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. 2000 May 1;14(9):1058-71.

The Drosophila brahma complex is an essential coactivator for the trithorax group protein zeste

A J Kal  1 , T Mahmoudi, N B Zak, C P Verrijzer
Affiliations

The Drosophila brahma complex is an essential coactivator for the trithorax group protein zeste

A J Kal et al. Genes Dev. .

Abstract

The trithorax group (trxG) of activators and Polycomb group (PcG) of repressors are believed to control the expression of several key developmental regulators by changing the structure of chromatin. Here, we have sought to dissect the requirements for transcriptional activation by the Drosophila trxG protein Zeste, a DNA-binding activator of homeotic genes. Reconstituted transcription reactions established that the Brahma (BRM) chromatin-remodeling complex is essential for Zeste-directed activation on nucleosomal templates. Because it is not required for Zeste to bind to chromatin, the BRM complex appears to act after promoter binding by the activator. Purification of the Drosophila BRM complex revealed a number of novel subunits. We found that Zeste tethers the BRM complex via direct binding to specific subunits, including trxG proteins Moira (MOR) and OSA. The leucine zipper of Zeste mediates binding to MOR. Interestingly, although the Imitation Switch (ISWI) remodelers are potent nucleosome spacing factors, they are dispensable for transcriptional activation by Zeste. Thus, there is a distinction between general chromatin restructuring and transcriptional coactivation by remodelers. These results establish that different chromatin remodeling factors display distinct functional properties and provide novel insights into the mechanism of their targeting.

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Figures

Figure 1
Figure 1
Zeste directs remodeling and transcription on nucleosomal arrays. (A) Recombinant FLAG-tagged Zeste was expressed in baculovirus infected Sf9 cells and, after extract preparation, immunopurified on an anti-FLAG column. The Zeste protein fractions and the purified Drosophila core histones were analyzed by SDS-PAGE followed by Coomassie blue staining. The positions of Zeste, core histones, and the molecular masses (kD) of protein standards are indicated. (B) Outline of the experimental design of the transcription experiments described in this article. Nucleosomal templates were generated using either the Drosophila embryo assembly system or by salt gradient dialysis. After sarcosyl treatment, in the case of S-190 assembly, and purification by sucrose gradient sedimentation, the templates were used in reconstituted transcription reactions. The transcription machinery and chromatin remodeling factors were provided by a partially purified nuclear extract (H0.4). Following a preincubation of 20 min in the presence (or absence, see Fig. 1F) of ATP and either no activator or purified Zeste, transcription was started by the addition of the remaining NTPs and allowed to proceed for 30 min. Transcription products were visualized by primer extension. (C) Transcriptional activation by Zeste. Transcription on chromatin templates (plasmid pAK156) generated using either the S-190 assembly system (lanes 1,2) or by salt gradient dialysis (lanes 3,4) was tested either in the absence (lanes 1,3) or presence of Zeste (lanes 2,4). (D) Zeste directs localized chromatin remodeling. S-190 assembled chromatin templates lacking (lanes 1,3) or bound by Zeste (lanes 2,4) were digested with MNase and analyzed by Southern blot hybridization using oligonucleotides corresponding to either promoter sequences (lanes 3,4) or distal plasmid sequences (the Amp gene; lanes 1,2).(E) ATP-dependent remodeling is a prerequisite for Zeste activation. A nuclear extract (H0.4) with either no activator (lane 1) or Zeste (lanes 2,3) was added to chromatin templates assembled by salt dialysis and purified over a sucrose gradient. Following a 20 min incubation in the presence (lanes 1,3) or absence of ATP (lane 2), transcription was initiated by the addition of NTPs. The analysis of transcription was performed as described in Fig. 1B.
Figure 2
Figure 2
Activation by Zeste requires the BRM complex, but not the ISWI factors. (A) DNaseI footprinting analysis of Zeste on either naked DNA (lanes 1,2) or on S-190 assembled chromatin (plasmid pAK156) using extracts that were either mock-depleted (lanes 3,4) or depletion with affinity purified anti-BRM antibodies (lanes 5,6) or anti-ISWI antibodies (lanes 7,8). After completion of assembly, chromatin was incubated for 1 hr either in the absence (odd numbered lanes) or presence of Zeste (even numbered lanes) followed by digestion with DNaseI. For digestion of the chromatin template, an 150-fold higher amount of DNaseI was used than for naked DNA. The DNaseI digestion pattern was visualized by primer extension. The Zeste footprints and DNaseI hypersensitive sites are indicated with bars and arrows, respectively. (B) The ability of H0.4 partially purified nuclear extract to support Zeste activation was tested after either mock depletion (lanes 1,2), depletion with affinity purified anti-BRM antibodies (lanes 3,4) or anti-ISWI antibodies (lanes 4,6). The chromatin templates were assembled using the S-190 system, sarcosyl treated and purified over a sucrose gradient prior to use in transcription reactions either in the absence (odd numbered lanes) or presence of Zeste (even numbered lanes). The experimental design was as described in Fig. 1B. (C) The efficiency and specificity of the immunodepletion of the H0.4 fraction with either mock (lane 1), anti-BRM (lane 2), or anti-ISWI antibodies (lane 3) was verified by Western blot analysis using antibodies directed against BRM, ISWI, dTAFII80, or NAP-1.
Figure 2
Figure 2
Activation by Zeste requires the BRM complex, but not the ISWI factors. (A) DNaseI footprinting analysis of Zeste on either naked DNA (lanes 1,2) or on S-190 assembled chromatin (plasmid pAK156) using extracts that were either mock-depleted (lanes 3,4) or depletion with affinity purified anti-BRM antibodies (lanes 5,6) or anti-ISWI antibodies (lanes 7,8). After completion of assembly, chromatin was incubated for 1 hr either in the absence (odd numbered lanes) or presence of Zeste (even numbered lanes) followed by digestion with DNaseI. For digestion of the chromatin template, an 150-fold higher amount of DNaseI was used than for naked DNA. The DNaseI digestion pattern was visualized by primer extension. The Zeste footprints and DNaseI hypersensitive sites are indicated with bars and arrows, respectively. (B) The ability of H0.4 partially purified nuclear extract to support Zeste activation was tested after either mock depletion (lanes 1,2), depletion with affinity purified anti-BRM antibodies (lanes 3,4) or anti-ISWI antibodies (lanes 4,6). The chromatin templates were assembled using the S-190 system, sarcosyl treated and purified over a sucrose gradient prior to use in transcription reactions either in the absence (odd numbered lanes) or presence of Zeste (even numbered lanes). The experimental design was as described in Fig. 1B. (C) The efficiency and specificity of the immunodepletion of the H0.4 fraction with either mock (lane 1), anti-BRM (lane 2), or anti-ISWI antibodies (lane 3) was verified by Western blot analysis using antibodies directed against BRM, ISWI, dTAFII80, or NAP-1.
Figure 3
Figure 3
Purification and characterization of the BRM complex. (A) Outline of the chromatographic scheme used to purify the Drosophila BRM complex. (B) Polypeptide composition of BRM-containing fractions from the final monoS column. Proteins were resolved by SDS-PAGE and visualized by silver staining (top) or Western blotting with an anti-BRM antibody (bottom). (C) Western blot analysis of the purified BRM complex. Nuclear extract (H0.4, odd numbered lanes) and a peak fraction from the monoS column (43, even numbered lanes) were analyzed by SDS-PAGE followed by Western blotting using antibodies directed against OSA (lanes 1,2), BRM (lanes 3,4), MOR (lanes 5,6), and ISWI (lanes 7,8). (D) Identification of the core BRM complex. A peak fraction from the monoS column (#42, lane 1) was incubated with beads coated with affinity purified anti-BRM antibodies. Proteins retained on the beads after extensive washes with a buffer containing 800 mm KCl and 0.01% NP-40, were resolved by SDS-PAGE on either an 8% polyacrylamide gel (lane 2) or a 12% polyacrylamide gel (lane 3) and stained with silver. (E) OSA is part of the core BRM complex. The input fraction (monoS #42, lane 1), bound material after the 800 mm KCl washes of the immunoprecipitation with anti-BRM beads (lane 2) and unbound material (lane 3), were analyzed by SDS-PAGE on a 6% polyacrylamide gel followed by Western blotting using a monoclonal antibody directed against OSA.
Figure 4
Figure 4
BRM complex mediates chromatin-specific transcriptional activation by Zeste. (A) The ability of the purified BRM complex (monoS #42) to restore Zeste-directed transcription in a BRM-depleted transcription system (lanes 3–6) was tested using either S-190 assembled chromatin (top), salt dialysis assembled chromatin (middle), or naked DNA (bottom) as a template. Mock-depleted H0.4 extract was used as a positive control (lanes 1,2). Transcription reactions were either in the absence (odd numbered lanes) or presence of Zeste (even numbered lanes). Approximately 20 fmoles of BRM complex was added to reactions 5 and 6 in which the nucleosome to BRM ratio was ∼50:1. The experimental design was essentially as described in Fig. 1. (B) Zeste-directed remodeling in a defined system. The Zeste-responsive template was assembled into chromatin by salt dialysis and incubated in the presence of varying combinations of BRM complex (about 20 femtomoles), Zeste, and ATP. After 30 min, the templates were digested with MNase and analyzed by Southern blot hybridization using an oligonucleotide corresponding to part of the promoter sequence.
Figure 5
Figure 5
Comparison of the nucleosome spacing activity of the ISWI factors and the BRM complex. Suboptimally spaced nucleosomal DNA was prepared by a stepwise salt dilution and dialysis protocol using pure histones. To test for ATP-dependent nucleosome spacing activity, these templates were incubated for 1 hr in the presence of ATP alone (lane 1), a partially purified ISWI fraction alone (lane 3) or both ATP and the ISWI fraction (lane 2) followed by MNase digestion. The MNase digestion pattern was vizualized by ethidium bromide staining of agarose gels. The ability of the ISWI fraction and an approximately equal molar amount BRM complex (about 40 fmoles) to order a nucleosomal array was compared in a similar experiment containing an approximated 180-fold molar excess of nucleosomes (lanes 4–6). Protein amounts were estimated by comparison of silver and Coomassie-stained SDS-PAGE gels containing known amounts of marker proteins and quantitative Western blotting (data not shown). The input template was incubated in the presence of ATP and either no remodeling factor (lane 4), the ISWI fraction (lanes 5), or the BRM complex (monoS #42) (lanes 6) and analyzed by MNase digestion.
Figure 6
Figure 6
Zeste directly binds selected BAPs within the BRM complex. (A) Zeste interacts with the BRM complex. Protein-A Sepharose resin (control, lane 2), anti-BRM affinity beads loaded with BRM complex (lane 3), or anti-ISWI affinity beads loaded ISWI complexes (lane 4) were incubated with 35S-labeled reticulocyte expressed Zeste. Protein complexes were washed, resolved by SDS-PAGE, and bound Zeste was detected by autoradiography. Lane 1 represents 5% of the input material used in the binding reactions. (B) Far-Western blotting analysis reveals that Zeste targets specific BAPs. The purified BRM complex (monoS #42) was resolved by SDS-PAGE and transferred to nitrocellulose. The nitrocellulose membrane was treated with 6 m guanidine-HCl, renatured, washed, and incubated with 35S-labeled reticulocyte expressed Zeste (lane 1) or MOR (lane 4). After extensive washing the filter was exposed to film. Filter 1 and 4 were reprobed with antibodies directed against BRM (lane 2) or MOR (lane 3), respectively. The positions of the BAPs bound by Zeste or MOR are indicated on the left (Zeste) or right (MOR) of the panels. (C) Mapping of the Zeste binding domain of MOR by GST pull-down assays. GST-MOR carboxyl terminus (residues 454–1174; lanes 2,10), GST-MOR amino terminus (residues 106–410; lanes 3,11), GST-MOR (residues 171–410; lane 8), GST-BRM (residues 230–736; lanes 4,12), or GST alone (lanes 5,7,13) were immobilized on glutathione-Sepharose beads and incubated with either 35S-Zeste (lanes 2–8) or 35S-MOR (lanes 10–13). Protein complexes were washed, resolved by SDS-PAGE and bound proteins were detected by autoradiography. Lanes 1, 6, and 9 represent 5% of the input material used in the binding reactions. The domain structure of MOR and its interaction domains with itself, BRM (asterisk indicates data from Crosby et al. 1999) and Zeste are indicated.
Figure 7
Figure 7
The leucine zipper of Zeste binds MOR. Mapping of the MOR-binding domain of Zeste by GST pull-down assays. GST alone (lane 2), GST-MOR carboxyl terminus (lane 3), GST-MOR amino terminus (lane 4), or GST-MOR residues 171–410 (lane 5) were immobilized on glutathione-Sepharose beads and incubated with 35S-methionine-labeled Zeste or various Zeste deletion mutants. Protein complexes were washed, resolved by SDS-PAGE, and bound proteins were detected by autoradiography. Lane 1 represents 5% of the input material used in the binding reactions. The domain structure of Zeste and the amino acid residues present in the various deletion mutants are indicated (DBD) DNA-binding domain; (L-zip) leucine zipper; (Q/A) region rich in glutamines and alanines; (AD) acidic domain; (P-rich) proline rich domain.
Figure 8
Figure 8
Summary and model. Our results suggest that the core BRM complex comprise 10 subunits (light grey) including trxG protein OSA and two putative novel BAPs of 170 and 26 kD. More loosely associated factors (dark grey) that are not part of the core complex such as p400, are indicated. Zeste binds to multiple sites in the promoter DNA as a large oligomer and directly binds selective subunits within the BRM complex (indicated by a bold outline). These include most significantly MOR, BAP170, and p400, whereas somewhat less strong binding to OSA and BAP111 was observed. MOR interacts with itself and is assembled into the complex via binding to BRM, BAP111, BAP60, and either (the most likely candidate) BAP47 or BAP45. Contacts between MOR and other BAPs are indicated by a bold interface. All other contacts depicted here are speculative. The results from our experiments suggest that the BRM complex is an essential coactivator that can be recruited to specific genes by Zeste. The BRM complex may create an open chromatin conformation, here indicated as naked DNA, that could facilitate the docking of other transcription factors.
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