The Drosophila BRM complex facilitates global transcription by RNA polymerase II

Abstract

Drosophila brahma (brm) encodes the ATPase subunit of a 2 MDa complex that is related to yeast SWI/SNF and other chromatin-remodeling complexes. BRM was identified as a transcriptional activator of Hox genes required for the specification of body segment identities. To clarify the role of the BRM complex in the transcription of other genes, we examined its distribution on larval salivary gland polytene chromosomes. The BRM complex is associated with nearly all transcriptionally active chromatin in a pattern that is generally non-overlapping with that of Polycomb, a repressor of Hox gene transcription. Reduction of BRM function dramatically reduces the association of RNA polymerase II with salivary gland chromosomes. A few genes, such as induced heat shock loci, are not associated with the BRM complex; transcription of these genes is not compromised by loss of BRM function. The distribution of the BRM complex thus correlates with a dependence on BRM for gene activity. These data suggest that the chromatin remodeling activity of the BRM complex plays a general role in facilitating transcription by RNA polymerase II.

Fig. 1. The BRM protein is associated with chromosome puffs and interbands of salivary gland polytene chromosomes. (A) Distribution of BRM protein on wild-type polytene chromosomes. Arrowhead indicates chromocenter. (B) The top panel shows indirect immunofluorescence using an anti-BRM antibody (green), the second panel is DAPI-stained DNA (blue), the third panel shows the merged images and the bottom panel is a split image. Note that BRM protein is predominantly found in the DAPI interbands. The distal region of chromosome arm 2L is shown.

Fig. 2. BAP55, an actin-related protein, is a subunit of the BRM complex. (A) BAP55 is physically associated with the BRM complex in embryo extracts. Immunoprecipitations were performed using the 12CA5 antibody against the HA tag and protein extracts derived from either OregonR embryos (lanes 1–3) or P[w⁺, brm-HA-6His]92C; brm²/Df(3L)th102 embryos (lanes 4–6). Western blotting was performed on one-tenth of the total input extract (I) and supernatant (S) and one-fifth of the total pellet (P) using antibodies against BRM, BAP111, BAP55 and RNA Pol IIc. The HMG-domain protein BAP111, another subunit of the BRM complex, is presented as a positive control (Papoulas et al., 2001). Note that BAP111 and BAP55 are immunoprecipitated with BRM, while Pol II is not (lane 6). Proteins in the pellet lane show slightly reduced mobility relative to start and supernatant due to differences in buffer conditions. (B) Western blot of fractions derived from a Superose 6 gel filtration column loaded with embryo extract and probed with antibodies against BRM, BAP111 and BAP55. Vertical arrows indicate void and elution volumes of molecular weight standards. Note that the majority of BAP55 co-elutes with BRM and BAP111, while some BAP55 appears to be monomeric. (C) Distributions of BRM (green) and BAP55 (red) on wild-type salivary gland polytene chromosomes. Chromosome arm 2L is shown. Note that the patterns of BAP55 and BRM proteins are predominantly overlapping.

Fig. 3. The BRM complex is associated with regions that are not bound by PC. Immunofluorescence detection of BRM (green) and PC (red) on wild-type salivary gland polytene chromosomes. Note that the distributions of BRM and PC proteins are dissimilar with few sites of overlap.

Fig. 4. The BRM complex is associated with sites of active Pol II transcription. (A) Immunofluorescence detection of BRM (green) and the 140 kDa IIc subunit of RNA Pol II (red) on wild-type salivary gland polytene chromosomes reveals a high degree of overlap. (B) Magnification of the distal region of 3L illustrates that BRM (green) marks active regions of the chromosome, although BRM protein is not always present at the same levels as Pol II (red). This is shown in the bottom right panel as a split image.

Fig. 5. The BRM complex co-localizes with both the promoter entry and elongational forms of Pol II. (A) Immunofluorescence detection of BRM (green) and RNA Pol IIo^Ser2 (red) on wild-type salivary gland polytene chromosomes reveals that most regions of transcriptional elongation are also bound by the BRM complex. The distal regions of the X and 3L chromosomes are shown. (B) Immunofluorescence detection of BRM (green) and RNA Pol IIa (red) on wild-type salivary gland polytene chromosomes reveals that the BRM complex is present at many sites of transcriptional initiation or promoter pausing. The distal region of the X chromosome is shown.

Fig. 6. The BRM bromodomain is not required for the recruitment of the BRM complex to active chromatin. Immunofluorescence detection of BRM (green) and RNA Pol II (subunit IIc) (red) on salivary gland polytene chromosomes derived from larvae expressing only BRM lacking the bromodomain (BRM^ΔBD). Note that BRM^ΔBD is localized to transcriptionally active regions. The distal region of the X chromosome is shown.

Fig. 7. Expression of dominant-negative BRM (BRM^K804R) in the salivary gland compromises Pol II transcription on polytene chromosomes. Immunofluorescence using antibodies directed against RNA Pol IIo^Ser2 (A and B, red) and RNA Pol IIa (C and D, green). Note that expression of dominant-negative BRM (UASbrm^K804R) results in a dramatic reduction in the levels of both Pol IIo^Ser2 (B) and Pol IIa (D) relative to that observed on chromosomes derived from salivary glands expressing LacZ (UASlacZ) (A and C).

Fig. 8. Loss of BRM function does not affect the binding of other chromatin-associated proteins to chromosomes. (A and B) Immunofluorescence detection of PC (green) and RNA Pol IIo^Ser2 (red). In marked contrast to RNA Pol IIo^Ser2 (red), the levels and distribution of PC (green) are comparable on polytene chromosomes derived from UASlacZ larvae (A) or UASbrm^K804R larvae (B). (C–F) The levels and distribution of ISWI (green) are comparable on polytene chromosomes derived from UASlacZ larvae (C) or UASbrm^K804R larvae (D). As an internal control, the chromosomes were simultaneously stained for RNA Pol IIo^Ser2 (red). The levels of Pol IIo^Ser2 are reduced on chromosomes from UASbrm^K804R larvae (F) relative to the levels observed on chromosomes from UASlacZ (E).

Fig. 9. Loss of BRM function does not non-specifically affect the level or activity of RNA Pol II. (A) Pol II protein levels are comparable in salivary glands expressing dominant-negative BRM and control glands expressing LacZ. Western blot of 10 salivary glands derived from UASlacZ larvae (lane 1) or UASbrm^K804R larvae (lane 2). The top panel is probed with antibody against the 140 kDa IIc subunit of RNA Pol II and the bottom panel is probed with antibody against α-tubulin. The ratio of protein levels of Pol II to α-tubulin is 1.5 in salivary glands expressing LacZ and 1.8 in salivary glands expressing BRM^K804R. (B) Unlike heat shock factor (HSF, red), BRM (green) is not recruited to heat shock loci following heat shock. DNA is stained with DAPI (blue). (C and D) The heat shock reponse is intact in salivary glands expressing dominant-negative BRM. UASlacZ larvae (B) or UASbrm^K804R larvae (C) were heat shocked and chromosomes were stained with anti-RNA Pol IIo^Ser2 (red) and DAPI (blue). Chromosome 3R is shown and the heat shock genes are indicated. Note that levels of Pol II associated with the heat shock genes are comparable in glands expressing LacZ or BRM^K804R.