 in wild-type or the mutant embryos.
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