GIANT KILLER.The floral, homeotic protein AGAMOUS coordinates multiple downstream genes through direct transcriptional regulation of the nuclear matrix attachment region binding protein  Arabidopsis homeotic protein AGAMOUS (AG), a MADS domain transcription factor, specifies reproductive organ identity during flower development. Using a binding assay and expression analysis, we identified a direct target of AG, GIANT KILLER (GIK), which fine-tunes the expression of multiple genes downstream of AG. The GIK protein contains an AT-hook DNA binding motif that is widely found in chromosomal proteins and that binds to nuclear matrix attachment regions of DNA elements. Overexpression and loss of function of GIK cause wide-ranging defects in patterning and differentiation of reproductive organs. GIK directly regulates the expression of several key transcriptional regulators, including ETTIN/AUXIN RESPONSE FACTOR 3 (ETT/ARF3) that patterns the gynoecium, by binding to the matrix attachment regions of target promoters. Overexpression of GIK causes a swift and dynamic change in repressive histone modification in the ETT promoter. We propose that GIK acts as a molecular node downstream of the homeotic protein AG, regulating patterning and differentiation of reproductive organs through chromatin organization.The  Multicellular development depends on proper expression of thousands of genes. Master regulators, such as homeotic proteins, code for transcription factors in both plants and animals and are thought to act by regulating other genes. Recent genomic studies in the plant Arabidopsis have shown that over 1,000 genes are regulated by homeotic proteins that directly control various target genes, including different classes of transcriptional regulators. It is not known, however, how expression of so many genes is coordinated by a single homeotic gene to form functional organs and tissues. Here we identified a transcriptional target of the plant homeotic protein AGAMOUS using bioinformatics analysis and showed that AGAMOUS directly controls GIANT KILLER, a multifunctional chromatin modifier. GIANT KILLER then binds to the upstream regions of multiple genes involved in patterning and differentiation in the AGAMOUS pathway and fine-tunes the expression of these genes. These data therefore provide a possible mechanism by which a homeotic gene coordinates multiple downstream targets in plants. APETALA1 (AP1) and APETALA2 (AP2), B class for APETALA3 (AP3) and PISTILLATA (PI), and C class for AGAMOUS (AG), have been extensively studied and have been shown to encode transcription factors AG encodes a transcription factor of the MADS-domain protein family, and AG is necessary for the specification of stamens and carpels, the floral reproductive organs ag-1 mutants, flowers undergo homeotic conversion to show a sepal-petal-petal reiteration instead of the normal sepal-petal-stamen-carpel structure. The complete lack of reproductive organs in ag-1 flowers places AG at the top of the hierarchy of genes controlling reproductive development. This conclusion is supported by microarray expression profiling of wild-type and ag mutant flowers showing that more than 1,000 genes are regulated downstream of AG During flower development, many key processes depend on tissue-specific regulation of gene expression achieved by the coordinated interplay of transcription factors. The classical ABC model was proposed nearly two decades ago to account for organ identity control in flower development SPOROCYTELESS  ArabidopsisGenome-wide studies by microarray using plant lines with controllable floral homeotic activities and chromatin immunoprecipitation (ChIP) led to the identification of direct target genes of the homeotic proteins SPL and DAD1, genetic studies in Arabidopsis have revealed a large group of genes that are necessary for proper patterning and differentiation of reproductive organs. ETTIN  acts redundantly with AUXIN RESPONSE FACTOR 4 (ARF4) to participate in abaxial-adaxial axis patterning of the floral meristem and reproductive organs, as well as in the apical-basal patterning of the gynoecium AG in petal primordia and also controls gynoecium fusion YABBY family gene CRABS CLAW (CRC) is expressed preferentially in the abaxial side of carpels and is involved in specification of the gynoecium and nectaries JAGGED (JAG) and NUBBIN (NUB), both encoding C2H2 zinc-finger transcription factors, function redundantly to promote proliferation of stamen and carpel primordia Along with GIANT KILLER (GIK), a gene coding for an AT-hook type DNA binding protein, as a target of AG. GIK belongs to a protein family consisting of 29 members in ArabidopsisGIK have been isolated using yeast one-hybrid screening as promoter-binding proteins as well as from activation tagging screens We report here the identification of GIK acts as a target of the floral homeotic protein AG and fine-tunes the expression of multiple genes involved in organ patterning and differentiation during reproductive development. Therefore, these data reveal one of the mechanisms by which homeotic genes regulate multiple downstream targets in plants.We propose that At2G35270 . First, we searched the entire Arabidopsis genome for the 16-bp consensus CArG box binding sequences of AG  http://www.arabidopsis.org/cgi-bin/patmatch/nph-patmatch.pl). We then identified 110 genes located near the putative binding sites of AG  and tested their expression in wild-type and ag mutant flowers using RT-PCR  showed AG-dependent expression patterns  ATHB40 (At4g36740) ATHB40, see below for GIK). We found a typical CArG box sequence located 732 bp downstream of the translational termination codon of GIK , AG-GR enters the nucleus and induces AG activity. GIK expression was upregulated 6 h after 10 µM DEX treatment compared to mock-treated inflorescences  using 35S::AG-GR ag-1 inflorescences treated continuously with DEX. The primer pair hybridizing to the 3′ region of genomic GIK DNA containing a putative CArG box showed enrichment over a primer pair hybridizing to the coding region of GIK  but that expression later than stage 6 may depend on AG during reproductive development. In ag-1 inflorescences, although GIK is noticeably expressed in inflorescence meristems and floral primordia in early stages, GIK expression is considerably reduced in developing organs , suggesting that GIK might play a role in root development. To determine whether GIK is a nuclear protein, we performed immunofluorescence staining using whole-mount seedlings and confocal microscopy. Staining was specifically detected in the nucleus  were examined during flower development . Similar reproductive phenotypes were observed at low frequency (3% to 4% of their flowers) in transgenic plants with a genomic copy of GIK, which showed 5- to 50-fold higher expression levels of GIK than wild-type plants (unpublished data), indicating that the 35S::GIK and 35S::GIK-GR constructs provide high levels of GIK activity. These results show that overexpression of GIK strongly interferes with normal reproductive development.To understand the role of elopment . At leas tissues , short v tissues , and exc tissues . These peatments . More th35S::GIK was introduced into the ag-1 mutant plants. 35S::GIK did not rescue the ag-1 organ identity defects: no stamen- or carpel-like organs were observed in 35S::GIK ag-1 flowers, even though there was occasional sepal-sepal fusion  was identified. It contains an insertion in the middle of the coding region, 450 bp from the start codon. Homozygous plants were verified by PCR-based genotyping and GIK expression analysis , but a small number of flowers (22 of 800) showed various degrees of defects in stamen and carpel development (GIK coding region (GIK transcripts were significantly reduced in flowers of GIK RNAi plants (unpublished data). To examine whether GIK has redundant functions with other GIK-like genes At4g17800 (67% amino acid identity) and created the transgenic plants on the gik mutant background. However, we did not observe any obvious enhanced effects in the transgenic plants (unpublished data). The GIK loss-of-function defects, albeit not at a high frequency, suggest some level of participation by GIK in reproductive development as a component of a fine-tuning mechanism.To further understand the role of analysis , B. Mostelopment –E. Stameelopment . In someelopment , D. Noneg region . In 5 ofettin  mutants GIK and ETT. ETT encodes a member of an auxin response factor family of DNA binding proteins, and loss of ETT activity results in severe reproductive defects 35S::GIK-GR-6HA plants with the weak ett-3 mutant. Overexpression of GIK in the heterozygous and homozygous backgrounds of the weak ett-3 allele showed strong ett mutant phenotypes  and then purified the matrix fraction by DNaseI treatment and extensive washing with high-salt buffer, which removes basic proteins and histones GIK contains an AT-hook DNA binding motif, which binds to the MAR of DNA sequences s is low , we usedETT promoter, we identified MARs in the upstream genomic region of ETT using SMARTest Software   was fused with a GUS reporter gene and the inflorescences were stained  in a time-dependent manner  after GIK induction  . As a co stained . ExpressUS lines . This ret manner . At day e to GIK . To exclactivity . To quantime PCR , C. In aectively . In contnduction . These rETT is associated with any known epigenetic histone modifications, we performed a ChIP assay using antibodies against modified histones in wild-type and 35S::GIK backgrounds  and reproductive differentiation that is regulated by KNU and JAG are under partial control of AG, and that GIK acts as a molecular organizer to orchestrate expression of these key regulators for floral reproductive patterning and differentiation  GIK overexpression mimics the phenotypes of ett mutants, (2) GIK and ETT show complementary expression patterns during late reproductive development, (3) ETT expression is increased in gik mutants, (4) GIK binds to ETT putative MARs in vivo, and (5) the putative ETT MARs are important in GIK-regulated ETT expression. GIK-mediated repression of ETT occurred relatively rapidly after GIK induction in floral tissues, and the stable repression of ETT required continuous GIK activity. We also showed that ETT silencing was associated with repressive histone dimethylation of H3K9 in the ETT promoter, especially at the distal putative MAR. It remains unclear whether GIK is directly involved in this histone modification. Because GIK lacks known domains typically found in chromatin-modifying enzymes, GIK may introduce structural changes to the genomic region through MAR binding and may thereby facilitate the binding of chromatin-modifying enzymes to carry out histone modifications. It is also possible that GIK serves as a center for organizing chromatin remodeling complexes in the nuclear matrix to regulate target gene expression. Alternatively, MAR binding by GIK may inhibit the binding of the transcriptional machinery to the proximal promoter, leading to gene silencing associated with dimethylated H3K9. However, our time-course analysis showed that the dynamic changes in H3K9 dimethylation levels appeared to precede negative regulation of ETT transcription, which does not support the later hypothesis. Dimethylation of H3K9 increased rapidly during the 2 h after GIK induction. A further increase in dimethylation at the 4 h time point corresponded with the steepest downregulation of ETT transcription at the 8 h time point. Conversely, a dynamic reduction of H3K9 dimethylation to a level lower than that seen prior to induction at the 8 h time point was followed by a steady recovery of ETT transcription at the later time points of 16 h and 24 h. Nevertheless, how this dynamic methylation pattern is achieved remains unknown. The mammalian AT-hook protein SATB1 has been shown to mediate gene repression by directly recruiting histone deacetylases We showed that ETT has recently been shown to be regulated by trans-acting short interfering RNAs (siRNAs) ETT expression may be refined by two different molecules, GIK and siRNA, to establish strict spatiotemporal expression boundaries. These events may also partially explain the modest effects of the GIK loss-of-function mutant and of deletion of putative MAR regions in the ETT-promoter reporter construct in the wild-type context. However, it remains to be determined whether GIK and siRNA have separate or overlapping roles in the control of ETT in reproductive development.Hox genes during skeletal development and osteoblast differentiation HOX genes in animals and MADS genes in plants Arabidopsis. Thus it is possible that AT-hook motif proteins have been independently incorporated into multicellular developmental processes in animals and plants, but with similar functions of orchestration and fine-tuning of tissue-specific expression of multiple genes.In mammals, MAR-binding proteins have been implicated in the control of expression of multiple genes. SATB1 in mice contains an AT-hook DNA binding motif and acts as a “gene organizer” to regulate temporal and spatial expression of multiple genes during thymocyte maturation and breast tumor growth and metastasis erecta background and were grown at 22°C under continuous light. DEX treatment was done by submerging inflorescences in a solution containing 10 µM DEX together and 0.015% Silwet L-77 for ∼1 min. Transgenic plants were generated by Agrobacterium-mediated infiltration All plants used in this study are on the Landsberg 35S::GIK and 35S::GIK-GR-6HA constructs, GIK cDNA was cloned into a pMAT137 vector and a composite pGreen vector containing a rat GR hormone binding domain and a 6×HA tag, respectively pMAT137 construct) and BASTA (for the pGreen construct) for two generations to obtain homozygous lines. 35S::GIK-GR-6HA plants were treated with DEX five times at 1 d intervals for phenotypic observation. More than 90% of DEX-treated flowers showed reproductive defects. A GIK insertion line was obtained from the TRAPPER collection (http://genetrap.cshl.edu/TrHome.html) . The enhancer trap was inserted into the middle of the coding region, 450 bp downstream from the start codon. Homozygous lines were verified by PCR-based genotyping. In total, ∼1% of gik mutant flowers showed reproductive defects. For the rescuing experiment of the gik mutant, a genomic copy of GIK, containing 4,660 bp of the 5′ upstream region, 858 bp of the GIK coding region, and 1,767 bp of the 3′ region, was cloned into the pDONR221 vector (Invitrogen) and later into the pBGW binary vector using gateway cloning GIK, thus showing the ectopic expression phenotypes. To generate the 35S::GIK-RNAi construct, a C-terminal fragment of the GIK coding region  was amplified using UltraPfu-High-Fidelity DNA polymerase (Stratagene) to produce BamHI-GIK-Cter-ClaI and XhoI-GIK-Cter-KpnI fragments. These fragments were cloned into the pKANNIBAL vector pKANNIBAL-GIK-RNAi was cut by NotI to produce a 35S::GIK-RNAi fragment, which was then cloned into the pMLBART binary vector GIK-RNAi transgenic plants were selected using BASTA. A few percentages of the examined flowers showed reproductive defects in the T1 and T2 generations. In the T3 generation, the lower ratio of the GIK-RNAi flowers showed reproductive defects. To generate the 35S::GIK2-RNAi construct, an N-terminal fragment of the GIK2  coding region was amplified using UltraPfu-High-Fidelity DNA polymerase to produce BamHI-GIK2-Nter-ClaI and XhoI-GIK2-Nter-KpnI fragments. These fragments were cloned into the pKANNIBAL vector and later into the pMLBART binary vector as described in the cloning process for 35S::GIK-RNAi. A T1 35S::GIK2-RNAi plant was crossed to gik and the GIK2 RNAi gik plants were obtained and confirmed following BASTA selection and PCR genotyping.To generate the GIK cDNA and cDNAs of the conserved N-terminal and AT-hook domains were cloned into the pQE30 vector (QIAGEN) to produce 6×His-GIK proteins. Recombinant protein was induced using 1 mM IPTG and purified on a nickel column (QIAGEN) under denaturing conditions. Protein was then partially refolded through buffer exchange and concentrated using a Centriprep Centrifugal Filter with an Ultracel YM-10 membrane (Millipore). Purified 6×His-GIK recombinant protein was injected intramuscularly into guinea pigs with Freund's adjuvant. Blood was withdrawn after the fourth and sixth immunizations. Whole blood was processed to obtain polyclonal anti-GIK serum.Full-length Arabidopsis roots, flowers, and leaves was ground in liquid nitrogen and re-dissolved in 80 µL SDS sample loading buffer . The samples were boiled for 10 min, and 25 µL of each sample was loaded onto a 12% SDS polyacrylamide gel for electrophoresis. Proteins were transferred onto a PVDF nylon membrane (Bio-Rad) and blocked with skim milk. The membrane was then incubated overnight with polyclonal anti-GIK at 4°C, washed with 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 0.1% [v/v] Tween 20 and further incubated with secondary anti-guinea pig coupled to horseradish peroxidase. Signal was detected using SuperSignal West Dura extended duration substrate (Pierce). A replicate membrane was stained with Coomassie Blue to show protein loading.Approximate 0.035 g each of Arabidopsis seedlings were rinsed with 1× phosphate buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 1 h. Seedlings were washed three times with PBS and incubated with 4% Driselase (Sigma) at 37°C for 30 min. After washing, seedlings were further incubated with PBS containing 10% dimethyl sulfoxide and 3% [v/v] NP-40 for 1 h at room temperature. Seedlings were washed three times with PBS and blocked with 3% bovine serum albumin for 30 min. Seedlings were then incubated overnight with polyclonal anti-GIK or monoclonal anti-trimethylguanosine . Cy3-conjugated anti-guinea pig and FITC-conjugated anti-mouse were used as secondary antibodies. TOPRO-3 was used as a fluorescent DNA dye. Immunostaining was analyzed with a laser scanning confocal microscope (Zeiss Meta LSM510).pQE30 expression vector carried by E. coli M15 cells. ETT MAR probes were generated by cloning SMARTest-predicted MAR sequences ETT upstream genomic region into the pCRII vector (Invitrogen). Probe 1  and Probe 2  fragments were generated by EcoRI digestion and were end-labeled with a digoxigenin probe synthesis mix (Roche) using Klenow fragment (New England BioLabs). South-Western analysis was performed as described ETT putative MAR probes in DNA binding buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 20 ng/mL salmon sperm DNA at room temperature, washed, and incubated with anti-dioxigenin coupled with alkaline phosphatase (Roche). Signal was detected using CDP-Star (Roche) as a substrate. P1 and P2 probes showed similar binding efficiencies to GIK-AT. The binding result with the P2 probe is shown.Recombinant proteins GIK-AT (residues 74-173) and GIK-MUT  were produced in the t-test.Total RNA was isolated from floral bud clusters at stage 10 or younger sp/Isp)/(Bctrl/Ictrl)} of Absp]/[{(Bsp/Isp)/(Bctrl/Ictrl)} of control IgG], where Bsp = amount of bound DNA measured by a specific primer pair; Isp = amount of Input DNA by a specific primer pair; Bctrl = amount of bound DNA by control primer pair (ACT); Ictrl = amount of Input DNA by control primer pair; and Absp = anti-AG or anti-HA. The control value was set at 1.0.The ChIP assay was performed as described sp/Isp) of X time points]/[(Bsp/Isp) of 0 h].The relative enrichment for the histone modification experiments was the ratio obtained from: [ and a fraction was kept as a total nuclear control. The remaining sample was digested with 50 U of DNaseI (Roche) in RSB containing 0.25 M sucrose and 1 mM CaCl2 for 2 h at room temperature. After centrifugation, pellets were resuspended in RSB and an equal volume of high-salt buffer I  and incubated for 10 min at 0°C. After centrifugation, the pellets were further extracted twice with high-salt buffer II . After high-salt extractions, the matrices were washed with RSB buffer containing 0.25 M sucrose and 0.25 mg/mL BSA and resuspended in the same buffer. The resuspended matrices and total nuclear lysates were used for western analysis. Anti-HA and anti-AG were used to detect GIK-GR-6HA and AG proteins, respectively.Nuclei matrix was isolated as described pETT::GUS construct, 8.7 kb of ETT upstream genomic sequence was first amplified using UltraPfu-High-Fidelity DNA polymerase with an extension time of 8 min and then cloned into the pDONR221 (Invitrogen) to create the entry clone. Similarly, the 4.9 kb pETTΔMAR::GUS construct was amplified using UltraPfu-High-Fidelity DNA polymerase with an extension time of 5 min and then cloned into the pENTR directional TOPO cloning vector (Invitrogen). Both clones were sequenced for confirmation. Subsequently, both entry clones were cloned into the pBGWFS7 binary vector 35S::GIK-GR-6HA plants to obtain pETT::GUS 35S::GIK-GR-6HA and pETTΔMAR::GUS 35S::GIK-GR-6HA double transgenic plants. DEX treatment was performed as described above continuously at 2 d intervals. Whole inflorescences were rinsed and stained to determine GUS activity for GUS expression analysis To generate the ETT cDNA and a 3′ specific region of GIK cDNA were amplified with PCR and cloned into pSK (Stratagene) and pCRII vectors, respectively, and used as templates for in vitro transcription.Nonradioactive in situ hybridization was performed as described Arabidopsis Genome Initiative locus identifiers of Arabidopsis genes used in this article are as follows: AGAMOUS , GIANT KILLER , ETTIN , CRABS CLAW , JAGGED , KNUCKLES , LEUNIG , TUBULIN 2 , MU-LIKE TRANSPOSASE , PHOSPHOFRUCTOSE KINASE , and LIPASE (At1g10740).Figure S1Semi-quantitative RT-PCR of 110 genes. PCR products approximately 500 bp in length were amplified using primer sets designed for 110 genes located near putative AG binding sites. Primer sets were designed to span intron sequences when possible to distinguish RT-PCR products from the amplification of genomic DNA. If no amplification was detected, primers were redesigned. If after the second round of PCR no amplification was observed, the gene was considered to be a pseudogene. PCR conditions were determined using a dilution series of control DNA  from 40 ng to 10 pg of genomic DNA equivalent to 4×105 to 100 copies of targets per reaction. We set the conditions as follows: to one cycle of 96°C for 15 min, followed by 40 cycles of 94°C for 50 s, 60°C for 50 s and 72°C for 90 s, followed by 72°C for 10 min using Hot StartTaq DNA polymerase. In this condition, target sites in the range of 1×105 and 1×103 copies/reaction can be quantified, and therefore abundant genes were disregarded. AG and SUP RT-PCR products roughly correspond to 1×105 and 4×103 copies in our flower samples (unpublished data). After this screen, 24 genes showed reduced expression in ag mutant flowers, as marked by asterisks next to gene names. Left lane, 100 bp ladder . Lanes show amplification products using cDNA synthesized from RNA isolated from wild-type roots, wild-type leaves, and flowers from wild-type and ag-1 mutant plants, from left to right. Accession numbers are as follows, 1-ABP, AT1G21530; 1-ANK, AT1G04780; 1-C3H, AT1G24580; 1-CON, AT1G61740; 1-DSO, AT1G05100; 1-ENP, AT1G09060; 1-EPO, AT1G74300; 1-ERP, AT1G80690; 1-EXG, AT1G14455; 1-HLH, AT1G73830; 1-HMR, AT1G48620; 1-HYP, AT1G43690; 1-INV, AT1G56555; 1-LIP, AT1G10740; 1-PEX, AT1G14540; 1-RIG, AT1G80400; 1-SEC, AT1G56660; 1-SKK, AT1G60940; 1-SRP, AT1G47710; 1-TIN, AT1G22810; 1-TNY, AT1G74930; 1-TRA, AT1G64150; 2-AG5, AT2G42830; 2-ATH, AT2G35270; 2-BRA, AT2G19460; 2-BZP, AT2G36270; 2-CHA, AT2G02710; 2-CON, AT2G15590; 2-CTH, AT2G04240; 2-CYP, AT2G28850; 2-DOB, AT2G41940; 2-DSK, AT2G17530; 2-INI, AT2G31430; 2-LIP, AT2G15230; 2-PHD, AT2G31650; 2-RLK, AT2G02220; 2-SIG, AT2G18770; 2-SPI, AT2G39260; 2-TFL, AT2G27550; 2-TTV, AT2G31990; 2-TYK, AT2G39740; 2-WRY, AT2G37260; 2-ZIN, AT2G32930; 3-AP2, At3g54990; 3-CAK, AT3G51850; 3-EDF, AT3G58680; 3-HAT, AT3G01470; 3-HUN, AT3G21690; 3-KIN, AT3G61410; 3-KIS, AT3G44050; 3-MYB, AT3G29020; 3-PET, AT3G01350; 3-RAS, AT3G11730; 3-RBL, AT3G50330; 3-REX, AT3G06140; 3-RIN, AT3G19950; 3-SIG, AT3G53920; 3-SUN, AT3G13180; 4-AG19, AT4G22950; 4-AG21, AT4G37940; 4-AIG, AT4G09950; 4-CEL, AT4G17615; 4-CHP, AT4G02180; 4-CLC, AT4G12550; 4-GL2, AT4G17710; 4-GLU, AT4G02290; 4-GLY, AT4G02480; 4-HOX, AT4G36740; 4-MYA, AT4G12350; 4-PEC, AT4G13210; 4-PIT, AT4G09160; 4-PRG, AT4G14965; 4-PRO, AT4G10510; 4-RHF, AT4G14220; 4-RIN, AT4G09100; 4-SAB, AT4G07320; 4-SAL, AT4G39070; 4-SEN, AT4G30430; 4-SKK, AT4G11460; 4-STK, AT4G25160; 4-TOP, AT4G22360; 4-TSP, AT4G27910; 4-TUB, AT4G14960; 4-UBQ, AT4G10570; 4-UBS, AT4G10590; 5-CDC, AT5G39420; 5-CHH, AT5G57520; 5-CHR, AT5G42920; 5-CLV, AT5G62230; 5-CO, AT5G41380; 5-CYT, AT5G57570; 5-DAG, AT5G44780; 5-DIS, AT5G45500; 5-DRO, AT5G47900; 5-GAL, AT5G26920; 5-GAS, AT5G15230; 5-HAP, AT5G67180; 5-HYP, AT5G40860; 5-KIN, AT5G25440; 5-MCR, AT5G55670; 5-MYB, AT5G49330; 5-NAL, AT5G39610; 5-NAM, AT5G39540; 5-PEC, AT5G66920; 5-REK, AT5G12000; 5-RLK, AT5G35390; 5-RLL, AT5G03140; 5-SET, AT5G43990; 5-SHG, AT5G14640; 5-WRK, AT5G22570.(1.60 MB TIF)Click here for additional data file.Figure S2Control ChIP assay using mock-treated ag-1 35S::AG-GR inflorescences. Chromatin immunoprecipitation (ChIP) was performed using ag-1 35S::AG-GR inflorescences at day 0 before DEX treatments. P1, P2, and P3 indicate primer pairs used for detecting different regions of GIK genomic DNA. Relative enrichment was obtained from the ratio of enrichment achieved by AG antibody to that of control IgG. Enrichment of a sequence amplified from PFK genomic DNA was used as a basal control and was set to 1.0. Standard deviation was obtained from PCR triplicates. No significant statistical differences among the relative enrichment ratios were found.(0.78 MB TIF)Click here for additional data file.Figure S3Western blotting and immunostaining using the GIK antibody. (A) Western blotting using whole protein extracts from Arabidopsis leaves, roots, and flowers. Bottom panel shows Coomassie Blue staining as a protein loading control. Several larger bands were observed in roots, which may be modified GIK proteins or GIK homologs. The band in leaves was barely detectable, indicating that GIK may be regulated at the protein level. (B) Immunostaining of wild-type Arabidopsis root cells with anti-GIK at low magnification. Bar, 5 µm.(1.63 MB TIF)Click here for additional data file.Figure S4Verification of the gik mutant and flower phenotypes. (A) Isolation of homozygous gik plants. Homozygous plants were confirmed with PCR genotyping using primer sets P1 and P2. Plant #1 is homozygous as shown by amplification with P1 but not P2, whereas plant #2 is heterozygous as shown by amplification with both P1 and P2. All ET14389 plants were grown on kanamycin MS-agar plates to select for the presence of the transposon before genotyping. A schematic diagram of the GIK coding region with the positions of the transposon insertion and the respective regions amplified by P1 and P2 are shown. (B) Expression analysis of GIK in the gik mutant using real-time PCR performed as described in gik mutant flowers showing bipartite anthers (* in C), a petalloid anther (D), and unfused carpels (F).(5.21 MB TIF)Click here for additional data file.Figure S5Overexpression of GIK enhances heterozygous and homozygous backgrounds of the weak ett-3 mutant. (A) The gynoecium of an ett-3/ett-3 mutant flower. (B) The gynoecium of an ett-3/+35S:GIK-GR-6HA flower after continuous DEX treatment. (C) The gynoecium of an ett-3/ett-3 35S:GIK-GR-6HA flower after continuous DEX treatment. Scale bars, 1 mm.(1.27 MB TIF)Click here for additional data file.Figure S6GIK expression is not upregulated in ett mutant flowers. Expression analysis of GIK in the ett-1 mutant using real-time PCR with RNA extracted from the inflorescences of wild-type and ett-1 mutant plants. Expression was normalized to the TUB expression. Relative expression level in the wild-type was set to 1.0.(1.30 MB TIF)Click here for additional data file.Figure S7AG positively regulates the expression of ETT. Time-course of ETT transcript expression after AG activation, as measured by real-time PCR. Inflorescences from ag-1 35S::AG-GR plants were treated with DEX four times at 1 d intervals and harvested at 0, 1, 2, 3, 4, 5, and 6 d after the first DEX treatment. ETT expression was normalized to the TUB RNA level. Relative expression at day 0 (0D) was set as 1.0.(2.99 MB TIF)Click here for additional data file.Figure S8Endogenous GIK binds to the putative MARs of the ETT promoter in wild-type plants. Wild-type inflorescences were harvested for ChIP experiments. Anti-GIK was used for immunoprecipitation. P1, P2, P3, P4, and P5 are primer pairs used to detect different regions of the ETT genomic DNA (as illustrated above). For details, please see the legend of (1.88 MB TIF)Click here for additional data file.Figure S9Effect of a deletion of distal MARs after GIK induction and real-time PCR analysis of GUS reporter gene expression. (A) A plant transgenic for promoter constructs of pETTΔMAR::GUS with a deletion of distal MARs  and pETTΔMAR::GUS 35S::GIK-GR (C) transgenic plants shown in GUS reporter gene were used for quantitative analysis. Each expression level at day 0 was set to 1.0. Paired student's t-test was used to analyze the differences between D0 and D3 (**p<0.01) and between D0 and D4 (**p<0.01) in (B).(2.24 MB TIF)Click here for additional data file.Figure S10Analysis of histone modifications at ETT genomic loci. Wild-type and 35S::GIK inflorescences were used for the ChIP assay with antibodies for dimethylated H3K9, trimethylated H3K27, and trimethylated H3K4. Primer pairs P2, P3, and coding are shown at the top. Relative enrichment was obtained from the ratio of bound/input achieved at the respective time points to that wild-type.(1.29 MB TIF)Click here for additional data file.Figure S11Real-time PCR analysis of 35S::GIK-GR inflorescences after mock, cycloheximide (CYC), DEX, and DEX+CYC treatments. Samples were harvested 2 h after the treatment and used for cDNA synthesis for the expression analysis of ETT (A), CRC (B), JAG (C), and KNU (D). Standard deviation was obtained from PCR triplicates.(1.41 MB TIF)Click here for additional data file.Figure S12Overexpression of GIK affects root development. Seedlings of Ler wild-type and 35S::GIK plants at day 5 post-germination. Seeds of Ler wild-type and 35S::GIK transgenic plants were planted on MS agar plates before observation of the phenotype.(2.87 MB TIF)Click here for additional data file.Table S1Genes located near the putative binding sites for AG with reduced or no expression in ag mutant flowers. Listed are the isolation name, accession number, position of CArG box sequences related to a gene-coding region (number of nucleotides from the initiation codon for the 5′ upstream region or from the stop codon for the 3′ downstream region), and gene description shown on the TAIR Web site (www.arabidopsis.org).(0.03 MB DOC)Click here for additional data file.Table S2Sequences of oligonucleotide DNA used in this study. All are shown in the 5′ to 3′ direction.(0.03 MB DOC)Click here for additional data file.Table S3Number of T1 transgenic plants for ETT promoter-GUS categorized by staining strength in inflorescences. Two lines for each construct show the results of two independent transformations.(0.06 MB PDF)Click here for additional data file.Table S4List of genes tested in the time-course analysis following GIK activation.(0.02 MB DOC)Click here for additional data file.


The resulting experimental signatures of responses to lactic acidosis and hypoxia are evaluated in a heterogeneous set of breast cancer datasets. A strong lactic acidosis response signature identifies a subgroup of low-risk breast cancer patients having distinct metabolic profiles suggestive of a preference for aerobic respiration. The association of lactic acidosis response with good survival outcomes may relate to the role of lactic acidosis in directing energy generation toward aerobic respiration and utilization of other energy sources via inhibition of glycolysis. This “inhibition of glycolysis” phenotype in tumors is likely caused by the repression of glycolysis gene expression and Akt inhibition. Our study presents a genomic evaluation of the prognostic information of a lactic acidosis response independent of the hypoxic response. Our results identify causal roles of lactic acidosis in metabolic reprogramming, and the direct functional consequence of lactic acidosis pathway activity on cellular responses and tumor development. The study also demonstrates the utility of genomic analysis that maps expression-based findings from in vitro experiments to human samples to assess links to in vivo clinical phenotypes.The tumor microenvironment has a significant impact on tumor development. Two important determinants in this environment are hypoxia and lactic acidosis. Although lactic acidosis has long been recognized as an important factor in cancer, relatively little is known about how cells respond to lactic acidosis and how that response relates to cancer phenotypes. We develop genome-scale gene expression studies to dissect transcriptional responses of primary human mammary epithelial cells to lactic acidosis and hypoxia  It is well recognized that tumor microenvironments play an important role in modulating tumor progression in human cancers. Although previous studies have highlighted the importance of hypoxia, there is limited knowledge on the effects of other components in tumor microenvironments. Therefore, we use gene expression to compare and analyze how cells respond to lactate, acidity, and hypoxia, as well as how these responses can be utilized to predict the clinical outcomes of patients with breast cancers. We uncover an unexpected association with better clinical outcome of the strong lactic acidosis and acidosis response in breast cancers as a result of their abilities to inhibit glycolysis and favor oxidative phosphorylation for energy generation. This effect is caused by not only the repression of the gene expression of glycolysis genes but also the inhibition of Akt activation of cells exposed to lactic acidosis and acidosis. In conclusion, we propose that lactic acidosis and acidosis to be considered as independent prognostic factors for human cancers. The tumor microenvironment is characterized by oxygen depletion (hypoxia), high lactate and extracellular acidosis (lactic acidosis) as well as glucose and energy deprivation The accumulation of lactic acid in solid tumors is often thought to be caused by tumor hypoxia – a by-product of glycolysis as the tumor cells shift to an anaerobic mode of energy production under hypoxia or due to the altered metabolic profiles of cancer cells. In spite of this apparent mechanistic link, these two factors exhibit significant disparities in their spatial and temporal distribution in tumors in vitro conditions of mammalian cell growth to re-create the relevant stresses experienced in vivo. This offers a means to explore the roles of individual environmental factors on cellular behavior and phenotype. Genetic and pharmacological manipulation can then be applied to explore the molecular mechanisms and genetic circuitry underlying cellular responses. Such studies typically observe in vitro cellular behavior and make inferences about contributions to tumor progression in vivo. For example, hypoxia, when applied in vitro, has been shown to promote angiogenesis, cellular migration and energy consumption, thus providing the potential mechanisms for its association with poor clinical outcome in vitro perturbations to in vivo cancer phenotypes is frequently challenging and indirect. Gene expression microarray signatures have provided a solution to this gap since they afford an opportunity to develop a surrogate phenotype of the in vitro state which can then be assessed in the in vivo state. This approach generates gene expression signatures from perturbations in cultured cells in vitro to represent a defined biological process in vivo. Using this approach, we have previously shown that the wound healing, hypoxia responses and various oncogenic mutations can play important roles in tumor progression in vitro culture cells and in vivo human cancers.Research on tumor microenvironments relies on our ability to manipulate 2) with neutral pH; 2) lactic acidosis – 25 mM lactic acidosis with pH 6.7; 3) hypoxia (2% O2) with neutral pH; 4) combined lactic acidosis and hypoxia. We did not find a significant change in media pH at the end of the 24 hour culture. The gene expression of these HMEC samples were interrogated with Affymetrix GeneChip U133 plus 2.0 arrays to measure the expression of more than 54,000 probe sets and at least 47,000 transcripts and variants. Gene expression profiles of cellular responses to hypoxia, lactic acidosis and combined stresses were first normalized by RMA, mean centered and filtered with the criteria of at least 2  observations with at least 1.75 fold changes to select 4722 probes sets. A clustering analysis on these genes revealed that hypoxia and lactic acidosis induced distinct sets of genes  brought to replicative arrest by growth factor/serum withdrawal for 24 hours. Since the HMEC represent normal epithelial cells with intact signaling components, the response elicited in HMECs is likely to reflect the cellular response not biased by genetic mutations present in cancer cell lines. We exposed HMECs to four different culture environments for 24 hours in triplicate samples: 1) control – ambient oxygen level  that has been utilized in a number of prior studies To survey the molecular pathways triggered by lactic acidosis and hypoxia, we analyzed the Gene Ontology (GO) enrichment in the genes induced and repressed by hypoxia and lactic acidosis using GATHER Previous studies have shown that lowering the extracellular pH from 7.4 to ∼6.7 will lead to a slight lowering of intracellular pH (pHi) from 7.4 to 6.9–7.0, which is likely to be mediated by monocarboxylate transporter (MCT) proteins To further assess the extent to which the hypoxia and lactic acidosis response overlaps, we used binary logistic regression to estimate the probability of activation for the overall hypoxia and lactic acidosis pathways revealed in gene expression under individual conditions. We first used the control vs. hypoxia (for hypoxia probability) and control vs. lactic acidosis (for lactic acidosis probability) groups to provide the training sets to generate respectively hypoxia  and lactHigh lactate (lactosis) and low pH (acidosis) often co-exist in tumor lactic acidosis, but these two factors are not necessarily present simultaneously. To determine the respective contributions of lactosis and acidosis in the lactic acidosis response, we created culture conditions to separate the lactic acidosis condition (pH 6.7 created by 25 mM lactic acid) into lactosis  and acidosis (pH 6.7 created by HCl) conditions. We analyzed the transcriptional responses of HMECs under lactosis and acidosis via microarrays. Gene expression profiles of the cellular responses to lactosis and acidosis were normalized by RMA, mean centered and filtered with the criteria of at least 4  observations with at least 1.75 fold change. 213 probes were identified and clustered in in vivo and predicts poor clinical outcome in vitro gene signature of hypoxia “response” or “pathway activity” into numerical scores on each tumor sample in the in vivo expression data to assess the corresponding predicted levels of hypoxia pathway activity in each tumor. Similar approaches have been used in other studies using in vitro generated signatures to infer in vivo responses or pathway activities in tumors We previously showed that the hypoxia response elicited in cultured epithelial cells provides a molecular gauge of hypoxia response for cancerous human tissues −11)  .Among the breast cancer patients included in the Sotiriou dataset, 64 patients were treated with tamoxifen while the remaining 125 patients were untreated. Both hypoxia and lactic acidosis pathway signatures are predictive of poor and favorable outcomes respectively in the patients treated with tamoxifen , but lesTo understand whether the prognostic value of lactic acidosis is evident in a cell autonomous manner, we tested the prognostic value of the lactic acidosis signature in various breast cancer cell lines grown in a controlled culture milieu. We used binary logistic regression to determine the probability of acidosis response in each of a set of breast cancer cell lines that vary in their potential for distant metastasis We also tested the prognostic value of gene signatures reflecting lactic acidosis response and hypoxia response in a multivariate survival analysis using the Sotiriou data set. When both gene signatures are included in the Cox survival model on all samples for which we have survival, ER status, tumor size, and data on node involvement, the p-values for lactic acidosis and hypoxia are 0.0379 and 0.0069 respectively. When we include these two pathway variables along with clinical variables indicating ER status, tumor size >2 cm and node involvement, the p-values are .07, .008, .85, .0032 and .77 respectively. Dropping ER status and node involvement (poor predictors for this dataset) gives p-values of .06, .008, and .002.We further evaluated parametric Weibull survival models involving different combinations of clinical variables and expression signatures. p values from 2.2E-7 to1.4E-15  There are two major pathways for ATP-generation in mammalian cells – glycolysis or aerobic respiration. One of the fundamental properties of cancer cells is their preferential utilization of glycolysis over aerobic respiration to produce ATP. The glycolytic phenotype of cancer cells is thought to offer selective advantages since the disruption of glycolysis phenotype (e.g. silencing of LDH-A) results in stimulation of mitochondrial respiration and significantly compromises their tumorigenicity and the proliferation under hypoxia in vitro, which may explain why lactic acidosis response can identify tumors with higher level of aerobic respiration.To investigate the possibility that lactic acidosis directly modulates the balance of energy production, we measured its influence on ATP production in cultured cells when aerobic respiration is inhibited by rotenone. In control conditions without hypoxia or lactic acidosis, we found that approximately 35 %of ATP production in Siha cells is sensitive to rotenone at 48 hours . Under hThese data reveal the distinct manner by which lactic acidosis and hypoxia redirect energy utilization – hypoxia favors the glycolytic pathways while lactic acidosis favors the aerobic respiration. This suggests these two stresses may impact cellular metabolism in distinct manner and have synergic effects on ATP inhibition when cells are exposed to simultaneous hypoxia and lactic acidosis. We measured ATP production under control, lactic acidosis, hypoxia and combined hypoxia and lactic acidosis. We found that lactic acidosis and hypoxia reduced the ATP production to ∼50% and 63% respectively in 48 and 96 hours . This reTo further understand how lactic acidosis and hypoxia modulate the balance between the aerobic respiration and glycolysis, we mapped their respective effects on gene expression onto the framework of metabolic pathways in energy metabolism . From thIn contrast, expression levels of these glycolytic genes were consistently repressed by lactic acidosis . For exain vivo relevance of these changes in energy production by hypoxia and lactic acidosis was tested by examining survival predictions based on signatures of these two small sets of genes representing glycolysis and the TCA cycle. When the hypoxia-induced changes of glycolysis are projected into the same breast cancer expression studies, we found that high glycolysis pathway activities have prognostic significance similar to the significance of the whole hypoxia gene signatures . This hypoxia-induced increase was also dramatically reduced when cells were placed under lactic acidosis and acidosis conditions  but not lactosis. Similar effects in decreasing glucose consumption and lactate production were also seen for SiHa  One of the principal metabolic properties of cancer cells is their preferential use of glycolysis for energy generation even in the presence of oxygen (aerobic glycolysis). Warburg has speculated that this is caused by defective or functionally impaired mitochondria. Recent studies suggest that mutations affecting mitochondrial DNA or enzymes of the TCA cycle might contribute to tumor formation, tumor progression and the Warburg effect. Two enzymes in the TCA cycle enzymes, SDH and FH, are found to be tumor suppressor genes in vivo and in vitro observations indicate that lactic acidosis induces extensive and coordinated changes in the mode of metabolism and energy utilization, which in turn may account for their effects on the clinical phenotypes of breast cancer.In addition to generation of ATP, glycolysis is responsible for the generation of acetyl-CoA, which feeds into the TCA cycle for aerobic respiration. Thus, the reduction in glycolysis under lactic acidosis may imply a reduction in the amount of acetyl-CoA derived from the glycolytic process. To maintain cellular energy, cells may increase the use of other energy sources, such as the β-oxidation of fatty acids as source of acetyl-CoA. Since Akt pathway activity is known to suppress β-oxidation and thus energy generation from fatty acids bona fide lactate and/or acidity in tumors or 3) inappropriate activation of the lactic acidosis response resulting from genetic and/or epigenetic alterations in cancers. Given these possibilities, it is important to note that several previous studies have suggested that high level of tumor lactate is associated with poor clinical outcomes in several solid tumors bona fide hypoxia These data suggest that the lactic acidosis-induced gene expression program may have a direct causal role in impacting tumor biology to affect clinical outcomes. It will be important to understand what specific lactic acidosis-driven biological processes underlie the phenotypic differences between groups of tumors separated by lactic acidosis pathway activity. The mechanisms underlying the variation in lactic acidosis responses in breast are still unknown. There are three reasonable possibilities; variations in the lactic acidosis-response program could reflect: 1) actual variations in lactate and/or acidity in the tumors 2) cell-type-specific variations in the magnitude of, or threshold for, the response to It is widely believed that the intimate relationship between tumor progression, tumor microenvironment and the response of the tumor to that environment, when combined with other genetic and clinical factors, offers promise for improving our understanding of the heterogeneity of the disease. Progress in this direction will require a substantial advance in our – currently limited – ability to dissect the roles played by multiple characteristics of the tumor microenvironment. We aim to develop this approach to further dissect other various microenvironmental factors in cancers, such as glucose starvation, reoxygenation and ATP depletion Human mammalian epithelial cells (HMEC) were cultured in MEGM (Cambrex) and growth factors were withdrawn for 24 hrs before being placed under different environmental stresses. DU145 cells were cultured in RPMI1640 with 10% FBS, 1% sodium pyruvate, 1% L-glutamine, 1% Hepes and 1% antibiotics . WiDr and SiHa cells were cultured in DMEM with 10%FBS. Lactic acidosis conditions were created with the addition of 25 mM lactic acid (Sigma) to pH 6.7. Hypoxia was created by lowering the oxygen level to 2%. Similarly, the lactosis condition was created with the addition of 25 mM sodium lactate, while acidosis conditions were created by titrating media to pH of 6.7 with HCl.RNAs were extracted by miRVana kits (Ambion) and hybridized to Affymetrix Hu133 plus 2 genechips with standard protocol. All microarray data are available on GEO (GEO accession number GSE9649). Hierarchical clustering with weighted average linkage clustering was performed after indicated data filtering based on spot quality and variations in signal intensity as described CAGGGGTGTAAAGGACCAGA, Reverse:CGCCAGTAGAGAAAAGTGCC), CD55, PLAU, SOD2, Actin-beta, B2M respectively following the manufacturer's protocol (Applied Biosystem).RNAs were reverse-transcribed to cDNAs with SuperScript II reverse transcription kit following the manufacturer's protocol (Invitrogen). cDNAs were then used as the substrate for gene expression level measurements by qPCR with Power SYBRGreen PCR Mix (Applied Biosystems) and primers specific for ErbB3(Forward:DU145 cells were serum-starved (0.2%FBS) for 24 hrs, followed by 24 hr of continuous incubation in serum starved (0.2%FBS) media (as the control) and media with 25 mM lactic acid. 20%, 10%, and 5% FBS were applied for 30 mins to induce Akt activation. Proteins were extracted with PARIS kit (Ambion) and equal amount of protein samples were loaded to SDS-PAGE gels and blotted with pSer473 Akt antibody  and other indicated antibodies.2). After 48hr incubation, media were collected for glucose  and lactate (ARKRAY) measurements and normalized against cell number to obtain the glucose consumption/lactate production per million cells.WiDr and SiHa cells were plated in six-well dishes . The next day fresh media of respective conditions, including control, 25 mM lactic acidosis, 25 mM sodium lactate, acidosis of pH 6.7, pH 6.5 and pH 6.0 were applied to cells with the continuous incubation of 48 hrs under either normoxia or hypoxia , would be applied. They will then be incubated under normoxia and hypoxia (1% oxygen) respectively. ATP was measured by ATPlite 1 step luminescence ATP detection assay system kit with the protocol provided by the manufacturer after 48 and 72 hours (Perkin Elmer). To prevent the interference caused by different colors of control versus lactic acidosis media, we replaced culture media with PBS right before the addition of substrate solution.SiHa cells were plated at the density of 2×10Figure S1The pathway activities of hypoxia (A) and lactic acidosis (B) signatures were estimated as probability (Y-axis) by binary regression models in the HMECs exposed to indicated treatments .(1.02 MB EPS)Click here for additional data file.Figure S2The change in gene expression in the indicated conditions for a set of genes whose expression is induced (A) or repressed (B) only in the presence of both hypoxia and lactic acidosis.(3.02 MB EPS)Click here for additional data file.Figure S3The confirmation of the induction of ERBB3 and SOD2 expression by acidosis using real time RT-PCR.(1.09 MB EPS)Click here for additional data file.Figure S4The prognostic values of gene signatures reflecting hypoxia  and lactic acidosis  response among the patients treated  and untreated  with tamoxifen in the Sotiriou datasets. The graphs show Kaplan-Meier curves for two patient subsets simply split on the median signature level in each case, for visual presentation. The p-values are for regression coefficients of the signature in the survival model analysis.(0.93 MB EPS)Click here for additional data file.Figure S5Prognostic values of predictive model based on different combination of gene signatures and clinical variables. The breast cancers are separated in (A) based on hypoxia, lactic acidosis response and nodal status; in (B) based on hypoxia, lactic acidosis response, tumor size, ER status and nodal status; in (C) tumor size, ER status and nodal status. The graphs show Kaplan-Meier curves for two patient subsets simply split on the median signature level in each case, for visual presentation. The p-values are for regression coefficients of the signature in the survival model analysis.(1.31 MB EPS)Click here for additional data file.Figure S6Genes in the TCA cycle gene set are highly enriched in the tumors with high lactic acidosis using GSEA.(0.02 MB PNG)Click here for additional data file.Figure S7Breast samples in the Pawitan data set arranged from left to right by descending hypoxia score (top row). The expression of genes in TCA cycles in these samples is shown in heat map, orange color being higher expression while blue color being lower expression, together with hypoxia score.(0.57 MB EPS)Click here for additional data file.Figure S8Higher hypoxia response of TCA cycle genes was associated with better prognosis, in contrast to the association with poor prognosis for hypoxia genelists (A). On the other hand, TCA cycle genes of lactic acidosis response were not associated with differences in clinical outcome (B).(1.87 MB EPS)Click here for additional data file.Table S1Estimated posterior probability and corresponding Bayesian p-value (1-probability) for gene probe sets showing responses to hypoxia, lactic acidosis and acidosis.(10.86 MB XLS)Click here for additional data file.Table S2GO terms enrichments  for genes which are upregulated and downregulated in HMECs under lactic acidosis.(0.06 MB XLS)Click here for additional data file.Table S3GO terms enrichments  for genes which are upregulated and downregulated in HMECs under hypoxia.(0.03 MB XLS)Click here for additional data file.Table S4The comparison between GO term enrichment under hypoxia and lactic acidosis.(0.02 MB XLS)Click here for additional data file.Table S5The expression mean-centered values of indicated MCT family proteins under hypoxia and lactic acidosis.(0.02 MB XLS)Click here for additional data file.Table S6The lists of genes which are strongly induced or repressed (top 1% probability) under lactic acidosis and hypoxia.(0.18 MB XLS)Click here for additional data file.Table S7Gene sets enriched in the four expression datasets of breast cancers with high vs. low lactic acidosis pathways from the GSEA analysis.(0.05 MB XLS)Click here for additional data file.Table S8The GO terms enriched for genes used for the analysis of glycolysis pathways.(0.02 MB TXT)Click here for additional data file.Table S9p-values for the changes in gene expression under lactic acidosis and hypoxia for the listed glycolysis genes.(0.02 MB XLS)Click here for additional data file.Table S10Connectivity map analysis of the lactic acidosis gene signatures.(0.09 MB XLS)Click here for additional data file.Table S11Relationship between lactic acidosis signature and Akt pathway activities in different tumor expression datasets.(0.04 MB XLS)Click here for additional data file.Text S1Statistical supplement. Details of Bayesian factor regression models and survival analyses.(0.14 MB PDF)Click here for additional data file.


Streptococcus pyogenes strongly suggests an origin in another species, acquired via replacement of the counterpart gene (ortholog) following a recombination event. An example of orthologous gene replacement is provided by the nra/rofA locus, which encodes a key regulator of pilus gene transcription. Of biological importance is the previous finding that the presence of the nra- and rofA-lineage alleles, which are ∼35% divergent, correlates strongly with genetic markers for streptococcal infection at different tissue sites in the human host .The evolutionary history of several genes of the bacterial pathogen nra/rofA locus is experimentally addressed. Replacement of the native nra-lineage allele with a rofA-lineage allele, plus their respective upstream regions, preserved the polarity of Nra effects on pilus gene transcription  in the skin strain Alab49. Increased pilus gene transcription in the rofA chimera correlated with a higher rate of bacterial growth at the skin. The transcriptional regulator MsmR, which represses nra and pilus gene transcription in the Alab49 parent strain, has a slight activating effect on pilus gene expression in the rofA chimera construct.In this report, the impact of orthologous gene replacement targeting the S. pyogenes.Data show that exchange of orthologous forms of a regulatory gene is stable and robust, and pathogenicity is preserved. Yet, new phenotypes may also be introduced by altering the circuitry within a complex transcriptional regulatory network. It is proposed that orthologous gene replacement via interspecies exchange is an important mechanism in the evolution of highly recombining bacteria such as  Streptococcus pyogenes  has a past history of extensive genetic recombination The human pathogen S. pyogenes have been documented for loci encoding transcription regulatory proteins  and the extracellular virulence factor streptokinase (ska) S. dygalactiae subsp. equisimilis – is a likely source for divergent nra/rofA and ska genes. The GAS alleles found at each of these loci comprise two  or three (ska1/ska2a/ska2b) discrete lineages. Furthermore, the mga, nra/rofA and ska loci occupy distant map positions on the GAS genome and are not physically linked. That orthologous gene replacement at these loci has biological significance is supported by the distribution of the lineage-specific alleles within the GAS population, whereby the mga, nra/rof and ska lineages each display strong linkage with a genetic marker for tissue site preference for infection at the throat or skin Orthologous gene replacements in emm region (denoted emm pattern), and are used to define throat specialist (emm pattern A–C), skin specialist (emm pattern D) and generalist (emm pattern E) strains, with each group having a predilection for causing infection at their respective tissue sites emm pattern group and streptococcal disease at superficial tissue sites – pharyngitis and impetigo – finds strong support in numerous population-based surveillance studies  was chromosomally replaced with the rofA gene from the classic throat strain D471 , using the Δnra mutant as a recipient . The T-serotype is based, at least in part, on the pilus structure As reported previously henotype , S1. AddaphA3 in the Δnra mutant, with rofA plus the modified rofA-cpa intergenic region (plus aad9), results in the restoration of pilus gene transcription. Furthermore, the Alab49 rofA::aad9 construct exhibits a substantial increase both in transcription of the cpa through fctB series of genes and pilus-like protein biosynthesis, relative to the Nra-dependent levels observed for the wt Alab49 strain. Importantly, RofA provides a functional substitute for Nra activation of pilus gene expression in this particular S. pyogenes strain background.Taken together, the data demonstrate that replacement of the kanamycin-resistance gene The humanized mouse model for GAS impetigo provides a highly sensitive and specific measure for an infectious process that closely mimics the pathological changes observed in natural human disease rofA::aad9 construct was tested for virulence in the humanized mouse model for skin infection  displayed a net increase in bacterial growth at the skin, indicative of “virulence” (rofA::aad9 (lower panel), were not statistically significant. In sharp contrast, inactivation of nra in Alab49 led to a significant decrease in the number of cfus recovered from the skin nra defect with rofA leads to restoration of the wt phenotype.The Alab49 nfection . Tissue rulence” . The difrulence”  versus ArofA::aad9 was similar, irrespective of the inoculum dose, yielding an overall mean average of 2.2×107 cfus . Since 7 d post-inoculation appears to be a saturating time point for bacterial growth at the skin by virulent strains, an earlier biopsy time was also examined.The total number of cfu recovered from skin grafts at 7 d for wt Alab49 versus Alab49 rofA::aad9 grown to mid-log phase were used to inoculate skin grafts that underwent biopsy 48 hours (h) later; with this size inoculum, approximately 13 population doublings are projected for saturated bacterial growth at the skin. At 48 h, wt Alab49 had undergone a mean average of 1.7±1.1 population doublings . Thus, the rofA::aad9 construct displays greater short term fitness at the skin than the wt.Equivalent doses of ∼2,500 cfus of wt and Alab49 oublings . In controfA::aad9 construct (data not shown).As a control, the bacterial growth curves in enriched broth were found to be nearly identical for wt Alab49 and the rofA::aad9 chimera, which over-expresses the polymeric pilus proteins, displays a higher growth rate during the initial stages of infection at the skin.In summary, the Alab49 rofA::aad9 at the skin relative to wt . The quantity of hyaluronic acid capsule, a key virulence factor whose regulation of expression is complex, was also close to equivalent for the rofA chimeric construct, yielding 18.0 and 20.2 fg of hyaluronic acid per cfu for wt Alab49 and rofA::aad9, respectively. The data indicate that in strain Alab49, the substitution of nra with rofA has no detectable effect on several other key virulence phenotypes that are known to be critical for skin infection by GAS.The effect of replacement of t Alab49 . As furtrofA::aad9 chimeric construct were examined in greater depth. The 522 bp rofA-cpa intergenic region of Alab49 rofA::aad9 has an additional set of potential promoter sites for pilus gene transcription , showing that the genomic DNA sequences are similar in this region. The results indicate that the PprtF1 site may contribute to the elevated level of pilus gene transcription in the Alab49 rofA::aad9 construct . Using a 5′end phosphorylated oligonucleotide primer hybridizing within the cpa ORF, the cpa transcriptional start site was mapped to 214 bp upstream from the TTG start codon in wt Alab49 .The activation of pilus gene transcription by MsmR becomes even more apparent when the relative transcript abundance of each t Alab49 . In eachnra (wt) versus rofA (rofA::aad9) is also evident when the 5′-untranslated region upstream of cpa is evaluated by qRT-PCR. Both the UP-3 and UP-4 primer sets show an increase in transcript abundance of ∼2- to 4-fold when the ΔmsmR mutant is compared to wt Alab49  in the RofA-positive construct is dependent on MsmR, indicating that MsmR modulates the synchronization of the TR-TG interaction. The regulatory effect of MsmR on the pilus gene target is opposite in the Nra versus RofA background , indicative of an important shift in the qualitative nature of the TR-TG connection. The msmR, nra and cpa genes form a feedforward loop network motif in wt Alab49  organisms nra protein products of Alab49 and the M49 strain differ at only a single amino acid residue, via an Asn-Lys conserved substitution. Yet, both Nra and RofA present in the Alab49 strain background function as activators of pilus gene expression. Thus, not only is the connection between the TR gene locus (nra/rofA) and the pilus TG maintained following orthologous gene replacement in Alab49, but the qualitative nature of that connection  is also preserved, despite the high level of sequence divergence (∼35%) between Nra and RofA. Experimental manipulation of the TR genes, as demonstrated in this report, underscores the plasticity and robustness of the circuitry in complex regulatory networks.Many previous studies on Nra function and nra and rofA swapped in the Alab49 background, but so were their divergent upstream regions, which are required for auto-regulatory control rofA::aad9 construct may be due directly to RofA activity, cis elements and/or other regulatory factors having input function. In addition to MsmR and Nra/RofA, the transcription regulatory proteins Mga and RALP3 affect transcription of FCT-region genes in at least some strains ralp3 gene and there is no evidence for cross-regulatory effects between Nra and Mga Not only were the ORFs of S. pyogenes, the experimental replacement of the Escherichia coli regulatory gene pmrD, with a divergent form derived from Salmonella enterica, results in differential regulation of homologous genes and alterations in a probable niche-determining phenotype The importance of orthologous gene replacements in bacterial evolution remains to be fully established. In studies analogous to the work presented on S. pyogenes are devoid of pathogenicity islands rofA::aad9 construct acquired a higher level of (short term) fitness at the skin as a result of orthologous gene replacement. Thus, an intermediate genotype harboring rofA in place of nra may be more likely to undergo positive selection and persist, and thereby become more readily available as the recipient of a subsequent genetic event. In general terms, orthologous genes evolve in separate species over long periods and adapt to different ecological niches. Therefore, orthologous gene replacements may play a critical role in the evolution of S. pyogenes, ultimately leading to quantum leaps in phenotype.Unlike many bacterial pathogens, 2 in Todd-Hewitt broth supplemented with 1% yeast extract (THY). Bacterial growth was monitored at OD600 nm.Unless otherwise specified, the M53 strain Alab49 and its isogenic mutants were grown at 30 or 37°C with 5% COaphA3 and aad9, respectively). Linear DNA cassettes were constructed by PCR-based fusion assembly and used to transform Alab49 by electroporation Mutants were constructed by allelic exchange mutagenesis following transformation of bacteria with purified linear DNA containing the kanamycin or spectinomycin resistance genes  nra, ΔmsmR and ΔfctA mutants and Alab49 nra::aad9 were previously described rofA::aad9 ΔmsmR construct was achieved by replacement of msmR with aphA3 on the rofA::aad9 background, using methods previously described Chromosomal replacement in the ΔrecA. The A260/A280 ratio of each RNA sample was measured to determine concentration and assess purity .Bacteria were grown overnight in THY broth, then diluted 1∶100 in fresh THY broth and grown to mid-logarithmic, late log or stationary phase. RNA was purified as previously detailed recA gene was used as an internal reference transcription control to normalize expression data for each target gene. Each gene target was tested in duplicate or triplicate, as specified, from ≥2 RNA templates prepared from independent bacterial cultures. Relative expression of each gene was determined by the 2−(ΔΔCT) method cDNA was synthesized from 1 µg RNA using the SuperScript III First-Strand Synthesis Supermix for qRT-PCR with random oligonucleotide primers (Invitrogen), as described previously 5′-CCC CGT TGC AAT ATC AGG TTC TAT ATT ATC ACC ATA ATC ATA ACT ATC CGG CGG); they include UP-F1 (5′-GAA TTC ACT ATT AGA ATA GC), UP-F2 (5′-GAA GGA CCC CAA ATG ACA TAA TC), UP-F3 and UP-F4 (5′- GAA GGT GAC TAC TCT AAA CTT CTA GAG GGA GCA AC), to amplify an internal fragment of the cpa gene.Genomic DNA and cDNA, generated from purified RNA as described above, were used as templates for PCR amplification with Taq polymerase according to standards methods nd UP-F4 . Primer 5′-(P)CGA ACG CTC TGA TAG; RACES1, 5′-GGA AGC GCT AAC AAC AAA CG; RACES2, 5′-CGA CGA TCG GAT TAC TGA AAG; RACEA1, 5′-GCA ATA TAT CCT CTC CCT CC; and RACEA2, 5′-CCA CAT AAC AGA CTT AGC TGT C. Primer RACERT hybridizes to sequences corresponding to the cpa ORF and was used with reverse transcriptase for cDNA synthesis from a purified RNA template. Primers RACEA1 and RACES1 were used for the first round of PCR amplification, and RACEA2 and RACES2 were used for the second round. The final PCR-generated amplicons were purified and underwent nt sequence determination.5′-end RACE was performed using a kit (TaKaRa Bio Inc.) according to the manufacturer's instructions. Oligonucleotide primers used are: RACERT, 600 = 0.350±0.05) or stationary phase (16 h).Cell wall extracts of GAS were prepared using mutanolysin as described E. coli and originally derived from strain Alab49 was used at a dilution of 1∶1000.SDS-PAGE was performed on gradient gels (4 to 15% acrylamide) under reducing conditions. Rabbit sera raised to recombinant fusion polypeptides The T agglutination test was performed following trypsin treatment of whole bacteria, as described previously scid mice, which fail to reject the xenografts. Healed skin grafts were gently scratched with a scalpel blade and inoculated with 50 µl of bacteria in THY broth. The inoculated bacteria had been freshly grown to mid-logarithmic or stationary phase and diluted as appropriate. The actual inoculum doses were ascertained by serial dilutions performed in duplicate and the number of cfus averaged. Mid-logarithmic phase was defined as the point of ∼half-maximal OD600. Stationary phase cultures were incubated for 24 h. Inoculated skin grafts were occluded with a bandage. At specified time points post-inoculation, the human skin grafts were surgically removed from mice, split and each portion weighed. One weighed portion of the graft was evaluated for the number of cfus released following a vigorous vortex, with serial dilutions performed in duplicate.The human skin-SCID mouse model for streptococcal impetigo was implemented as previously described in extensive detail The HA content of GAS, attributable to the polysaccharide capsule, was measured according to previously described methods Statistical significance was calculated using the t-test, either paired or unpaired, as specified, or the Mann-Whitney U-test . Both tests are conservative and may slightly underestimate the significance of differences between groups having a small sample size.Figure S1nra mutant (lanes 3 and 4), Alab49 nra::aad9 construct (lanes 5 and 6), and the Alab49 rofA::aad9 construct (lanes 7 and 8). Immunoblots following SDS-PAGE were reacted with antiserum raised to rFctA (panel A) or rFctB (panel B). Extracts from cells grown to mid-logarithmic phase (4 h at 30°C) are shown in lanes 1, 3, 5 and 7; extracts from cells grown to stationary phase (16 h at 30°C) are shown in lanes 2, 4, 6 and 8. Molecular weight markers are shown .Immunoblots of bacterial cell extracts. Mutanolysin extracts were prepared from wt Alab49 (lanes 1 and 2), Alab49 Δ(0.20 MB TIF)Click here for additional data file.Figure S2Absence of assembled pili has no effect growth rate at the skin during early stages of infection. The number of bacterial population doublings (log2 change in cfu) at the skin by 48 or 72 h post-inoculation with mid-logarithmic phase broth cultures of the Alab49 ΔfctA mutant (diamonds). Bars depict average mean values. The mean average inoculum dose and standard deviations are also indicated.(0.06 MB TIF)Click here for additional data file.Table S1(0.08 MB PDF)Click here for additional data file.Table S2(0.08 MB PDF)Click here for additional data file.


Drosophila myogenic network. Mutations in either Myocyte enhancing factor 2 (Mef2) or the zinc-finger transcription factor lame duck (lmd) lead to very similar defects in myoblast fusion, yet the underlying molecular mechanism for this shared phenotype is not understood. Using a combination of ChIP-on-chip analysis and expression profiling of loss-of-function mutants, we obtained a global view of the regulatory input of both factors during development. The majority of Lmd-bound enhancers are co-bound by Mef2, representing a subset of Mef2's transcriptional input during these stages of development. Systematic analyses of the regulatory contribution of both factors demonstrate diverse regulatory roles, despite their co-occupancy of shared enhancer elements. These results indicate that Lmd is a tissue-specific modulator of Mef2 activity, acting as both a transcriptional activator and repressor, which has important implications for myogenesis. More generally, this study demonstrates considerable flexibility in the regulatory output of two factors, leading to additive, cooperative, and repressive modes of co-regulation.Understanding how complex patterns of temporal and spatial expression are regulated is central to deciphering genetic programs that drive development. Gene expression is initiated through the action of transcription factors and their cofactors converging on enhancer elements leading to a defined activity. Specific constellations of combinatorial occupancy are therefore often conceptualized as rigid binding codes that give rise to a common output of spatio-temporal expression. Here, we assessed this assumption using the regulatory input of two essential transcription factors within the  Drosophila, Mef2 mutant embryos display a block in myoblast fusion, which is very similar to what is observed in mutant embryos for lmd, a zinc-finger transcription factor. To understand the underlying nature of these defects we used ChIP-on-chip analysis to obtain a global view of their co-regulated enhancers, and we used expression profiling of mutant embryos to reveal their downstream transcriptional response. The results indicate that Lmd acts as a tissue specific modulator of Mef2 activity. Using in vivo and in vitro reporter assays, we show that co-binding to the same enhancer element can lead to diverse regulatory responses. The presence of Lmd has an additive, cooperative, or repressive effect on Mef2 activity, demonstrating that it acts as a molecular switch for gene expression during muscle differentiation. More broadly, our results highlight the difficulty in translating information on combinatorial binding data into a functional regulatory response.While genetic studies are essential to reveal the phenotypic relationships between genes, it is often very difficult to disentangle the molecular mechanism of two genes that phenocopy each other. In this study, we used global scale and single gene analysis to investigate the relationship between two transcription factors whose mutant embryos have a similar defect in myogenesis. In  CRMs are typically bound by multiple transcription factors, whose concentrations and interactions change dynamically over time. It is this combinatorial and dynamic property of CRM occupancy which makes regulatory output difficult, if not impossible, to predict based on information from a single transcription factor (TF) Drosophila development. Although the phenotypic defects in myoblast fusion are almost identical in Mef2 or lmd loss-of-function mutant embryos Development is driven by precise patterns of spatio-temporal gene expression, which are regulated through the action of transcription factors and cell signaling cascades converging on Members of the Mef2 family of MADS-box proteins were first characterized in vertebrates as important regulators downstream of the MyoD family of transcription factors, and have since been identified as part of an evolutionarily ancient regulatory network driving myogenesis from flies to man Drosophila embryos, the single Mef2 ortholog is expressed exclusively in mesoderm and its muscle derivatives. Even in this relatively simple model system, Mef2 regulates distinct batteries of target genes in precise spatial patterns  was recently identified as a potential repressor of Mef2-dependent transcriptional activation via the recruitment of the general co-repressor Groucho In Drosophila body wall muscle or somatic muscle is formed from two heterogeneous populations of cells- the founder cells (FCs), which represent 30 distinct cells in each hemisegment of the embryo, and the fusion competent myoblasts (FCMs) Mef2 expression in FCMs, and like Mef2, is also essential to regulate a program of muscle differentiation, the first step of which is myoblast fusion lmd mutant embryos have defects in the specification or maintenance of FCMs Mef2 itself The lmd and Mef2 and the similarity in the myoblast fusion phenotype observed in their loss-of-function mutants, we hypothesized that Lmd may act as an FCM-specific modulator of Mef2 activity. To assess this, we have systematically compared the in vivo enhancer occupancy of Lmd and Mef2 and identified a large number of combinatorially bound enhancers during myogenesis. Expression profiling of loss-of-function lmd and Mef2 mutants revealed that, although these TFs co-occupy the same enhancer region, they have different regulatory effects on the expression of the target genes. We used a combination of in vivo and in vitro approaches to demonstrate differential integration of regulatory input from Lmd and Mef2 at individual CRMs. Taken together, these data emphasize the diversity of transcriptional responses that can be generated by two transcription factors and identify Lmd as a new context-specific modulator of Mef2 activity.Given the extensive co-expression of lmd mutant embryos and its potential combinatorial regulation with Mef2, we set out to identify Lmd-bound enhancer regions and directly regulated target genes. To identify Lmd-bound enhancers within the developing embryo, we used chromatin immunoprecipitation followed by microarray analysis (ChIP-on-chip) during defined stages of muscle development. Lmd-associated DNA was precipitated from tightly staged embryos at two consecutive developmental time points, spanning most of the developmental stages when lmd is expressed (stages 10–13). To obtain data with high sensitivity and specificity we performed a total of eight independent chromatin immunoprecipitations per time-point using two different anti-Lmd antibodies for each time point (Drosophila genome As a first step towards understanding the phenotype of me point . The enrMef2 locus (sns (lmd for its expression in FCMs sns expression. The expression of the bHLH transcription factor twist persists longer in lmd loss-of-function mutants than in wild-type embryos twist locus (data not shown). Although we cannot exclude low-level Lmd occupancy below the detection limit of our assay, this result suggests an indirect regulatory connection between Lmd and twist.Lmd binding was detected at 154 unique genomic regions at one or both developmental time points , includif2 locus . In addicus .The recovery of enhancers of both in vivo  and in vttk (tramtrack) enhancer at stages 10–11, but not later, while the blow (blown fuse) and CG5080 enhancers are co-bound at stages 12–13, and not earlier  to our previously reported ChIP-on-chip data for Mef2 performed at the same developmental time points  earlier .in vivo and in vitro, to address these questions. First, we used global expression profiling to determine which genes require Lmd and Mef2 activity for their correct expression in vivo  proportion of Lmd direct target genes are also directly regulated by Mef2 , but are either unchanged or have slightly decreased or increased levels in Mef2 mutants  and tramtrack (ttk), have decreased expression at the late time points in lmd mutants and increased expression in Mef2 mutants, suggesting activation by Lmd and repression by Mef2   , and conon (ISH) . This anmd alone . As expemd alone . A thirdf2 alone . Howeverf2 alone . Ubiquitblow and sug were also strongly reduced in lmd mutants, and weakly reduced in Mef2 mutants , suggesting that the transcription factors co-occupy a single enhancer element. In addition, each refined sequence was found to contain at least one Mef2 consensus binding site conserved in Drosophila pseudoobscura (data not shown).Individual enhancer regions in in vivo by generating transgenic reporter lines. All tested enhancer regions specifically activated GFP-reporter expression in the developing muscle  and the fork head domain transcription factor biniou are recruited to this enhancer in vivogol enhancer required lmd activity , however in the majority of cases Lmd- and Mef2-binding could be mapped to the same genomic location  transcription factors are generally expected to have very similar spatio-temporal expression profiles. In fact, this assumption has been used by many studies to computationally predict the location of enhancer elements by searching for common TF binding motifs in the vicinity of clusters of co-expressed genes (or synexpression groups) Act57B locus. Ectopic Mef2 expression in the ectoderm is sufficient to induce Act57B expression, while providing Lmd alone is not  per time point. DNA amplification, labeling and hybridizations were performed as described previously Embryo collections and chromatin immunoprecipitations were performed as described previously 1lmdlmd mutant and stage-matched Canton S embryos were collected and aged. Homozygous mutants were selected with an automated embryo sorter The assayed 2>0.7 or <−0.7) were considered to be differentially regulated (2 >0.58 or <−0.58) were considered to be significantly enriched  egulated . Immunopenriched .Mef2-mutant or lmd-mutant expression profiling data. For a small number of regions, data from this study indicated a more likely target gene than had been assigned previously with Mef2 data alone lmd expression profiling (E-TABM-894). Lmd- and/or Mef2-bound regions and mutant expression data can be visualized at http://furlonglab.embl.de/data/.Automatic assignment of ChIP-enriched fragments to target genes was performed as described previously actin57B), chr2R:20197035-20197429 (betaTub60D), chr3R:6619371-6620063 (CG14687), chr3R:27529661-27530409 (ttk), chr2R:3472616-3473387 (blow), chr3R:27538572-27539618 (ttk), chr2R:8813219-8814579 (sug), chr2R:20966587-20969610 (gol). For all constructs at least two independent transgenic lines were obtained and assayed. The UAS-Mef2 line used in this study has been described previously lmd line was previously referred to as UAS-gflFragments within the following coordinates (based on BDGP genome release 5) were cloned into the pH-stinger (AF242365) vector for germline transformation in situ hybridizations were done as described previously in situ hybridizations were done in parallel and the confocal imaging was performed with identical laser and gain settings for each gene in the four genetic backgrounds. The following ESTs were used to generate digoxigenin or fluorescein-labeled probes: RE53159 (betaTub60D), LD04994 (act57B), LD34147 (CG5080), LP02193 (blow), LD36528 (sug), RE74890 (CG14687), RE28322 (CG9416), GH20973 (gol), AT15089 (twi) and RE02607 (wg). The full-length sns cDNA was a kind gift from S. Abmayr. GFP expression in transgenic animals was detected by immunohistochemistry with rabbit α-GFP antibody (Torrey Pines Biolabs) at a concentration of 1:500. Biotinylated secondary antibodies were used in combination with the Vector Elite ABC kit (Vector Laboratories).Double fluorescent Drosophila S2 cells were transiently transfected using Cellfectin (Invitrogen). Lmd and Mef2 were expressed from full-length ESTs  in pAc5.1 vector (Invitrogen). The enhancers (coordinates given above) were assayed in a pGL3 luciferase reporter vector (Promega) with an Hsp70 minimal promoter and the luciferase activity was normalized to Renilla standard (Promega). The total amount of transfected DNA was kept constant by supplementing empty pAc5.1 vector. The measurements were performed according to the supplier's recommendations  with a PerkinElmer 1420 Luminescence Counter.Figure S1in vivo. (A–D) Schematic overviews of the tiling array probes covering the (A) act57B, (B) Tub60D, (C) CG9416, and (D) CG14687 loci. These previously described regulatory regions (black bars) show significant binding of Lmd (blue) and Mef2 (green). (A″–D″) Immuno-histochemistry using an anti-GFP antibody to detect reporter gene expression. All four enriched sequences are able to specifically activate GFP-reporter expression in transgenic embryos in the mesoderm as early as (A′–D′) stage 11, with persistent GFP-presence at (A″–D″) stage 13.Previously identified enhancer regions are co-occupied by Lmd and Mef2 and reproduce target gene expression (2.79 MB TIF)Click here for additional data file.Figure S2wg gene (red). Ectopic expression (using the engrailed GAL4 driver) should be visible juxtaposed to the wg stripe. The wg expression was therefore used to ensure that the confocal imaging was performed with identical laser and gain settings for each gene within the four genetic backgrounds. (A–I) FISH of wild-type embryos with probes specific for (A) βTub60D, (B) act57B, (C) CG5080, (D) blow, (E) sug, (F) sns, (G) CG14687, (H) CG9416, and (I) gol, detecting specific expression in the mesoderm. No specific staining was observed in the ectoderm (white brackets). An engrailed-Gal4 driver line was used to ectopically express (A′–I′) UAS-Lmd, (A″–I″) UAS-Mef2-HA or (A″–I″) both UAS-Mef2-HA and UAS-Lmd in ectodermal stripes. Lmd and Mef2 show differential ability to activate ectopic target gene expression (white brackets). Note: The area indicated by the white brackets highlights the ectopic expression.Ectopic expression of Lmd and Mef2 reveals differential regulatory input on target gene expression. Double fluorescent in situ hybridisation (FISH) of the gene of interest (green) and endogenous (9.37 MB TIF)Click here for additional data file.Figure S3lmd and Mef2 loss-of-function mutant embryos. In situ hybridisations described in twist mRNA  or lacZ mRNA expressed from a balancer  to identify homozygous mutant embryos. Twist expression persists longer in lmd mutants  than in wt embryos . LacZ expression is associated only with heterozygous, balancer containing embryos  .Enhancer activity in (4.22 MB TIF)Click here for additional data file.Table S1ChIP-on-chip enriched fragments.(0.08 MB XLS)Click here for additional data file.Table S2Overlap of ChIP data with previously characterised enhancer regions.(0.04 MB PDF)Click here for additional data file.Table S3Lmd expression profiling timecourse.(0.15 MB XLS)Click here for additional data file.Table S4Direct targets genes of Lmd and Mef2.(0.04 MB XLS)Click here for additional data file.Table S5Expression profiling of Lmd direct target genes in Lmd and Mef2 mutant embryos.(0.05 MB XLS)Click here for additional data file.