The Brakeless co-regulator can directly activate and repress transcription in early Drosophila embryos

Developmental Biology 407 (2015) 173–181

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The Brakeless co-regulator can directly activate and repress transcription in early Drosophila embryos Filip Crona, Per-Henrik Holmqvist, Min Tang, Bhumica Singla 1, Helin Vakifahmetoglu-Norberg 1, Katrin Fantur, Mattias Mannervik n Stockholm University, Department of Molecular Biosciences, The Wenner-Gren Institute, Arrheniuslaboratories E3, Stockholm, Sweden

art ic l e i nf o

a b s t r a c t

Article history: Received 19 May 2015 Received in revised form 22 July 2015 Accepted 6 August 2015 Available online 7 August 2015

The Brakeless protein performs many important functions during Drosophila development, but how it controls gene expression is poorly understood. We previously showed that Brakeless can function as a transcriptional co-repressor. In this work, we perform transcriptional profiling of brakeless mutant embryos. Unexpectedly, the majority of affected genes are down-regulated in brakeless mutants. We demonstrate that genomic regions in close proximity to some of these genes are occupied by Brakeless, that over-expression of Brakeless causes a reciprocal effect on expression of these genes, and that Brakeless remains an activator of the genes upon fusion to an activation domain. Together, our results show that Brakeless can both repress and activate gene expression. A yeast two-hybrid screen identified the Mediator complex subunit Med19 as interacting with an evolutionarily conserved part of Brakeless. Both down- and up-regulated Brakeless target genes are also affected in Med19-depleted embryos, but only down-regulated targets are influenced in embryos depleted of both Brakeless and Med19. Our data provide support for a Brakeless activator function that regulates transcription by interacting with Med19. We conclude that the transcriptional co-regulator Brakeless can either activate or repress transcription depending on context. & 2015 Elsevier Inc. All rights reserved.

Keywords: Transcription Transcriptional co-regulator Drosophila embryo Mediator complex Brakeless Scribbler Master of thickveins

1. Introduction Differential gene expression generates diverse cell types from an identical DNA sequence during embryo development. Gene expression is controlled at multiple levels, but regulation of transcription is a common control step. Critical for transcription are sequence-specific transcription factors that bind to cis-regulatory DNA and interact with the cell's transcriptional machinery, resulting in gene repression or activation. Such interactions are mediated by co-regulators, i.e. transcriptional co-repressors or coactivators that bridge transcription factors with the basal transcription apparatus (reviewed in Mannervik et al. (1999)). The Drosophila melanogaster Brakeless protein has previously been shown to function as a transcriptional co-repressor (Haecker et al., 2007). It was identified in a genetic screen for regulators of Drosophila embryo patterning, and was shown to function as a corepressor for the transcription factor Tailless. Brakeless can associate with another co-repressor, Atrophin, and both Brakeless and Atrophin are required for Tailless repressor function (Haecker n

Corresponding author. Fax: þ 46 8 156756. E-mail address: [email protected] (M. Mannervik). 1 Present address: Karolinska Institutet, Stockholm, Sweden.

http://dx.doi.org/10.1016/j.ydbio.2015.08.005 0012-1606/& 2015 Elsevier Inc. All rights reserved.

et al., 2007; Wang et al., 2006). In brakeless or atrophin mutant embryos, expression of the Tailless-regulated segmentation genes Krüppel and knirps (kni) expand to more cells than normal. Genetic evidence is mostly consistent with Brakeless functioning as a co-repressor. Brakeless was initially identified as required for correct termination of photoreceptor axon projections in the optic lobe of the Drosophila brain (Rao et al., 2000; Senti et al., 2000), where it represses Runt expression in photoreceptor R2 and R5 neurons (Kaminker et al., 2002). In parallel, it was isolated as scribbler, since when mutated it causes a larval turning behavior phenotype in the absence of food (Yang et al., 2000). The gene has also been named master of thickveins (mtv), since it represses expression of the Dpp receptor Thickveins in wing imaginal disks (Funakoshi et al., 2001). The brakeless locus encodes two proteins, Brakeless-A and Brakeless-B, generated by alternative splicing. The only sequence similarity to known functional domains is a single C2H2 zinc finger located in the unique region of Brakeless-B. One additional domain (D2) is highly conserved and present also in sequences from other metazoa. In vertebrates, two Brakeless homologs are present, ZNF608 and ZNF609. ZNF608 controls Rag1 and 2 expression during mouse thymocyte development, and has been associated with body mass index in humans (Speliotes et al., 2010; Zhang et al., 2006). ZNF609 is part of a protein network for

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pluripotency in mouse embryonic stem cells as a component of a Dax1 complex (van den Berg et al., 2010). Although Brakeless has been suggested to participate in mediating SUMO-dependent transcriptional repression (Stielow et al., 2008), the mechanisms by which Brakeless controls transcription are not understood. Identification of proteins that interact with Brakeless could provide insight into such mechanisms. The Mediator complex is a multi-subunit co-regulator of RNA polymerase II transcription and is a key link for transducing regulatory signals from enhancer to promoter (reviewed in Kornberg (2005)). It is evolutionarily conserved from yeast to man and contains approximately 25 subunits. Electron microscopy has revealed a four domain structure: head, middle, tail and kinase domains (Tsai et al., 2014). The Med19 subunit forms a hook at the end of the middle domain (Tsai et al., 2014), and in the absence of the Med19 subunit, the middle module can be released and a stable complex consisting of only head and tail isolated under stringent conditions (Baidoobonso et al., 2007; Tsai et al., 2014). However, purification of Mediator from a Med19 mutant yeast strain under more physiological conditions results in an intact Mediator complex, but that interacts less efficiently with Pol II than wild-type Mediator (Baidoobonso et al., 2007; Tsai et al., 2014). Med19 has been implicated both in repression and in activation of transcription. In the yeast Saccharomyces cerevisiae, Med19 is encoded by Rox3, and was first identified as a negative regulator of the CYC7 gene (Rosenblum-Vos et al., 1991). It is also known as SSN7, since it suppresses the requirement of the Snf1 kinase for expression of glucose-repressed genes (Song et al., 1996). Med19 has also been shown to negatively regulate sporulation-specific genes and a Swi4p-dependent reporter (Friesen et al., 1998; Tabtiang and Herskowitz, 1998). On the other hand, it is also necessary for full galactose induction of GAL1 expression, Ino2-dependent transcriptional activation, as well as for heatshock induced gene expression (Brown et al., 1995; Dettmann et al., 2010; Kremer et al., 2012), and when tethered to DNA it activates transcription (Song et al., 1996). In human cells, MED19 is also known as LCMR1 (Lung Cancer Metastasis Related Protein 1), and is overexpressed in a variety of cancers and cancer cell lines (Chen et al., 2011; Ding et al., 2012; Ji-Fu et al., 2012; Li et al., 2011; Liu et al., 2012; Sun et al., 2011; Wen et al., 2012; Zhang et al., 2012). It is recruited by the RE1 silencing transcription factor (REST) together with MED26, and mediates repression of neuronal gene expression (Ding et al., 2009). Med19 is also necessary for androgen receptor transcriptional activity in Drosophila S2 and human prostate cancer cells (Imberg-Kazdan et al., 2013), and for gene activation by Hox transcription factors in Drosophila imaginal disks (Boube et al., 2014). Here, we perform transcriptional profiling of brakeless mutant embryos and identify directly regulated target genes. Unexpectedly, we find that most genes are down-regulated in brakeless embryos, indicating that Brakeless can function both as a co-repressor and as a co-activator. From a yeast two-hybrid screen, we identify Med19 as interacting with the highly conserved Brakeless D2 domain. Interestingly, down-regulated but not upregulated Brakeless target genes are also affected in embryos depleted of Med19 and Brakeless. Together, these data provide support for a Brakeless co-activator function that regulates transcription through Med19 in Drosophila.

2. Materials and methods 2.1. Fly stocks, Brakeless and Med19 knock-down, and generation of germline clones D. melanogaster strains were kept at 25 °C. w1118, w; FRT2RG13 c

px sp, or offspring from female w; α-Tubulin67C-Gal4-VP16/ þ crossed to w1118 males were used as controls. UAS strains containing a long hairpin targeting Brakeless (y v; P{TRiP.JF02375}attP2 derived from hairpin TR02171P.1 in the VALIUM10 vector) or a short hairpin microRNA targeting Med19 (y sc v; P{TRiP.HMS00588} attP2 derived from hairpin SH00843 in the VALIUM20 vector) were kindly provided by Norbert Perrimon. Embryos where the maternal Brakeless and/or Med19 contribution has been knockeddown were obtained from w; α-Tubulin67C-Gal4-VP16/þ; P{TRiP}/ þ females crossed with males of the same genotype. Over-expression of maternal Brakeless in early embryos was achieved by crossing w; α-Tubulin67C-Gal4-VP16/þ ; UASp-BksB/þ or w; α-Tubulin67C-Gal4-VP16/þ; UASp-BksB-VP16/þ females with males of the same genotype. For brakeless germline clones, FRT2RG13 ovoD1/T(1;2)OR64/CyO (Bloomington stock #4344) was used instead of FRT2RG13 ovoD1/Dp (?;2)bwD, S1wgSp-1Ms(2)M1bwD/CyO (stock #2125) for the FLP-FRT dominant female sterile technique previously described (Chou and Perrimon, 1996). The bks278 allele was used, which has a 345 bp deletion that causes a frame shift at aa 741 resulting in addition of 79 novel amino acids (Haecker et al., 2007). It was outcrossed with a w1118 strain to remove potential second-site mutations. FRT2RG13 bks278/CyO females were crossed with males of the genotype hsFLP/Y; FRT2RG13 ovoD1/CyO, and offspring larvae heat-shocked for 3 h at 37 °C on days 3, 4 and 5 after egg-laying to induce expression of the FLP recombinase. Cy þ females were crossed to FRT2RG13 bks278/CyO males and embryos were collected, dechorionated using bleach, and directly frozen in Trizol at  80 °C for RNA isolation. 2.2. Generation of transgenes The BksB cDNA was cloned into the pUASp vector using XbaI. For fusion with the VP16 activation domain, an AscI site was introduced in place of the stop codon by PCR. The PCR product and BksB cDNA were digested with RsrII (cuts in Bks) and AscI (cuts in modified pBS vector), and used to replace the 3′ end of the BksB cDNA. The VP16 sequence was PCR amplified from pSlax-VP16 (a gift from Johan Ericson, Karolinska Institutet) with a 5′ primer containing an AscI site, and a 3′ primer containing XbaI and AscI sites. The PCR product was digested with AscI and ligated into the modified pBS-Bks plasmid. XbaI was then used to lift out the BksBVP16 fusion from pBS and cloned into the XbaI site of pUASp. The constructs were injected into w1118 flies following standard procedures (Rubin and Spradling, 1982). 2.3. Microarray The microarray data, statistical and gene ontology analyses are described in Crona et al. (2015). 2.4. Quantitative RT-PCR Total RNA was extracted from embryos using Trizol (Invitrogen) according to the manufacturer's protocol. For one biological replicate 20 μl embryos were collected. A total of 1 μg from each RNA sample was subjected to DNase digestion (Sigma) and cDNA synthesis (Applied Biosystems) according to the manufacturer's protocol. Samples were confirmed for quality and quantity using agarose gel electorphoresis and Nanodrop. Out of 200 μl (10  diluted) total cDNA, 2 μl was subsequently used for qRT-PCR in a total volume of 20 μl using CFX96 (BioRad instruments, Sweden) with EvaGreen supermix (Solis BioDyne). Resulting Ct-values were converted into raw quantities with the ΔCt method using Genorm (Vandesompele et al., 2002), which were used to calculate the geometric mean of two reference genes β-tubulin and RpL32 or

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GAPDH and RpL32, serving as normalization factors for each cDNA sample. The stability measure M (Vandesompele et al., 2002), for the mean of the reference genes was below 1.5 for all the conditions tested. Relative expression levels were calculated by dividing the raw quantities of the genes of interest for each sample by the corresponding normalization factors. 2.5. Antibodies GST-Bks (834-1151) (Haecker et al., 2007) and GST-Bks (450620) were produced and purified from Escherichia coli BL21 cells, and used to immunize guinea-pigs and rats. Guinea-pig GST-Bks (450-620) serum was used in this study. 2.6. Immunoprecipitation, Western blot and RNAi Embryos 2–4 h old were crushed in 10 times the volume of RIPA buffer, and then shaken vigorously at 4 °C for 20 min. Extracts were centrifuged at 13,000 rpm for 5 min. The supernatant was pre-cleared with protein A and G Dynabeads mix (Invitrogen) blocked with BSA. BSA blocked protein A and G Dynabeads preincubated with anti-Bks serum was mixed with 500 μl pre-cleared extract. Protein extract and beads were rotated at 4 °C over-night, and then washed twice with RIPA buffer, once with TEN (10 mM Tris pH 8.0, 1 mM EDTA, 100 mM NaCl), and once with 50 mM Tris pH 6.8. Beads were resuspended in 10% the original volume of RIPA buffer and 1/4 volume of 5  Laemmli loading buffer and ran on an 7.5% polyacrylamide-SDS gel together with input supernatant mixed with 1/4 volume of 5  Laemmli loading buffer. S2 cells, or S2 cells stably transfected with HA-tagged BksB under a metallothionein promoter induced for 5 h with 1 mM CuSO4, were collected at 1400 g and resuspended in RIPA buffer at 2*107 cells/ml. Lysis was carried out by vigorous shaking for 10 min at 4 °C. Lysate was centrifuged at 10,000 rpm 5 min at 4 °C, and the supernatant recovered. Immunoprecipitation used 0.3 ml protein extract and was performed as described for the embryo extract. For double-stranded RNA treatment, S2 cells were split the day before treatment. Cells were collected and washed twice in S2 cell medium without fetal calf serum (FCS). For each treatment 2*106 cells were resuspended in 0.75 ml FCS-free medium and added to one well of a 6-well plate. 37 nM dsRNA was added to the cells and mixed by shaking the plate. Cells were incubated for 1 h at 25 °C, and then 1.5 ml S2 cell medium with 15%FCS was added. Cells were maintained at 25 °C for four days before harvesting. Proteins were transferred to a PVDF membrane and blocked in 1% milk powder in PBST. Guinea-pig GST-Bks (450-620) serum was used at a 1:400 dilution, a rabbit anti-alpha-Tubulin (ab18251, Abcam) at 1:1000, and rabbit anti-HA (ab9110, Abcam) at 1:2500. The membrane was incubated with primary antibody for 1 h at room temperature or overnight in a cold room. HRP-coupled rabbit anti-guinea-pig or goat anti-rabbit (1:10,000) secondary antibody (Dako) in 1% milk/PBST was incubated with membrane for 1 h at room temperature. Enhanced chemiluminescence (ECL) was used for signal detection (GE Healthcare), and the membrane exposed to a Luminescent Image Analyzer (LAS-100plus, Fujifilm). We used MultiGauge (Fujifilm) for quantification of intensities and normalized against Tubulin in the input. 2.7. Chromatin immunoprecipitation Chromatin immunoprecipitation was carried out as described (Holmqvist et al., 2012) from three independent chromatin extracts. In brief, two to four hour old dechorionated embryos were dounce homogenized and cross-linked in 1.8% formaldehyde for 15 min at room temperature. Washed, cross-linked nuclei were sonicated on a Bioruptor (Diagenode). For immunoprecipitation, a

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mix of Protein A and G Dynabeads (Invitrogen) blocked with BSA were either used directly as a mock control, or pre-incubated with guineapig anti-Bks 450-620 antibody. Beads were resuspended in chromatin extract corresponding to 40 ml of embryos followed by incubation at 4 °C over night. After washing of beads, the crosslinks were reversed at 68 °C for at least 4 h, Proteinase K treated, and DNA purified by phenol–chloroform extraction and ethanol precipitation. The DNA was resuspended in 100 ml 0.1  TE. Two microliters of the ChIP material was analyzed by qPCR (EvaGreen, Solis BioDyne) in duplicate using a CFX96 system (BioRad Instruments, Sweden). 2.8. Yeast-two-hybrid and GST-pulldown A Drosophila embryo 0 12 þ12–24 h old cDNA library was screened with a LexA-Bks (834-1151) bait at Hybrigenics, Paris, France. Eleven high confidence interactions were obtained from 78.5 million analyzed. One of these corresponds to Med19. Two independent Med19 clones were isolated. The interaction was confirmed by a GST-pulldown assay, where GST-Bks (834-1151) was incubated with in vitro translated Med 19. GST and GST-Bks (834-1151) were expressed in E. coli BL21 and purified on glutathione sepharose beads. 35S-labeled Med19 was in vitro translated with the TnT coupled reticulocyte lysate system (Promega), and precleared with GSH-sepharose. It was then incubated with GST-fusion proteins on GSH sepharose in NETN buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40) for 1 h. Sepharose beads were collected by a brief centrifugation at 2000 rpm and washed 4 times in NETN before elution by boiling in loading buffer. Proteins were separated on a 10% polyacrylamide-SDS gel and exposed to a phosphoimager (FLA-3000, Fujifilm). 2.9. Accession number The microarray data complies with MIAME guidelines and is deposited in GEO under accession number GSE60048.

3. Results 3.1. Identification of Brakeless-regulated genes Our previous work showed that Brakeless is a co-repressor required for Tailless function in early Drosophila embryos (Haecker et al., 2007). To identify additional target genes in the early embryo, RNA was isolated from 2–4 h old embryos derived from brakeless (bks278) germline clones, lacking maternal Brakeless function. As a control, germline clones were created with the unmutagenized FRT chromosome on which the bks278 allele was generated (Luschnig et al., 2004). The RNA was converted to cDNA and hybridized to an Affymetrix array. After applying a cutoff at 1.5 fold change in expression level, 66 up-regulated and 174 downregulated genes were identified (Table 1 in Crona et al. (2015)). Since Brakeless was previously shown to be a co-repressor, we were surprised to find more down-regulated than up-regulated genes. The genes were subjected to functional annotation analysis using DAVID (Dennis et al., 2003), which groups genes into clusters based on co-association with gene ontology (GO) terms. Among the top clusters for the up-regulated list of genes were terms associated with DNA binding, metal binding, RNA processing, transcription, transcription regulator activity, and pyrimidine metabolism (Crona et al., 2015). This indicates that Brakeless represses genes encoding transcription factors and other regulators of gene expression. The top clusters for the down-regulated list of genes include pigmentation, endopeptidase activity, synaptic

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transmission, polysaccharide metabolism, glutathione transferase activity, immune response and aminoglycan metabolic processes (Crona et al., 2015). This shows that many of the genes that are activated by Brakeless are involved in metabolic and physiological processes, and demonstrates that repressed and activated genes fall into distinct categories. Since Brakeless is a maternal protein, and around 80% of all Drosophila genes are maternally contributed, Brakeless could be regulating the identified genes either during oogenesis or zygotically in the embryo. We therefore compared our gene list to an RNA-seq dataset that distinguishes maternal from zygotic transcripts in Drosophila embryos using polymorphisms (Lott et al., 2011). The Brakeless-regulated genes were categorized as being maternally, zygotically, or maternally and zygotically (matzyg) derived. The comparison revealed 34 maternal, 14 matzyg, and 3 strictly zygotic transcripts among the up-regulated genes (Table 1 in Crona et al. (2015)). Of the down-regulated genes, 62 were maternal, 15 matzyg, and 38 zygotic. The rest were genes without polymorphisms in the datasets, and could therefore not be classified. Since only zygotic transcripts are going to be direct Brakeless targets in the early embryo, we focused our analysis on genes with matzyg or zygotic RNAs. Of note, there are more downregulated than up-regulated genes with early zygotic transcription. 3.2. Brakeless occupies both up- and down-regulated genes The relatively large number of down-regulated zygotic genes raised the possibility that some of these are directly regulated by Brakeless. We raised antibodies against two Brakeless parts in both guinea-pig and rat. None of them could reliably detect Brakeless protein in total cell or embryo extracts. However, the guinea-pig serum raised against Brakeless amino-acids 450-620 recognized two large and one small protein isoform in a Western blot following immunoprecipitation (IP) using the same serum (Fig. 1A). Expression of HA-tagged BksB resulted in a stronger Brakeless signal, and the protein was recognized by HA antibody after Brakeless IP (Fig. 1A). Treating S2 cells with brakeless doublestranded RNA reduced the Brakeless signal after IP (Fig. 1B). We used this serum in chromatin immunoprecipitation (ChIP) from S2 cells, and detected higher occupancy at three putative target genes compared to an intergenic control locus and compared to an IgG control (Fig. 1C). In brakeless RNAi-treated cells, the signal was reduced, although not statistically significant (Fig. 1C). Taken together, these data show that the serum is able to IP and ChIP Brakeless. We then generated Brakeless ChIP-seq data from S2 cells (manuscript in prep.). We further compared the Brakelessregulated genes in embryos with the Brakeless peaks in S2 cells to search for potential direct targets, and designed quantitative PCR (qPCR) primers for a subset of genes to be tested in ChIP from early embryos (Table S1, location of primers in relation to S2 peaks are shown in Fig. S1). As a positive control, we used primers from the knirps (kni) enhancer to which Brakeless is recruited by Tailless (Haecker et al., 2007). We performed ChIP from three biological replicates of 2–4 h old embryos and examined occupancy of Brakeless at both up-regulated and down-regulated genes. We plotted occupancy as fold enrichment over a negative control locus (background), an intergenic region devoid of any known factor. As a control, we performed ChIP with no antibody. As expected, we observed Brakeless occupancy at the kni enhancer (Fig. 2). Brakeless also showed statistically significant occupancy relative background at regions close to Copper transporter 1A (Ctr1A) and plexus (px) (up-regulated), as well as shroud (sro), Suppressor of cytokine signaling at 36E (Socs36E), CG30428, and highwire (hiw) among the down-regulated genes (Fig. 2). For some genes, we did not detect significant Brakeless enrichment (pxb, Dad and shn in Fig. 2). These

Fig. 1. Characterization of a Brakeless serum. (A) Extracts from Drosophila S2 cells or S2 cells expressing an HA-tagged Brakeless B isoform were immunoprecipitated with a guinea-pig anti-Brakeless (450-620) serum or control IgG, transferred to membrane and probed with the same Brakeless serum (Bks), re-probed with Tubulin as a loading control, and anti-HA antibody. Two BksB and one BksA isoforms were detected, and only tagged BksB was recognized by the HA antibody. The expected molecular weight of BksB is 230 kDa and BksA 90 kDa. (B) S2 cells were treated with GFP (control) or Brakeless double-stranded RNA (Bksi), immunoprecipitated with Brakeless serum, blotted and probed with the Brakeless serum and Tubulin as a control for the amount of starting material. The BksB bands were reduced to 45% of control, and BksA reduced to 32%. (C) Chromatin immunoprecipitation using the Brakeless serum and control IgG from S2 cells treated with GFP or Brakeless double-stranded RNA (Bksi). Percent input values are plotted for an intergenic negative control locus (IG2c), and three putative target genes, pxb, Ctr1A, and sro. Error bars indicate S.D. (n¼3 biological replicates).

genes could either be occupied by Brakeless at other, tissue-specific regulatory regions, or they could be indirect target genes. Taken together, our ChIP and microarray data indicate that Brakeless may directly control expression of both some up- and some down-regulated genes. 3.3. Brakeless target genes respond to Brakeless over-expression We thereafter focused our study on 7 genes directly regulated by Brakeless. These are zygotically or matzyg expressed, show a statistically significant change in the expression array, and have occupancy of Brakeless at the locus. The exceptions are kni, which was not identified in the array, and hiw that was classified as maternal. However, recent GRO-seq experiments showed that very few genes are strictly maternal and that zygotic hiw transcripts could be detected with this sensitive method (Saunders et al., 2013). To validate the microarray results, we used an alternative approach to reduce the maternal Brakeless load from early embryos. We used a knockdown method that is effective in the female germline (Ni et al., 2011). Flies were generated that contain the Gal4-VP16 activator expressed in the female germline under control of the α-Tubulin67C promoter (α-Tubulin67C-Gal4-VP16), as well as a UAS-hairpin construct producing Brakeless doublestranded RNA, and embryos collected. We prepared RNA from 2– 4 h old brakeless knockdown embryos (tub 4bks RNAi) and from

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Fig. 2. Identification of direct Brakeless targets. Determination of Brakeless occupancy at Brakeless-regulated genes by Chromatin immunoprecipitation-quantitative PCR in 2–4 h old w1118 embryos. Brakeless serum (anti Bks) and no antibody control ChIP signals were calculated at up- and down-regulated genes, and the values were normalized against an intergenic locus devoid of known factors (IG2c). As a positive control the knirps enhancer (kni) was used. Error bars indicate S.D. (n¼ 2 and 3 biological replicates) and * indicates Po 0.05, one-tailed unpaired Student's t-test.

embryos derived from α-Tubulin67C-Gal4-VP16 heterozygous females as a control (tub), and performed quantitative RT-PCR (RTqPCR). Up-regulation of px was observed upon Brakeless knockdown, whereas kni up-regulation did not reach statistical significance, and Ctr1A showed no difference to control embryos in the RT-qPCR (Fig. 3A). However, upon Brakeless over-expression, all three genes were down-regulated (Fig. 3B). In these experiments, the α-Tubulin67C-Gal4-VP16 activator was crossed to a UASp-BksB transgene and embryos collected from w; α-Tubulin67C-Gal4-VP16/þ ; UASp-BksB/ þ females (tub 4 BksB). Fig. 3B shows that an increased maternal contribution of Brakeless leads to down-regulation of target genes that are up-regulated in brakeless mutant embryos. Next, we analyzed down-regulated genes. Expression of sro and hiw was reduced upon Brakeless knockdown, whereas Socs36E and CG30428 were unaffected (Fig. 4A). In embryos derived from females over-expressing Brakeless in the germline (tub 4BksB), sro and CG30428 showed a statistically significant up-regulation, whereas Socs36E and hiw were only marginally affected (Fig. 4B). Taken together, these data demonstrate that Brakeless can function as an activator of direct target genes, since loss of Brakeless reduces transcription and over-expression of Brakeless results in increased transcription. 3.4. Brakeless activator function is maintained by fusion to an activation domain To test if the function of Brakeless could be modulated, we fused the VP16 activation domain to the C-terminus of the Brakeless-B isoform. We provided early embryos with this fusion protein by expressing it in the female germline. As can be seen in Fig. 3B, over-expression of unmodified BksB causes repression of kni, Ctr1A, and px whereas more variation in expression of these genes was observed upon expression of the BksB-VP16 fusion protein. However, sro, Socs36E, CG30428, and hiw expression was up-regulated by the BksB-VP16 fusion protein compared to control (Fig. 4B). This indicates that the activation function of Brakeless is intact in the BksB-VP16 fusion protein and that activation of these genes is not due to repression of upstream regulators. It also provides further evidence that Brakeless directly regulates expression of these genes.

Fig. 3. Brakeless is a repressor of some genes. Expression of the Brakeless-target kni, and two genes (Ctr1A, px) showing statistically significant up-regulation (41.5 fold, Po0.05) in a bks expression array. (A and B) Quantitative RT-PCR of Brakeless target gene expression in 2–4 h embryos. (A) Brakeless knock-down leads to stronger expression of up-regulated targets. Columns depict average expression levels relative to a mean of two reference genes in Brakeless knock-down embryos derived from females expressing a long hairpin targeting Brakeless in the germline under control of an αtubulin Gal4-VP16 driver (tub4bks RNAi). Embryos from females with only the Gal4 driver (tub) were used as controls. S.D. (n¼ 3–4) relative to a mean of two reference genes (β-tubulin and RpL32). Values from the control (tub) were set to 1. (B) Brakeless over-expression leads to gene repression, whereas a Brakeless-VP16 fusion protein fails to repress expression of these target genes. Embryos were derived from females expressing BksB (tub4BksB) or BksB-VP16 (tub4BksB-VP16) in the germline under control of the α-tubulin Gal4-VP16 driver. Embryos from females with only the Gal4 driver (tub) were used as controls. Columns depict average expression levels with S.D. (n¼3–6) relative to a mean of two reference genes (GAPDH and RpL32). Values in the control (tub) were set to 1. * indicates Po0.05, two-tailed unpaired Student's t-test.

3.5. Brakeless interacts with the Mediator subunit Med19 The mechanisms by which Brakeless represses and activates transcription are not understood. Identification of proteins that

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detected in Brakeless knock-down embryos (Fig. 5D). This suggests that Brakeless auto-regulates its own expression. Consistent with this notion, Brakeless protein occupancy was observed at the brakeless locus (Fig. 5E). Brakeless target genes were then assayed by RT-qPCR. Both upand down-regulated genes respond to Med19 knock-down in a manner almost identical to Brakeless knock-down (compare Fig. 5F and G with Figs. 3A and 4A). Interestingly, in embryos where both Med19 and Brakeless were knocked down, there was no further up-regulation of target genes repressed by Brakeless, but instead a return to wild-type expression levels (Fig. 5F). By contrast, most Brakeless activated genes that were down-regulated by Med19 knock-down were reduced in their expression even further in the double knock-down (Fig. 5G). Although autoregulation and cross-regulation between Brakeless and Med19 complicates the interpretation of these results (Fig. 5C), they indicate that the interaction between Brakeless and Med19 may be more important for Brakeless' ability to activate transcription than for Brakeless-mediated repression (Fig. 6). Fig. 4. Brakeless can activate gene expression. Expression of four genes (sro, Socs36E, CG30428, hiw) showing a statistically significant down-regulation ( 41.5 fold, Po 0.05) in the bks expression array. (A and B) Quantitative RT-PCR of Brakeless target gene expression in 2–4 h embryos. (A) Down-regulation of direct target genes in Brakeless knock-down embryos. Columns are depicted as in Fig. 2. (B) Activation of target genes upon Brakeless or Brakeless-VP16 overexpression. Embryos were derived from females expressing BksB (tub 4BksB) or BksB-VP16 (tub 4BksB-VP16) in the germline under control of the α-tubulin Gal4-VP16 driver. Expression levels are depicted as in Fig. 2.

interact with Brakeless may provide insight into such mechanisms. We therefore performed a yeast-two hybrid screen with the most conserved part of Brakeless as a bait, the D2 domain, and also included the conserved zinc-finger that is present in BksB but absent from BksA (Fig. 5A). We screened a Drosophila embryo cDNA library, and identified 11 genes from 78.5 million tested interactions. One of the positives corresponds to the Mediator subunit Med19. Two independent Med19 cDNA clones were obtained, and the common part in these cDNAs, amino acids 66-202, is the selected interaction domain that binds to Brakeless (Fig. 5A). To confirm this interaction, we used the GST-pulldown assay. GST and GST-Bks (834-1151) were purified from E. coli and incubated with full-length Med19 cDNA in vitro translated in the presence of 35 S-Methionine. The GST-Bks fusion protein could interact with Med19, whereas the GST control did not (Fig. 5B). We next wanted to investigate if the Brakeless-Med19 interaction observed in vitro had relevance for Brakeless function in vivo. We generated a Med19 null mutant by imprecise excision of a P-element transposon, and recombined this allele to an FRT chromosome in order to allow elimination of the maternal Med19 contribution to early embryos. However, females with germline clones of the Med19 null allele failed to produce any eggs, indicating that Med19 is required for oogenesis. We therefore turned to the knockdown approach (Ni et al., 2011), using a short hairpin microRNA targeting Med19. Since α-Tubulin67C-Gal4-VP16 does not drive expression during early oogenesis in the germarium (Staller et al., 2013), we were able to knock-down maternal Med19 without blocking oogenesis. Embryos derived from w; α-Tubulin67C-Gal4-VP16/þ ; UAS-Med19 RNAi/ þ females (tub 4Med19 RNAi), from w; α-Tubulin67C-Gal4-VP16/þ; UAS-bks RNAi/UASMed19 RNAi females (tub 4bks RNAi, Med19 RNAi), or from w; αTubulin67C-Gal4-VP16/ þ control females were collected. As shown in Fig. 5C, Med19 expression is reduced in both Brakeless and Med19 knockdown embryos, as well as Brakeless Med19 double knockdown embryos. Surprisingly, higher bks RNA levels were observed in Brakeless knock-down embryos (Fig. 5C). Despite the increased bks RNA levels, decreased Brakeless protein levels were

4. Discussion The Brakeless protein has many important functions during Drosophila development (Funakoshi et al., 2001; Haecker et al., 2007; Rao et al., 2000; Senti et al., 2000; Yang et al., 2000), but how this protein controls gene expression is not understood. Previous work from our laboratory has shown that Brakeless can function as a transcriptional co-repressor (Haecker et al., 2007). We now demonstrate that Brakeless can both repress and activate transcription, and that an interaction with the Mediator subunit Med19 may be important for gene activation. We found that embryos devoid of maternal Brakeless misregulate expression of 240 genes. Although potential second-site mutations on the brakeless mutant chromosome may affect gene expression, several targets were confirmed by brakeless RNAi experiments, indicating that many of the genes are affected by lack of Brakeless. Surprisingly, the majority of mis-regulated genes were down-regulated in mutant embryos. A large fraction of these genes are maternally contributed, and may therefore be regulated by Brakeless during oogenesis. Since we wanted to identify targets for Brakeless in the embryo, we focused on genes with early zygotic transcription. Also among this class, the majority of Brakeless-regulated genes were down-regulated in mutant embryos (Crona et al., 2015). That some of these genes are direct Brakeless targets is supported by several observations. First, we could detect occupancy of the Brakeless protein at genomic regions in close proximity to the affected genes (Fig. 2). Secondly, over-expression of Brakeless caused a reciprocal effect on expression of these genes, demonstrating that Brakeless levels influence their expression (Fig. 4). Thirdly, Brakeless fused to the VP16 activation domain behaves as the unmodified Brakeless in that it up-regulates expression of these target genes (Fig. 4). These results argue against a model where Brakeless-mediated repression indirectly leads to gene activation, for example by controlling expression of non-coding RNAs (van Werven et al., 2012). Taken together, our data suggest that Brakeless can behave either as a repressor or as an activator depending on context. Transcriptional co-regulators are believed to mediate the repressive or activating functions of DNA-binding transcription factors and therefore serve as dedicated co-repressors or co-activators, but recent experiments demonstrate that co-regulators may switch transcriptional activity (reviewed in Mannervik (2014)). In the case of the CtBP co-repressor, the oligomeric state determines its function. The CtBP monomer is a co-activator whereas the dimer acts as a co-repressor (Bhambhani et al., 2011). How Brakeless

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Fig. 5. Brakeless interacts with Med19. (A) A yeast-two hybrid screen used the evolutionarily most conserved part of Brakeless (depicted as darker gray) and the zinc-finger unique to the Brakeless-B isoform (white) as a bait. Med19 was identified from a Drosophila embryo cDNA library. The schematic drawing shows the evolutionarily conserved part of Med19 in darker gray. Med19 amino acids 66-202 correspond to the selected interaction domain that binds to the Brakeless bait. (B) GST-pulldown assay where GST and GST-Bks (834-1151) were purified from E. coli and incubated with full-length in vitro translated 35S-labeled Med19. (C, F, G) Quantitative RT-PCR of gene expression levels in 2–4 h old Brakeless or Med19 single knockdown and in Brakeless Med19 double knock-down embryos. Columns depict average expression levels relative to a mean of two reference genes (β-tubulin and RpL32) in embryos derived from females expressing hairpins in the germline under control of an α-tubulin Gal4-VP16 driver. Embryos from females with only the Gal4 driver (tub) were used as controls. Values in the control (tub) were set to 1. S.D. (n¼ 3 and 4) is shown, and * indicates Po 0.05, two-tailed unpaired Student's t-test. (C) Expression of brakeless and Med19 in knockdown embryos. (D) Western blot after Brakeless immunoprecipitation in extracts from control (tub) or Brakeless knock-down embryos. The lanes are spliced together from the same membrane. The input material was loaded on a separate gel and probed with Tubulin antibody. Quantification of Brakeless bands in relation to Tubulin in the input demonstrated a decrease to 84% BksB and 56% BksA in bks RNAi embryos. (E) Brakeless ChIPqPCR (anti Bks) and no antibody control at the bks locus. Values were normalized against an intergenic locus devoid of known factors (IG2c). (F) Brakeless-repressed genes are up-regulated in Med19 knock-down embryos, but expression normalized in double knock-down embryos. (G) Brakeless-activated genes are down-regulated in Med19depleted embryos, and expression further reduced in double knock-down embryos, indicating that Brakeless and Med19 collaborate in activating these genes.

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Bks A

Med19

R1

R2

Med19 A

Bks

Med19

Bks A1

A2

Fig. 6. Schematic model of Brakeless function. (A) Brakeless (Bks) functions as a corepressor at the kni, Ctr1A and px genes, interacting with DNA-binding repressors (R1). These genes are also repressed by Med19, which may interact with different repressors (R2). Brakeless and Med19 together may in addition be needed for expression of activators (A) of these genes, so that in double knock-down embryos, transcription is restored to wild-type levels. (B and C) At other genes, such as sro, Socs36E, CG30428, and hiw, Brakeless functions as a co-activator by associating with DNA-binding activators (A). In some cases, a direct interaction with Med19 could be responsible for Brakeless-mediated activation, whereas Brakeless and Med19 may associate with different activators at some genes.

can function as a co-activator at some genes, but as a co-repressor on others will be an important future question. Although Brakeless is believed to associate with transcription factors as a way of recruitment to cis-regulatory DNA, the interactions that result in gene repression or activation are not known. We therefore performed a yeast two-hybrid screen with the most evolutionarily conserved part of Brakeless (Fig. S2). Med19, which is a subunit of the Mediator complex, was identified as one of the interacting proteins. The part of Med19 that interacts with Brakeless is also evolutionarily conserved (Fig. S3). The Mediator complex is present in all eukaryotes and pivotal for transcription by RNA polymerase II (reviewed in Kornberg (2005)). By interacting both with enhancer-bound transcription factors and with the C-terminal domain (CTD) in the largest subunit of RNA polymerase II, it functions as a co-regulator that recruits RNA polymerase II to the promoter (reviewed in Malik and Roeder (2010)). Mediator can also stimulate CTD phosphorylation and enhance transcriptional elongation (reviewed in Carlsten et al. (2013)). Interestingly, the Med19 subunit has been identified in multiple genetic screens in yeast (Brown et al., 1995; Dettmann et al., 2010; Friesen et al., 1998; Kremer et al., 2012; Rosenblum-Vos et al., 1991; Song et al., 1996; Tabtiang and Herskowitz, 1998), suggesting that this subunit has a crucial function in transmitting transcription factor activity to RNA polymerase II. Importantly, in cells lacking Med19, the Mediator complex can remain intact (Baidoobonso et al., 2007). In contrast to subunits whose absence leads to Mediator complex disassembly, Med19-depleted cells are therefore not expected to cause global gene expression changes, but rather cause specific transcriptional defects. Med19 is implicated both in gene repression and in gene activation in yeast (Brown et al., 1995; Dettmann et al., 2010; Friesen et al., 1998; Kremer et al., 2012; Rosenblum-Vos et al., 1991; Song et al., 1996; Tabtiang and Herskowitz, 1998). In Drosophila, Med19 is required for gene activation by Hox transcription factors, but not for Hox-mediated repression (Boube et al., 2014). We found that Brakeless-activated, but not Brakeless-repressed genes, are affected by Med19 and Brakeless double knock-down (Fig. 5). This

indicates that the interaction of Brakeless with Med19 may be important for Brakeless-mediated activation, but less so for Brakeless repressor function (Fig. 6). Consistent with this idea, the ability of Brakeless to repress a reporter gene in S2 cells when artificially tethered to DNA was not affected by Med19 knockdown (Fig. S4). Taken together, our data suggests that Brakeless activates transcription by interacting with Med19. Predicted proteins with sequence similarity to Brakeless can be found not only in insects, but also in other Ecdysozoa, in Deuterostomes, in Cnidaria, and even in sponges (Fig. S2). However, we have not found homologous sequences in unicellular eukaryotes, indicating that Brakeless may have participated in evolution of the complex gene regulatory networks required for multicellularity. In this regard, it is interesting that the Brakeless homolog ZNF609 is part of a protein network for pluripotency in mouse embryonic stem cells (van den Berg et al., 2010). Learning more about Brakeless function could provide novel insights into metazoan gene regulation.

Acknowledgments We thank Norbert Perrimon for sharing shRNA flies prior to public release, Bernard Moussian for providing flies with the unmutagenized FRT2RG13 chromosome, and Johan Ericson for a plasmid with the VP16 activation domain. This work was supported by grants from the Swedish Cancer Society.

Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ydbio.2015.08.005.

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