Drosophila, it has been shown thatmaternal morphogen gradients and ubiquitously distributed general transcriptionfactors may cooperate to upregulate zygotic genes that are essential for patternformation in the early embryo. Here, we show that DrosophilaSTAT (STAT92E) functions as a general transcription factor that, together withthe transcription factor Zelda, induces transcription of a large number ofearly-transcribed zygotic genes during the MZT. STAT92E is present in the earlyembryo as a maternal product and is active around the MZT. DNA–bindingmotifs for STAT and Zelda are highly enriched in promoters of early zygoticgenes but not in housekeeping genes. Loss of Stat92E in theearly embryo, similarly to loss of zelda, preferentiallydown-regulates early zygotic genes important for pattern formation. We furthershow that STAT92E and Zelda synergistically regulate transcription. We concludethat STAT92E, in conjunction with Zelda, plays an important role intranscription of the zygotic genome at the onset of embryonic development.In many organisms, transcription of the zygotic genome begins during thematernal-to-zygotic transition (MZT), which is characterized by a dramaticincrease in global transcriptional activities and coincides with embryonic stemcell differentiation. In  Drosophila STAT (STAT92E)functions as a general transcription factor that, together with Zelda, inducestranscription of a large number of early-transcribed zygotic genes during theMZT. We further show that STAT92E and Zelda synergistically regulatetranscription. Thus, multiple transcription factors, such as STAT92E and Zelda,cooperate to control transcription of the zygotic genome at the onset ofembryonic development.In the initial phase of the early embryo, transcription is inactive anddevelopment is supported by maternally derived gene products. During a timewindow termed the maternal-to-zygotic transition (MZT), the maternal geneproducts are degraded and the zygotically expressed genes required forembryogenesis initiate their transcription. How the dramatic upregulation ofzygotic genes during the MZT is achieved is not completely understood, althoughit has been shown that the transcription factor Zelda plays a critical role. Inthis manuscript, we show that  Drosophila, maternally derivedmorphogen proteins form broad gradients along the major body axes to define bodypolarities hunchback (hb), Krüppel (Kr),knirps (kni), and tailless (tll) are transcribedzygotically in broad and mostly non-overlapping domains along the anteroposterior(A/P) body axis. The boundaries of these zygotic genes are determined by morphogengradients that are set up by maternal gene products, such as Bicoid (Bcd) andmaternal Hb Embryonic pattern formation is a complex and progressive process. In manymulticellular organisms, the initial period of embryogenesis relies on gene productsinherited from the mother. In zelda  To date, only a few transcription factors have been implicated in transcription ofthe zygotic genome during the MZT. For example, the maternal morphogens Bcd andDorsal activate target genes along the anteroposterior (A/P) and dorsoventral (D/V)axis, respectively insilico analyses, taking advantage of the large amount of informationavailable in public databases on transcriptional regulation of zygotic genesexpressed during early embryogenesis in Drosophila. This approachled to the identification of STAT92E, in addition to Zelda, as a plausibletranscription factor important for the upregulation of multiple genes during theMZT. Global expression profiling studies indicate that loss of STAT92E, similarly toloss of Zelda, preferentially causes down-regulation of zygotic genes essential forearly embryogenesis. We further demonstrate that STAT92E is indeed involved intranscription of the developmentally important genes dpp, tailless(tll), and Kr during early embryogenesis. Ourresults suggest that STAT92E is essential for upregulation of a multitude ofzygotically transcribed genes during the MZT, and thus is important for transitionof the early embryo from a totipotent embryonic stem cell state to a state ofcellular differentiation.To identify additional ubiquitous transcription factors that are important fortranscription of the zygotic genome during the MZT, we first conducted hunchback (hb), huckebein (hkb),Giant (Gt), Krüppel (Kr), knirps (kni), and tailless(tll); the pair-rule genes: even skipped (eve), fushitarazu (ftz), hairy (h), odd paired (opa), paired (prd), sloppy paired 1(slp1), and runt (run); the segmental polarity andother genes: engrailed (en) and Sex lethal(Sxl), as well as genes expressed along the D/V axis:decapentaplegic (dpp), zerknüllt (zen), rhomboid (rho), shortgastrulation (sog), snail (sna), and twist(twi).To identify general transcription factors that are required for transcription ofa large number of zygotic genes at early embryonic stages, or during the MZT, weperformed a meta-analysis to search for candidate transcription factors requiredfor activation of multiple zygotic genes. To this end, we first selected a listof developmentally important zygotic genes transcribed during the MZT (referredto as “zygotic genes”), whose expression patterns altogether coverthe entire embryo, and whose transcriptional activation has previously beenstudied. We analyzed a total of 21 early zygotic genes, including the gap genes:http://flybase.org) and PubMed (http://www.ncbi.nlm.nih.gov), and compiled a list of allcurrently known or potential transcriptional activators or signaling pathwaysinvolved in their transcriptional induction  http://genomeenhancer.org/fly) As a second step, for each of these genes, we searched Flybase , Bicoid (Bcd), STAT92E, Torso, Caudal (Cad),Dorsal, and Twist (Twi) . Zelda hDrosophila genome is slightly AT-rich, with 57.4% ATand 42.6% GC base pairs p) for random occurrence of one STAT binding site(with 6 fixed nucleotides) at any position is 3.08x10−4 (0.2874x0.2132),and its frequency of occurrence within the 4 kb upstream regulatory regions of21 genes  at random is 25.9. However, when we searched for STATbinding sites within the 4 kb upstream region of the 21 zygotic genes, we found43 in total  (p = 0.001).To test whether STAT92E is important for transcription of early “zygoticgenes”, we first assessed the occurrence of consensus STAT92E bindingsites (TTCnnnGAA) in the promoter region, defined as 4 kb genomic sequenceupstream of the transcription start site, of the 21 zygotic genes in this study.The d value) . Assuminrp49, GAPDH,Actin5C, and those encoding ribosomal proteins and RNA polymerases,we found a total of 13 STAT-binding sites , including ng sites , which ing sites .Moreoveng sites , whereasng sites .It has been shown that Zelda-binding sites (the TAGteam motif) are enriched inthe promoter regions of “zygotic genes” Stat92E gene products  at the samestage.To determine whether STAT92E functions as a general transcriptional activator ofthe zygotically expressed genes in the early embryo, we determined theexpression profiles of early stage embryos  of wild-type control and of thoselacking the maternal mat–Stat92E embryos, 657 geneswere down regulated and 558 genes up-regulated by at least 1.5 fold, comparedwith wild-type control  on the Gene Chip, while the majority(91.1%) of the genes exhibited no significant changes  were alsodown-regulated in mat–Stat92Eembryos,suggesting that these genes might be co-regulated by STAT and Zelda (We found that in  control . In Stat changes .Consistc genes” . In contc genes” .This obnd Zelda .hb) were all significantly down-regulated, with a 4.3 folddown-regulation on average, whereas the 21 housekeeping genes showed nosignificant changes in expression, with the exception of DNase II , which is acomputational method that determines whether an a prioridefined set of genes shows statistically significant, concordant differencesbetween two biological states  mat–Stat92E embryos when compared withwild-type control .To validate our gene profiling results from the microarray studies, weinvestigated the effects of over-activation and loss of STAT92E on transcriptlevels of a number of early “zygotic genes”. We chose to examineexpression levels of dpp, Kr,tll, and eve in the early embryo(1–2 h after egg laying) using semi-quantitative reverse-transcriptionpolymerase chain reaction (RT-PCR) in Stat92E gain- orloss-of-function genetic backgrounds. We found that in hopGOFembryos, in which STAT92E is overactivated mat–Stat92E embryos,these four genes were expressed at approximately 50% of the wild-typelevels  experimentswith early embryo extracts using anti-STAT92E antisera. Binding of STAT92E tothe eve enhancer and of Zelda to the TAGteam sequences enrichedin “zygotic genes” have been previously shown dpp, Kr, andtll , or amutant version with both STAT-binding sites mutated (DM), with an enhancedyellow fluorescent protein (EYFP), and transfected S2 cells , but notthe double mutant (DM) construct  or Zelda, or both STAT92E (with Hop) and Zelda, in thepresence of WT-lucdpp ordpp™-luc, and carried out luciferaseassays. When assayed at 72 h after induction of transgene expression, we foundthat STAT activation alone induced WT-lucdpptranscription by 22 fold, and Zelda alone caused upregulation ofWT-lucdpp by 48 fold, whereas in thepresence of both Zelda and activated STAT, WT-lucdppwas up-regulated by 230 fold  have reduceddpp expression by 5.7 fold dpp transcription to a limited extent, whereas the presenceof both Zelda and STAT activation synergistically promote dpptranscription.The apparent synergy between STAT92E and Zelda could explain the results from thegene profiling experiments. Microarray results show that embryos without STAT92E(in which Zelda presumably remains active) exhibit a 3.1 fold decrease inpression , and thadpp, Kr, and tll in theearly embryo, by in situ hybridization, while the effects ofStat92E mutation on eve expression havepreviously been documented Having shown that STAT92E, possibly acting synergistically with Zelda, isimportant for expression levels of many early “zygotic genes”, wenext investigated whether loss of STAT92E also affects the spatial expressionpatterns of the early “zygotic genes”. We examined the expression ofdpp expression domain spans nearly the entire A/P axis inthe dorsal regions of the early embryo dpp transcription in the ventral region is repressed byDorsal, a Rel family transcription factor dpp expression in the dorsal region  morphology of the larva or late embryo Finally, we investigated the biological consequences of reducing expressionlevels, without altering spatial domains, of multiple zygotically expressedearly genes, as with loss of STAT92E. The correct expression of the earlyzygotic genes during the MZT is essential for formation of different tissues andbody parts at the correct positions, i.e., pattern formation thabdominal segment and the Filzkörper .To understand the role of STAT92E in individual signaling pathways important forpattern formation, we investigated whether loss of STAT92E could furthercompromise pattern formation in sensitized genetic backgrounds. To this end, weexamined cuticles of tll is essential for the development of terminalstructures tll mutant homozygous embryos do not have A8 and theFilzkörper   . This suKr is required for development of the thoracic and anteriorsegments, and these segments are missing in–/–Kr embryos , the posterior-most denticle belt extendedsignificantly dorsally to cover approximately 80% of the circumference(mat–Stat92E and+/–dpp embryos (n>500). Thus,in the absence of STAT92E, a half dose of dpp is no longersufficient for dorsoventral patterning, consistent with the notion that STAT92Enormally regulates dpp expression levels.The dorsally expressed l region  [37]. No embryos , they di embryos , suggestcuticles ,suggestmference, arrow.tll, Kr, and dpp,uncovered the role of STAT92E in regulation of specific early zygotic genesimportant for pattern formation.In summary, loss of STAT92E caused heterogeneous patterning defects, as revealedby varying cuticle defects, consistent with an insufficiency of multiplepathways. A further reduction in the dosage of genes in different pathways, suchas dpp,Kr, and tll, that are important for patternformation and/or cell fate specification in the early embryo. Our studies suggestthat STAT92E cooperate with Zelda to control transcription of many “zygoticgenes” expressed during the MZT. While STAT mainly regulates transcriptionlevels, but not spatial patterns, of dpp, tll, andKr, and possibly also other “zygotic genes”, Zeldais essential for both levels and expression patterns of these genes We have undertaken a bioinformatics approach to investigating the mechanismscontrolling transcription of the zygotic genome that occurs during the MZT, and haveidentified STAT92E as an important general transcription factor essential forup-regulation of a large number of early “zygotic genes”. We havefurther investigated the role of STAT92E in controlling transcription of a fewrepresentative early zygotic genes, such as The transcriptional network that controls the onset of zygotic gene expression duringthe MZT has remained incompletely understood. It has been proposed thattranscription of the zygotic genome depends on the combined input from maternallyderived morphogens and general transcription factors. The former are distributed inbroad gradients in the early embryo and directly control positional information, whereas the latter are presumably uniformlydistributed regulators that augment the upregulation of a large number of“zygotic genes”. Other than Zelda, which plays a key role as a generalregulator of early zygotic expression Stat92E or hopmutant embryos, expression of eve stripes 3 and 5 are significantlyreduced but not completely abolished Sxl expressionduring the MZT GOF-induced ectopic tllexpression but not essential for the spatial domains of tllexpression in wild-type embryos under normal conditions Our conclusion that STAT92E is important for the levels but not the spatial domainsof target gene expression in the early embryo is consistent with several previousreports. It has been shown that in Drosophila embryo. Moreover, it would beinteresting to investigate whether STAT plays similar roles in embryonic stem celldifferentiation in other animals.Finally, the MZT marks the transition from a totipotent state to that ofdifferentiation of the early embryo. As a general transcription factor at thistransition, STAT, together with additional factors . To generateStat92Emat– embryos, 82B Stat92E6346/TM3hsp70-flp;FRT females werecrossed to 82B /TM3hsp70-Flp; FRT males. Their 3rd instar larvalprogeny were heat-shocked at 37°C for 2 hrs daily for 3–4 days, andresulting adult females of the genotype 82BStat92E6346/FRT82B hsp70-flp; FRT were used to produce embryos that lackmaternal Stat92E gene products, as described in the dominantfemale-sterile “germline clone” technique All crosses were carried out at 25°C on standard cornmeal/agar medium unlessotherwise specified. Fly stocks of tll transcription based on thereports that tll is not expressed in torsoloss-of-function mutants and is overexpressed in torsogain-of-function mutants The following rules were used for assigning a score to known or putativeactivators of each of the “zygotic genes”. We placed top importanceon genetically demonstrated activation during early embryogenesis, with such anactivator receiving an activation score of 10. For instance, Torso was assigneda score of 10 as an activator of Databases and programs used in this study:http://flybase.org); PubMed (http://www.ncbi.nlm.nih.gov); RedFly ; FlyEnhancer (http://genomeenhancer.org/fly).Flybase  and luciferase. Mutagenesis of two STAT92Ebinding sites within the dpp promoter was done by PCR, and wasverified by sequencing. V5-Hop and V5-STAT92E are gifts from S.X. Hou The script A .This geIn situ hybridization for detectingdpp, Kr, and tll mRNA wasperformed according to a standard protocol using digoxigenin-incorporatedantisense RNA probes made from dpp, Kr, andtll cDNA, respectively, according to the supplier'sprotocol. A standard protocol was used for antibody staining of embryos, and abiotinylated secondary antibody and the Vectastain ABC kit  were used according to the manufacturer's instructions. Stainedembryos were mounted in DAPI-containing mounting medium for accurate staging,when necessary. Mounted embryos were photographed using Normaski optics on aZeiss Axioscope and images were analyzed using Photoshop or ImageJ software.Cuticle preparations were performed according to a standard protocol with minormodifications. Embryos were dechorionated with 50% Clorox, washedextensively with 0.1% Triton, mounted in Hoyer's, and photographedusing dark-field optics. Total RNA was isolated from embryos (from flies raised at 25°C) collected at1–2 h after egg laying (corresponding to nuclear division cycles8–14) using trizol (Invitrogen) or the RNeasy Kit (QIAGEN) according tothe manufacturer's instructions. RNA quality was assessed using the Agilent2100 Bioanalyzer and the RNA 6000 Nano kit .rp49transcript levels and to control genotypes. Data shown are averages and standarddeviations from at least three independent experiments. The following primerpairs were used.For RT-PCR analysis, first strand complementary DNA (cDNA) was generated from 5µg of purified total RNA using Superscript III reverse transcriptase(Invitrogen) and oligo(dT)12–18 in 50 µl total reaction volume. ThecDNA (at 1∶100 dilution) was used as template for either semi-quantitativePCR reactions or real time PCR analysis using SYBR green based detection on aBioRad iCycler. Reactions were carried out in triplicate, and melting curveswere examined to ensure single products. Results were quantified using the“delta-delta Ct” method to normalize to rp49: TCCTACCAGCTTCAAGATGAC, CACGTTGTGCACCAGGAACT.dpp: AATCAATCTTCGTGGAGGAGCCGA, TTGGTGTCCAACAGCAGATAGCTC.eve: TGCACGGATACCGAACCTACAACA, GTTCTGGAACCACACCTTGATCGT.Kr: CAAGACGCACAAACGCGAACCTTA, TTGACGGTTTGCAGCCAGAAGTTG.tll: AATACAACAGCGTGCGTCTTTCGC, ACATTGGTTCCTGTGCGTCTTGTC.E. coliRNase H and DNA ligase) to convert single-stranded cDNA into a double-strandedDNA (dsDNA). The dsDNA was then used as a template for in vitro transcription ina reaction containing biotin-labeled UTP and T7 RNA Polymerase to generatebiotin-labeled antisense RNA (aRNA). Twenty µg of labeled aRNA wasfragmented and fifteen µg of the fragmented aRNA was hybridized toAffymetrix Drosophila Genome 2.0 Array Chips according to themanufacterer's Manual . Array Chips werestained with streptavidin-phycoerythrin, followed by an antibody solution(anti-streptavidin) and a second streptavidin-phycoerythrin solution, performedby a GeneChip Fluidics Station 450.For microarray analysis, 200 ng of total RNA was used to prepare biotin-labeledRNA using Ambion MessageAmp Premier RNA Amplification Kit . Briefly, the first strand of cDNA was synthesized usingArrayScript reverse transcriptase and an oligo(dT) primer bearing a T7 promoter.Then DNA polymerase I was used  and normalized using the recommendeddefaults. The signals from 11 perfect matched oligonucleotides for a specificgene and 11 mis-matched oligonucleotides were used to make comparisons ofsignals. Genes were identified as present when the present (P) assignment wassignificant (p<0.05).http://www.broadinstitute.org/gsea) was used to determinewhether the predetermined gene sets  online software , Rabbit anti-V5 , goat anti-STAT92E , rabbitanti-Kr , and anti-phospho-STAT92E. Common secondary antibodies were usedin whole-mount immunostaining or Western blots.Drosophila Schneider L2 (S2) cells were cultured at 25°C inDrosophila Serum-Free Medium  supplementedwith 10% Fetal Bovine Serum  and 0.5xAntibiotic-Antimycotic (Invitrogen). Cells were cultured at2.5×106/ml prior to transfection. Transfections wereperformed with FuGene 6 (Roche) according to the manufacturer'sinstructions. Cu2SO4 (Sigma) was added to the medium at afinal concentration of 0.5 mM 16 hours after transfection, and cells wereharvested 48 hours after induction. To stimulate JAK/STAT activation in S2cells, 2 mM H2O2 and 1 mM sodium vanadate (pervanadate)were added to the medium and cells were harvested at desired times aftertreatment. Treated S2 cells were harvested in cell lysis buffer  for Western blotting or ChIP experiments.ChIP experiments were carried out according to standard protocols with thefollowing modifications. 200 µl of early embryos (1–2 h AEL) or1×107 S2 cells were treated with 1% formaldehyde at roomtemperature for 20 min (embryos) or 10 min (cells) to crosslink protein withchromatin/genomic DNA. Embryos or cells were homogenized and lysed in 300µl of RIPA lysis buffer with 2 mM EDTA and protease inhibitors on ice. Thelysate was sonicated to shear the genomic DNA to lengths between 500 and 1000bp. An aliquot (50 µl) of sonicated sample was saved as the input control.5 µg goat anti-STAT92E  or rabbit anti-V5 antibodies wereadded to 200 µl experimental samples in RIPA buffer with 2 mM EDTA andprotease inhibitors, and the mixture was incubated overnight at 4°C withrotation. Protein G beads (Sigma), pre-blocked with sonicated salmon sperm DNA(Stratagene), were added to precipitate the antibody-bound chromatin and theprecipitate was washed extensively. After reversing the crosslink, DNA wasrecovered by using a Qiagen PCR purification kit and quantified by PCR. Thefollowing forward and reverse primers (flanking two STAT-binding sites in therespective promoter regions) were used for PCR reactions.dpp: AATTCCGGATAGCGCCTGG, AAAGATGGCACACGCTGGG.Kr:CATGCGTTTGCATACTGGAG,CTATTCGAATCGCCCTTGTC.tll:AGTGCTTTGAGGTCGGAATG,AAGAAACCGTGGTGTCCTTG.Stat92E:TGACTGCCCGCTTTTATACC,CAAACGGCGGTCAATAGTTT.Figure S1Distribution of STAT and Zelda-binding sites in promoter-distal enhances.Dashed horizontal lines represent genomic DNA sequences surrounding thepromoter regions from-4000 bp to +1 (transcription start site) of theindicated early zygotic genes. Known enhancers . Gray arrowheads indicate thepositions of Zelda-binding consensus sequences (CAGGTAG).(GIF)Click here for additional data file.Figure S2Stat92E mutant versus wild-typecontrol. Total RNA isolated from 1–2 h wild-type andmat–Stat92E embryos were subjectedto microarray analysis. The expression level of each gene is plotted as thelog of the average ratio of fluorescent intensity on themat–Stat92E chip to the wild-typecontrol chip. Note that expression levels of the majority of the genes werenot changed (centered at 0). The gene number is from the Agilent microarraychip.Gene expression profile of (GIF)Click here for additional data file.Figure S3Zelda transcription levels in different genetic backgrounds.Total RNA was isolated from staged early embryos (1–2 h after egglaying) of the indicated genotypes, and mRNA levels ofzelda and rp49 (control) were measuredby real-time RT-PCR. Zelda expression levels are shown as relative to rp49and normalized to wild-type control. Three independent experiments wereaveraged. Error bars are standard deviations.(GIF)Click here for additional data file.Figure S4dpp genomic region and enhancer sequence. (A) Horizontalline indicates the genomic region of dpp, which can bedivided into three regions based on functional requirements fordpp, as indicated on top. Transcript A ofdpp is shown; filled boxes indicate coding, and grayboxes non-coding, regions. The position of the 1.3 kb promoter region isshown. (B) A 500 bp sequence within the 1.3 kb promoter is shown. STAT92Econsensus sites are marked in blue, Zelda site in red. (C) Comparison of theputative STAT92E and Zelda binding sites in the dpppromoter with the consensus sequences is shown. Numbers indicate positionsof the sites relative to the start of dpp transcript A.(GIF)Click here for additional data file.Figure S5dpp reporter gene expression S2cells. (A) Drosophila S2 cells were transfected withSTAT92E-V5 and were stimulated with H2O2/vanadate.Cells were lysed 30 min after stimulation and were subjected to SDS-PAGE.STAT92E phosphorylation was detected with anti-pSTAT92E, and transfectedSTAT92E was detected with anti-V5. Anti-Tubulin was used as a loadingcontrol. (B) S2 cells were transfected withWTdpp-EGFP orDMdpp-EGFP, and pervanadate treatment wasused to activate endogenous STAT92E. EGFP was imaged by confocal microscopyat the same settings for both constructs at different time points afterstimulation. Note that EGFP expression inWTdpp-EGFP transfected cells was detected 1.5 hfollowing pervanadate treatment, but not inDMdpp-EGFP transfected cells. Right panelsare higher magnifications of the white square in the left panel.STAT activation induces (GIF)Click here for additional data file.Figure S6dpp expression in late stageembryos.  In GOF/+hop embryos,dpp expression is increased, but remains excluded fromthe ventral-most region (arrow in A). The cuticle morphology appears mostlynormal (B).  dpp expression in parasegment 7  of stage 14 embryos.  Stage 16 embryos were stained withanti-Crumb to reveal the gut epithelia.  In wild-type embryos,dpp is expressed bilaterally at ps7 and other tissues(not marked), as has previously been shown dpp expression, as has previously been shown GOFHopembryos, dpp expression at ps7 is increased in intensity,although the midgut appears mostly normal in morphology, albeit slightlyover-constricted compared to wild type.  Inmat–Stat92E embryos,dpp expression at ps7 is much reduced or undetectable.The midgut lacks constriction.JAK/STAT signaling regulates (GIF)Click here for additional data file.Figure S71/+Kr cuticle showing minoranterior defects such as a weakened A2 (arrowhead). (C)+/–dpp larvae exhibit mostlynormal cuticle morphology, with correct D/V polarity, albeit occasionaldenticle defects.Larval cuticle morphology. (A) A wild-type larval cuticle, with eightabdominal denticle belts seen in the ventral region. (B) A(GIF)Click here for additional data file.Table S1Early zygotic genes and their activators. Activators are based on publishedliterature and may not be transcription factors or directly act on targetgenes.(XLSX)Click here for additional data file.Table S2Activators of zygotic transcription and their activation score.(XLSX)Click here for additional data file.Table S3Housekeeping genes and STAT-binding site distribution in their promoters.(XLS)Click here for additional data file.Table S4mat–Stat92E earlyembryos.Genes down-regulated in (XLS)Click here for additional data file.Table S5mat–Stat92E earlyembryos.Genes up-regulated in (XLS)Click here for additional data file.Table S6Zygotic and housekeeping gene sets.(XLSX)Click here for additional data file.


Drosophila nervous system. Threshold Gcm/Glide levels control the glial versus neuronal fate choice, and its perdurance triggers excessive gliogenesis, showing that its tight and dynamic regulation ensures the proper balance between neurons and glia. Here, we present a genetic screen for potential gcm/glide interactors and identify genes encoding chromatin factors of the Trithorax and of the Polycomb groups. These proteins maintain the heritable epigenetic state, among others, of HOX genes throughout development, but their regulatory role on transiently expressed genes remains elusive. Here we show that Polycomb negatively affects Gcm/Glide autoregulation, a positive feedback loop that allows timely accumulation of Gcm/Glide threshold levels. Such temporal fine-tuning of gene expression tightly controls gliogenesis. This work performed at the levels of individual cells reveals an undescribed mode of Polycomb action in the modulation of transiently expressed fate determinants and hence in the acquisition of specific cell identity in the nervous system.The Gcm/Glide transcription factor is transiently expressed and required in the  Drosophila glial determinant and transcription factor Gcm/Glide. Thus, PcG proteins also finely tune gene expression, and this is independent of memory mechanisms, suggesting that “transient” promoters may have a different affinity to PcG proteins compared to “stable” promoters. PcG proteins negatively affect Gcm/Glide autoregulation, thereby promoting neurogenesis at the expense of gliogenesis. Thus, PcG genes act in the fate choice between two types of differentiated cells, implying that distinct cell populations have specific requirements for general chromatin modifiers.Epigenetic mechanisms are essential to define cell identity, and the Polycomb and the Trithorax Group proteins  control the body plan by maintaining the epigenetic state of homeotic genes. PcG and TrxG act by triggering stable chromatin modifications that are “remembered” after cell division and keep gene expression in an OFF or ON state. Recent genome-wide analyses call for additional targets of PcG proteins, but the role of these chromatin factors in dynamic transcriptional states and/or in specific cell fates is difficult to apprehend, mostly because very sensitive readouts are required. This in vivo study performed at the single-cell level shows that PcG proteins affect the levels and the kinetics of the transiently expressed  Drosophila melanogaster Glial cell missing/Glial cell deficient transcription factor  is transiently expressed and is key to decide between glial and neuronal fates in the multipotent neural precursors One of the most challenging issues in biology is to elucidate the mechanisms underlying cell fate determination and maintenance. The Two major classes of proteins that modify the chromatin structure and its condensation state, the Polycomb group (PcG) and the Trithorax group (TrxG), are known as critical regulators of HOX transcription factors, which act as molecular switches that are maintained in a silent or in an active state Recent chromatin immunoprecipitation studies have shown that PcG and TrxG binding is also associated with dynamic transcriptional states modulating different processes including mitogenic pathways and progression from multipotency to differentiation  carries a stereotyped number of sensory organs called macrochaete or bristles. Pyxgcm/+ flies ectopically express gcm in the larval notum, which triggers the differentiation of supernumerary sensory organ precursors (SOPs) and bristles  animals. Using large overlapping deficiencies , we performed a primary screen and identified 42 genomic regions that dominantly enhance or suppress the Pyxgcm dominant phenotype when deleted  and peripheral (PNS) nervous systems, neuroblasts (NB) and SOPs, respectively Pyxgcm suppressors and indeed, the large and the small deficiencies covering three genes – escargot (esg), worniu (wor) and snail (sna) – expressed in most embryonic NBs act as Pyxgcm suppressors  and crooked legs (crol), identified in a microarray as induced by Gcm To evaluate the specificity and the sensitivity of the screen, we asked whether the selected deficiencies eliminate genes expected to interact with pressors . Testinghenotype . Finallygcm interactors were identified validates our screen and shows that the dominant bristle phenotype is a reliable and very sensitive readout.The fact that known and predicted trxG gene brahma (brm), which encodes a transcriptional coactivator related to yeast SWI/SNF proteins and plays a role in ATP-dependent nucleosomal remodeling brm, Df(3L)brm11, Df(3L)th102 and, most importantly, a null brm allele, enhance the Pyxgcm phenotype  suppresses it ) and the E(bx) mutation itself enhance the Pyxgcm phenotype (E(bx)  encodes a transcription coactivator that belongs to the ISWI chromatin remodeler complex, another TrxG complex, and negatively regulates the JAK-STAT pathway gcmFurthermore, a deficiency covering henotype . InteresDrosophila COMPASS-like complex trx null mutation acts as a suppressor of the Pyxgcm phenotype (Pyxgcm phenotype (Drosophila CREBS-binding protein (dCBP) encoded by nejire (nej) is responsible for H3K27 acetylation nej null mutation suppresses the Pyxgcm phenotype   and PRC2 ((esc), E(z)), as well as the PcG protein recruiter pipsqueak (psq). Mutations in all four genes enhance the Pyxgcm phenotype  studies on Drosophila embryos and different cell lines gcm and gcm2, which are organized head to head in a 30 kb region gcm-gcm2 5′ regulatory region. As expected, the profile of H3K4methylation complements that of H3K27me3 , or an adjacent region (GlacAT) and a negative control (Rp49) (H3K27me3 . Pc bindl (Rp49) .gcm gene bound by PcG proteins is able to recruit PcG proteins in transgenic assays. For this, we examined PcG binding to a transgene containing the upstream region of the gcm locus on salivary gland chromosomes by Immuno-FISH experiments. Similar to the endogenous gcm locus, which associates with both Pc and Ph proteins (gcm construct including 9 kb from the promoter region (9 kb gcm) induces the recruitment of PcG proteins to an ectopic site (gcm) is not able to efficiently recruit PcG proteins , as transgenes carrying PREs/TREs in property . Transgeic lines .gcm promoter region contains a PRE and suggest that PcG proteins directly regulate gcm expression.Altogether, these data indicate that the Pc gcm interaction in a physiological asset, i.e., in loss of function conditions for both genes. The gcm-Gal4 line, an insertion in the gcm locus, is a hypomorphic semiviable allele in homozygous conditions and can be used to follow gcm activation and glial cells using a UAS-green fluorescent protein (GFP) line gcm-Gal4,UAS-GFP  animals and in homozygous gcm-Gal4 animals that are also heterozygous for Pc. As a control, we used heterozygous gcm-Gal4 animals.We next scored for Drosophila wing contains two major nerves, L1 and L3, covered by glia that depend on gcmThe gcm-Gal4 homozygous flies show the glia to neuron transformation observed in gcm null clones gcm-Gal4 homozygous animals, the GFP+ cell expresses Elav rather than Repo (9% penetrance) (etrance) . By 24 hetrance) . The penetrance) , indicatPc downregulation rescues the phenotype of homozygous gcm-Gal4 wings. Indeed, no evidence of stable glia to neuron transformation was found in homozygous gcm-Gal4 wings that carry only one Pc functional allele  also produces a significant value . These data indicate a partial rescue of the gcm-Gal4 proliferation phenotype by Pc, the moderate difference likely depending on the fact that only one dose of Pc is deleted.In order to extend the above findings, we analyzed late gliogenesis upon lowering the dose of Pc. Differentiated me stage . Moreovel4 wings , confirmPc mutant animals in an otherwise wt background and asked whether the mutation affects the number of glia , a stronger, significant, increase is observed in E(z)/+ wings , which have a compromised PRC2, and an even stronger phenotype is observed in double heterozygous Pc/E(z) animals , which display compromised PRC2 and PRC1 (E(z)/+ or Pc/E(z) double heterozygous animals, which show the most significant increase in glial cell number, show a significant increase in the number of wings with proliferating glia, whereas Pc/+ animals, in which the increase in glial cell number very small, do not. Thus, PcG proteins likely synergize and affect both glial differentiation and proliferation.To understand the role of Pc in gliogenesis, we also analyzed  of glia . Since rand PRC1 . Finallyand PRC1 . E(z)/+ gcm expression profile. Positional cues first trigger initiation of transcription, then Gcm positively autoregulates gcm expression progressively decreases so that its transcripts are no longer present in mature glia gcm transcription in gcm-Gal4/+; Pc/+ wings. Previous analyses showed that the gcm RNA becomes detectable by 8–9 hr APF . We therefore analyzed 7–8 hr APF wings and found that the GFP appears at the same time as in wt animals (data not shown). Since the binary Gal4 system may not faithfully reproduce the temporal pattern, we analyzed wings carrying one dose of Pc and the P-mediated insertional rA87gcm allele expressing the LacZ reporter and confirmed that the β-Gal labeling starts as in wt animals , which produce numerous cells that migrate and form the glia of the lamina visual ganglion  gcm. The gcm2 2 kb proximal promoter contains four GBSs and was previously shown to be activated by Gcm in transfection assays gcm 2 kb promoter itself, which only contains one GBS. As for repo, the cotransfection with Gcm and Pc reduces the activation of the gcm2 promoter. Thus, Pc represses the expression of Gcm stably and transiently expressed targets.We first analyzed the Pc represses gcm autoregulation and Gcm downstream targets, thereby inhibiting glial development.In sum, the above data support the hypothesis that Drosophila model unveil the role of the Polycomb chromatin modifier in the generation of glial cells upon fine modulation of the transiently expressed fate determinant gcm.Cell fate determination and maintenance require pathways that finely modulate gene expression and hence ensure the proper balance of cell types in metazoa. The pleiotropic and genome-wide effects of such pathways still hamper clear understanding of their impact and mode of action at single cell level. Our screen and genetic analyses in the Pyxgcm phenotype. The screen also provided hints onto the function of the interactors, suppressors or enhancers of a given phenotype. For example, sna and esg act as Pyxgcm suppressors, in line with the fact that Pyxgcm triggers the expression of NB-specific genes Pc, in the second are osa and Ash1 (Notch (N), which controls gcm expression N itself, we identified Suppressor of Hairless (Su(H)), which regulates the transcription of the N targets, and Lethal (2) giant disc 1, which negatively regulates N receptor trafficking  and negative (Pc) factors in the establishment of the glial fate is also provided by a rare phenotype observed in a gcm-Gal4; Pc/+ background (1/17 wings) in which the GFP+ cell expresses Repo and Elav, indicating an intermediate glial/neuronal state , see Supplementary Material and Methods.Pyxgcm allele and a deficiency were scored for the supernumerary bristle phenotype and compared to sibling females carrying the Pyxgcm allele and the balancer from the deficiency stock. This allowed us to classify each deficiency as Pyxgcm modifier or not modifier , double heterozygous females carrying the modifier a. 75 defmodifier b; for eaesg or osa, respectively, w; EP(2)0684/CyO or w; P{w[+mC] = UAS-osa}s2/CyO females were crossed with w; Pyx/Spgcm; hs-Gal4/Sb males. A 30 minute heat-shock pulse on 2nd instar larvae was performed at 37°C.To overexpress qChIP was performed as in gcm TSS were used, see these assays were performed as in gcm2 promoter construct is pBLCAT6-1.96 from repo promoter pPAC-gcm is described in UAS-gcm is described in pPAC-Pc and UAS-Pc were obtained by cloning the entire Pc cDNA in backbone vectors. pPAC-lacZ was a gift from T. Cook. Transient transfection of Drosophila S2 cells gcm2 reporter construct (pBLCAT6-1.96), cells were harvested 48 hr after transfection and normalized for ß-Gal activity. CAT levels were determined using the CAT ELISA kit (Roche). For repoRFP, images of cells were acquired 48 hr after transfection, and the green (UAS-GFP)/red (repoRFP) cells, were quantified automatically using the ImageJ software.The Figure S1Pyxgcm modifiers and validation of candidate genes. (A) Flow-chart of the screen: Pyx/CyOgcm, twist-LacZ flies were crossed to Bloomington Deficiency kit strains. The bristle phenotype was compared between sibs: control  and experimental females (Pyxgcm/Df). The screen was performed in three steps  and followed by gene validation. The number of analyzed deficiencies and the quantitative data are presented. Bal = balancer, Dfs = Deficiencies. (B) Primary quantitative screen deficiencies summary. Top: chromosome arms, names and cytology of deficiencies selected as strong modifiers of the bristle phenotype: suppressors (S) and enhancers (E). Bottom: total number of modifier deficiencies on each chromosome arm, number of suppressor and enhancer deficiencies. (C) Histograms present the average number of bristles per heminotum (y-axis) in different genotypes (x-axis). Large (Df(2L)TE35BC-24) and small Df(2L)ED1050) deficiencies cover esg, wor and sna genes. Pyx/esg GOF stands for Pyxgcm/EP(2)0684; hs-Gal4. Phenotype observed upon heat shocking Pyxgcm animals that carry the hs-Gal4 driver and the EP(2)0684 insertion expressing esg in response to Gal4 induction. Note that both deficiencies eliminate wor, esg and sna, but only the large one covers the Su(H) mutation, which acts as suppressor. This may explain why the large deficiency seems to act as a stronger suppressor. (C) Deficiencies deleting pim and crol genes as well as their single mutations. In each graph, average values are indicated +/− SEM (bars); P-values from t-test are indicated in the following way: *** (P≤10−3), ** (P≤10−2), * (P≤5×10−2). P-values vs. Pyxgcm/+: Pyxgcm/Df(2L)TE35BC-24 ; Pyxgcm/Df(2L)ED1050 ; Pyxgcm/esg ; Pyxgcm/sna ; Pyx/esg GOF . Pyxgcm/Df(2L)J2 ; Pyxgcm/pim (5×10−5); Pyxgcm/Df(2L)esc-P2-0 ; Pyxgcm/crol .Genetic screen for (TIF)Click here for additional data file.Figure S2Pyxgcm. From left to right, columns indicate the name of the gene, the cytology, the heterozygous phenotype over Pyxgcm , the phenotype of the large deficiency over Pyxgcm (nt – the gene region is not covered by the tested deficiencies), the function of the gene, previous identification as a Gcm target/regulator, references. TF = transcription factor, TrxG = Trithorax group, PcG = Polycomb group; JAK-STAT = Janus kinase/Signal Transducer and Activator of Transcription.Summary of the genes analyzed over (PDF)Click here for additional data file.Figure S3gcm) on polytene chromosomes carrying a transgene including a 2 kb region upstream of the gcm transcription start site.The Pc binding region and the polytene chromosomes of the 2 kb transgenic line. (A) List of primers used for immuno-FISH and qCHIP. (B) Immuno-FISH staining Click here for additional data file.Figure S4Pc/+ wings or in Pc overexpressing wings, using two different Gal4 drivers. (E) Quantitative analysis of gcm expression in the optic lobe ; brm/+; brm,trx/+.Mutant phenotypes in wings and brains. (A) Percentage of 24 hr APF wings of the described genotypes carrying different numbers of Repo+ cells on the L3 vein.  Summary tables showing the number of Repo+ cells observed on the L3 (B) and L1 veins (C) in each pupal wing analyzed. The observed minimum and maximum value in samples of the different genotypes are respectively highlighted in pale-blue and red. (D) Number of wings scored for the fate transformation phenotype in heterozygous lobe see : histogr(TIF)Click here for additional data file.Figure S5gcm expression in Pc/+ wings. (A–G) Immunolabeling of 7 hr APF wings from the P-mediated insertional rA87gcm allele expressing the LacZ reporter, anterior to the top, distal to the right. By this stage, the β-Gal labeling is still not present onto the L1 vein in most of the wings (11/12); in one wing (A–D), one β-Gal labeled cell is visible at the distal tip  in green). This cell (arrow) is close to a neuron (arrowhead), (D) shows a magnification from the boxed region. L1 and L3 indicate the position of the L1 and L3 veins, respectively. In wings heterozygous for Pc (n = 13) (E–G), no precocious β-Gal labeling was observed on the L1 vein.  In situ hybridization with a gcm-specific probe on 29 h APF wings from wt (H) and from Pc/+ (I) animals. Note that, in both backgrounds, gcm is no more expressed.Initiation and extinction of (TIF)Click here for additional data file.Figure S6Pc/Pc (B), scabrous-Gal4/UAS-Pc animals (C).Repo expression in wild type, Pc LOF and GOF. Immunolabeling to show Repo protein in st. 14 embryos. Ventrolateral view in wild type (A), (TIF)Click here for additional data file.Figure S7repo promoter. (A) Levels of Pc binding and H3K27me3 at the repo locus in Drosophila embryos were determined by quantitative ChIP (qChIP) experiments, the bxd locus was used as a positive control. Results are represented as percentage of input chromatin precipitated. The standard deviation was calculated from two independent experiments. (B) ChIP-on-chip binding profiles of indicated PcG proteins and histone marks in Drosophila embryos at the repo regulatory region obtained as reported by −4) are shown in red. Black bars indicate the location of primers used for qChIP analysis. The graph (C) shows the activation of a reporter construct carrying 4 kb from the repo upstream regulatory sequence displaying eleven GBSs (D). The ratio between reporter activity upon Gcm/Pc coexpression and that observed when only Gcm is expressed indicates that the repo promoter is activated when Gcm is expressed in S2 cells and repressed upon Gcm and Pc coexpression.Pc binds to and acts on the (TIF)Click here for additional data file.Figure S8gcm/gcm2 and bx/bxd PREs. ChIP-on-chip binding profiles of indicated PcG proteins and histone marks in Drosophila S2 cells from  or Drosophila embryos from . Nomenclature as in Comparison between (TIF)Click here for additional data file.Text S1Supplementary materials and methods.(DOC)Click here for additional data file.


Congenital generalized lipodystrophy (CGL) or Berardinelli–Seip congenital lipodystrophy (BSCL) is a rare genetic syndrome characterized by the absence of adipose tissue. As CGL is thought to be related to malfunctions in adipocyte development, genes involved in the mechanisms of adipocyte biology and maintenance or differentiation of adipocytes, especially transcription factors are candidates. Several genes (BSCL1-4) were found to be associated to the syndrome but not all CGL patients carry mutations in these genes.In vitro analyses revealed that the mutation facilitates the formation of a novel and specific protein/DNA complex. Using mass spectrometry we identified the proteins of this novel complex. Cellular investigations demonstrate that the wild type c-fos promoter can reconstitute the signaling defect in the patient, excluding further upstream signaling alterations, and vice versa the investigations with the c-fos promoter containing the identified mutation generally reduce basal and inducible c-fos transcription activity. As a consequence of the identified point mutation gene expression including c-Fos targeted genes is significantly altered, shown exemplified in cells of the patient.In a patient with CGL and insulin resistance we investigated the known candidate genes but the patient did not carry a relevant mutation. Analyses of the insulin activated signal transduction pathways in isolated fibroblasts of the patient revealed a postreceptor defect altering expression of the immediate early gene c-fos. Sequence analyses revealed a novel homozygous point mutation  in the patients’ c-fos promoter. The point mutation was located upstream of the well characterized promoter elements in a region with no homology to any known cis-elements. The identified mutation was not detected in a total of n=319 non lipodystrophic probands. The immediate-early gene c-fos is one essential transcription factor to initiate adipocyte differentiation. According to the role of c-fos in adipocyte differentiation our findings of a mutation that initiates a repression mechanism at c-fos promoter features the hypothesis that diminished c-fos expression might play a role in CGL by interfering with adipocyte development. Lipodystrophy can be acquired or inherited and results in partially or complete loss of adipose tissue. In the most severe form, congenital generalized lipodystrophy (CGL) or Berardinelli–Seip congenital lipodystrophy (BSCL), total absence of adipose tissue is associated with altered development, fatty liver, muscular hypertrophy, hypertriglyceridemia, acanthosis nigricans, hyperinsulinism and type-2-diabetes ,2.CGL is rare with estimated 1:10 million births and thought to be a genetic syndrome with autosomal recessive trait . In humaLipodystrophy resemble syndromes of disturbed adipocyte biology or metabolism but the severe congenital forms are thought to be related to malfunctions in adipocyte development ,5. ThereSince a mutation in the known BSCL genes have not been found a patient with CGL and insulin resistance we examined insulin signaling as central metabolic signal transduction pathways and show the identification of a homozygous point mutation in the c-fos promoter  in this patient. This mutation causes a novel protein/DNA complex which ubiquitously lowers basal and inducible c-fos promoter activity. According to the role of c-fos in adipocyte differentiation our investigations provoke the hypothesis that diminished c-fos expression interferes with adipocyte development and might play a role in CGL.-7 M insulin, 10-8 M IGF-1, 1.5×10-8 EGF or 3.3×10-9 M PDGF. Preadipocytes (3T3-L1) and muscle cells (A7r5) were cultured in DMEM and liver cells (HepG2) in RPMI 1640 (Life Technologies) supplemented with 10% FCS. Institutional research ethics approval (PV3641) in line with the Helsinki Declaration was obtained for this study.Fibroblasts initiated from skin biopsies of patient and healthy caucasian volunteer controls were expanded for 4 cycles and stored in liquid nitrogen. Cells were recultured  and expanded for a maximum of 3 passages before harvesting. For exogenous stimulation fibroblasts were grown to 70% confluence and serum starved (1% FCS) for 40 h  prior to induction with 10Nuclear extracts from fibroblasts of the patient and controls, or HepG2 cells were prepared as described .MAPK activity assays and western blotting with polyclonal anti-Akt or anti-phospho Akt antibody  or polyclonal anti-MAPK antibody  were performed as described .Total RNA was extracted with RNeasy Mini Spin Kit . RT- PCR analyses were performed in triplicates with c-fos gene-specific probes and 18S RNA internal standard  as described . ExpressC-fos promoter  according to K00650) was PCR amplified from genomic DNA of controls and patient using following primers: -734 to −712: 5’-GCGAGGAACAGTGCTAGTATTGCT-3’/ +43 to +12: 5’-CGGCTCAGTCTTGGCTTCTCAGTTGCTCGCT-3’. Wild type c-fos promoter fragment and the corresponding fragment of the patient were inserted in sense orientation into pGL3basic vector  to bring the reporter gene luciferase under control of wild type c-fos promoter (pc-fos-wt) or the mutated promoter (pc-fos -439T>A). The expression vector pFA-Elk-1 containing the regulative domains of transcription factor Elk-1 (aa 307 to 427) fused to the heterologous DNA binding domains of Gal4 (aa 1 to 147) under control of a MLV-promoter was used (Life Technologies). For monitoring transactivation the reporter plasmid pGal4-Luc5 containing the luciferase gene under control of 5× Gal4 binding elements was cotransfected in these experiments (Life Technologies). As reference of transfection efficiency the β-galactosidase expression vector pEF-ßGal was used. Independent plasmid preparations and cells were used for replicate experiments.5 cells/well) of 3T3-L1, A7r5, HepG2 cells or fibroblasts of patient and control were mixed with vectors as indicated in figure legends and pulsed for 18 msec  or 9 msec (fibroblasts). For exogenous induction, cells were serum-starved on day one following transfection for 40 h and incubated with 10-7M insulin or 3.3×10-9M PDGF for 3 h before harvesting. Transfection, monitoring of transfection efficiency and luciferase assays were performed as described dGTP using 5U Klenow fragment, prior to use. For completion experiments 100- or 10-fold molar excess of unlabeled sre fragments or unspecific SP-1 promoter fragment (5’-GTTAGGGGCGGGATGGGCGGAGTT -3’) were used.EMSA were performed according to  and reacATCTGCGCCG-3’) endlabeled with 5U PNK and 20 μCi [γ32P]dATP, prior to use. For competition experiments 10× or 100× unlabeled c-fos-wt or pc-fos-patient fragment were used. Experiments were performed with nuclear extracts (5 μg) of human liver cells (HepG2).Analyses of the novel protein/DNA interaction at the c-fos promoter were performed with c-fos-wt (−451 to −431: 5’-TGTTCTCTCTCATTCTGCGCCG-3’) or c-fos-patient  and fluorescence marked bands were cut from gels. The gel slices were placed on a 10% SDS-PAGE for separation of complex proteins. Four independent EMSA replicates were performed and cutted protein/DNA complex were separated on four SDS-PAGE each. Of all protein bands three different punch samples were excised and subjected to mass spectrometry analyses according to  for idenhttp://www.affymetrix.com/support/Technical/manual/expression_manual.affx) and used for expression analyses with Hu95A_v2 Arrays (Affymetrix UK Ltd). Syntheses of cRNA and fragmentation were quality controlled and monitored with a RNA 6000 nano kit . Detection of probe sets was performed using a GeneChip scanner . The original CEL files were directly implemented into Genespring 12.0 (Agilent) for analyses. The Genespring 12.0 Volcano Plot analyses workflow with default settings  of the gene expression data sets were used to identify genes with statistic significant expression (p < 0.05) and a minimum 1.5-fold difference among conditions. Full data sets are available under accession number GSE39825 (http://www.ncbi.nlm.nih.gov/geo/).Fibroblasts of patient and 6 individual controls were cultured to passage 6 each. Four replicate analyses of patient cells (initiated from two primary stored cell pools) and 6 individual controls (initiated from one primary stored cell pool each) were used. Equal amounts of total RNA were processed according to the GeneChip One-Cycle eukaryotic Target Labeling Assay  [http://www.ingenuity.com). IPA was carried out with p < 0.002 as cutoff point. Pathways indicating altered transcriptional regulation were deduced from fold change differences observed.For functional annotation and conserved promoter element site search web based tools were used (rf.gov/) ,16. For Data are given as means ± S.D. Students t-test was used to determine statistical significance.acanthosis nigricans was observed, being a hint for altered insulin signaling. Until the age of five years prediabetes, progressive hepatomegaly and lipoatrophy appeared with complete loss of adipose tissue. Physical examination revealed generalized decreased subcutaneous adipose tissue, distended abdomen with enlarged palpable liver and growth retardation from 10% (1 year) to 75% (7 years) of normal range. The patient had the typical appearance of congenital generalized lipodystrophy including hypertrichiosis, hepatomegaly, splenomegaly, but no mental retardation. The patient had marked muscularity probably due to missing subcutaneous adipose tissue. The main known candidate genes associated with congenital generalized lipodystrophy, i.e. BSCL1/AGPAT2, BSCL2/seipin, BSCL3/caveolin-1 and BSCL4/PRTF were analyzed. No sequence alteration specific for the lipodystrophic phenotype was identified (data not shown). At age of five years laboratory analyses showed elevated plasma cholesterol levels (450 mg/dl) and modestly elevated triglycerides (218 mg/dl). Glucose intolerance detected by oGTT showed an increase of blood glucose levels from 103 mg/dl  up to 176 mg/dl  and plasma insulin levels from 96 mU/l to 276 mU/l, respectively, indicating insulin resistance. The patient died at the age of eight during a hyper acute varicella infection as primary cause of death. An autopsy was not performed.The female caucasian patient was born at term with reduced birth weight . The parents were healthy, not consanguineous and gave birth to four further healthy children. At age of one year the patient retrieved to thrive and beginning lipodystrophy was diagnosed. Pronounced As the patient was the only known case of lipodystrophy in the family nothing remained but analyzing a possible defect in insulin signaling, we characterized known signaling pathways. For this purpose we utilized primary fibroblasts initiated from skin biopsies.in vitro insulin receptor binding capacity, auto- and substrate-phosphorylation was in normal range (data not shown). The insulin mediated signaling cascade including Akt abundance or phosphorylation  a novel homozygous point mutation in the promoter at position c.–439 Figure A upstrean Figure B.One functional possibility of a point mutation in a promoter is the direct interference with protein/DNA interactions. Utilizing the c-fos promoter in nuclease protection assays revealed novel protein binding sites P1-P3) only with DNA-fragments bearing the identified point mutation in P2 . Expression data analyses utilizing standard algorithmus was performed with Genespring 12.0 to identify genes with statistic significant expression (p< 0.05) and a minimum 1.5- fold difference. For consensus site prediction web based tools as Click here for filehttp://david.abcc.ncifcrf.gov/ were used.GO annotation of regulated transcripts. Transcripts are listed in identical order as in Additional file Click here for file


Trachemys scripta), a species with temperature-dependent sex determination (TSD), the expression of the aromatase gene during gonad development is strictly limited to the female-producing temperature. The underlying mechanism remains unknown. In this study, we identified the upstream 5′-flanking region of the aromatase gene, gonad-specific promoter, and the temperature-dependent DNA methylation signatures during gonad development in the red-eared slider turtle. The 5′-flanking region of the slider aromatase exhibited sequence similarities to the aromatase genes of the American alligator, chicken, quail, and zebra finch. A putative TATA box was located 31 bp upstream of the gonad-specific transcription start site. DNA methylation at the CpG sites between the putative binding sites of the fork head domain factor (FOX) and vertebrate steroidogenic factor 1 (SF1) and adjacent TATA box in the promoter region were significantly lower in embryonic gonads at the female-producing temperature compared the male-producing temperature. A shift from male- to female-, but not from female- to male-, producing temperature changed the level of DNA methylation in gonads. Taken together these results indicate that the temperature, particularly female-producing temperature, allows demethylation at the specific CpG sites of the promoter region which leads the temperature-specific expression of aromatase during gonad development.In the red-eared slider turtle ( Elevated expression of the aromatase gene is consistently observed in differentiating ovaries among taxa while its expression is usually suppressed during development of testes Temperature-dependent sex determination (TSD) is an example of developmental plasticity where the ambient temperature of the embryos leads to the development of the bipotential gonads into either testes or ovaries. In TSD species such as the American alligator and some turtle species, gonadal development can be modified by the concentration of sex steroid hormones in the surrounding system of the embryos. For example, an excess of either testosterone or estrogen during gonadal differentiation leads to testicular or ovarian development, respectively, regardless of the surrounding temperature Trachemys scripta) the level of aromatase expression in gonads depends on the ambient temperature; high expression is exhibited at female-producing temperature  while low expression is observed at male-producing temperature  In the red-eared slider turtle (Accumulating evidence suggests that epigenetic modification such as histone modification and DNA methylation mediate specific gene expression changes in response to the environment during development Alligator mississippiensis), chicken , quail (Coturnix coturnix), and zebra finch (Taeniopygia guttata). These species were selected because of their sequence similarities in the aromatase 5′-flanking region to the slider aromatase according to a nucleotide BLAST search . Homology analysis showed that the 5′-flanking sequence was particularly conserved the region within the approximately 800 bp upstream of the ATG , which was previously identified in the red-eared slider turtle  the ATG . The Ame the ATG .The gonad-specific transcription start site (TSS) of aromatase was identified on mRNAs that were purified from embryonic gonads at stage 16, 19, and 21 at FPT. During these stages, the expression of aromatase exhibits a rapid increase in the embryonic gonads that is specific to FPT Next, we identified the putative transcription factor binding sites (TFBSs) on the total 4113 bp of the 5′-flanking region of aromatase sequence. Previous studies show that the following transcription factors have conserved roles in vertebrate sex determination and exhibit temperature-specific expression patterns in slider gonads during TSP: steroidogenic factor 1 (Sf1), estrogen receptor (ER), forkhead box protein L2 (FoxL2), doublesex and mab-3 related transcription factor 1 (Dmrt1), and SRY-box 9 (Sox9) We also compared the pattern of TFBSs within the homologous sequence region among species . ApproxiDNA methylation level at the regulatory regions often regulates the temporal and spatial expression of development-related genes Second, we constructed Arom Set5 primers to amplify the promoter region containing 7 CpG sites that are potential targets for DNA methylation  and examSince DNA methylation patterns are often correlated to the particular CpG sites within or near TFBSs that, in return, affect the binding of transcription factors Aromatase is an essential factor for gonad determination and differentiation in non-mammalian vertebrates. Most studies to date have been limited to the gene expression profiles in differentiating gonads. In this study, we identified for the first time in TSD species the temperature-dependent epigenetic signatures within the gonad-specific promoter of the aromatase gene. In most mammals, birds, and amphibians, the aromatase gene has multiple TSS driven by tissue-specific promoters producing unique 5′-untranslated regions that are alternatively spliced along with a common coding region cis-regulatory mechanisms of aromatase gene expression. We found several SF1 sites located within the 500 bp upstream region of the translation start codon  and maintained in accordance with humane animal practices under IACUC protocol. The protocol was approved by IACUC (protocol # AUP-2011-00149) at the University of Texas at Austin. Eggs were stored at room temperature for 10 days to assess viability of the embryos and randomly assigned to incubators at either MPT (26°C) or FPT (31°C) with moistened vermiculite. Incubator temperatures were monitored daily with thermometers and HOBO data loggers . Eggs were incubated until Greenbaum's embryonic stage 16 2) for subsequent assays. Ten eggs were continuously kept at MPT or FPT while ten other eggs were shifted to the opposite temperature regime at stage 16. At stage 19 and 21, gonads (n = 10) from each treatment group were pooled, snap-frozen in LN2, and stored at −80°C until bisulfite treatment. These groups were MPT, FPT, MPT shifted to FPT (MPT→FPT), or FPT shifted to MPT (FPT→MPT). For the cloning of the 5′-flanking region of aromatase, tissues including the adrenal-kidney complex, liver, or head were separately collected from FPT hatchling, snap-frozen in LN2, and stored at −80°C for genomic DNA (gDNA) extraction.For the methylation assay, gonads (n = 10 gonads/temperature) at each temperature (MPT or FPT) were pooled at stage 16 as the base line control and snap-frozen in liquid nitrogen  containing 10 µg of RNase A at 37°C for 1 hr. Proteinase K (50 µg) was added to the lysate for an overnight incubation at 55°C. gDNA was extracted by the conventional phenol-chloroform method and precipitated by ethanol (EtOH). The purity of the gDNA was confirmed by the presence of smear on a 0.6% agarose gel after a digestion with restriction enzyme DraI. gDNA extracted from the head had the most distinct smear after DraI digestion, therefore was utilized for subsequent assays.The 5′-flanking region upstream of the ATG start codon of the aromatase gene was cloned using the Genome Walker Universal Kit  according to the manufacturer's protocol. Briefly, frozen turtle tissues at hatching were grinded in LNTM Adaptor. Two reverse primers, the aromatase gene specific primer (Arom GSP) 1 and Arom GSP2, were designed based on the known slider aromatase sequence of the first exon (GenBank accession no. AF178949). Primer sequences and locations are indicated in 2O to perform the second round of nested PCR using Arom GSP2 and ASP2 primers under the following PCR conditions: 5 cycles of 25 sec at 94°C and 3 min at 72°C, 20 cycles of 25 sec at 94°C, 3 min at 67°C, and 7 min at 67°C. The second PCR products were visualized on a 1.5% agarose gel, extracted using a gel extraction kit , and cloned in a pGEM-T vector  for sequencing. Each library produced different sizes of PCR products (data not shown) that were subsequently aligned for the identification of the novel 5′-flanking region sequence of aromatase. Additional Arom GPS reverse primers (sequences not shown) were designed based on this 5′-flanking region to repeat the PCR and sub-cloning processes on the existing gDNA libraries until approximately 4 kb of the newly identified 5′-flanking region was identified and sequenced. A whole 5′-flanking region was generated by PCR and eight clones were aligned for the best sequence prediction. The sequenced data was submitted to GenBank (accession no. KC554066). The TFBSs for the vertebrate steroidogenic factor 1 (SF1), estrogen response elements (ERE), fork head domain factors (FOX), DM domain-containing transcription factor (DM), sox/SRY-sex/testis determining and related HMG box factors (SOX), and heat shock factor (HEAT) on the 5′-flanking region of aromatase were detected with MatInspector . The CpG islands within the 5′-flanking region were examined using CpG Plot .To construct libraries, the gDNA was digested with restriction enzymes, DraI, EcoRV, PvuII, or StuI to produce four libraries. As a positive control, human gDNA supplied with the kit was digested with Pvu II. Digested gDNA was purified per the phenol-chloroform method and ligated with the Genome Walkeralligator (AY029233), chicken (D50335), quail (D50336), and zebra finch (AH008871) using PIPMaker Approximately 4 kb of the newly identified 5′-flanking region of the slider aromatase sequence was compared with the 5′ upstream region of known aromatase sequence in other species (GenBank accession no.) including the TTA ATA CGA CTC ACT ATA GGG CCC AGA ATT AAC CCT CAC TAA AGG GA) and the reverse compliment sequence of DiPromF, RCDPromR reverse primer were designed. One microgram of each primer were incubated together with 10X T4 DNA ligase buffer (NEB) at 95°C for 5 min and placed at 65°C heat block which was then left at room temperature to naturally cool to the ambient temperature (approximately 26°C) for the slow annealing process.To identify gonad-specific TSS in the 5′-flanking region of aromatase, we conducted a modified RNA ligase-mediated rapid amplification of cDNA ends (RLM-5′RACE) described by Bohm et al. in vitro-transcribed to RNA oligo using mMESSAGE mMACHINE T7 kit (Life technologies) according to the manufacturer's protocol. The final concentration of RNA oligo after T7 transcription was approximately 50 µg. The 10 µM of the RNA oligo adaptor was ligated with decapped RNA using 5 units of T4 RNA ligase (NEB) at 37°C for 1.5 hrs. Ligated products were reverse transcribed with 200 units of SuperScript III reverse transcriptase (Life technologies) using 2 pmol of Arom GSP5 reverse primer  of the slider aromatase gene was examined using EZ DNA Methylation-Direct kit . Ten pooled gonads taken from embryos at stage 16, 19, and 21 were incubated at MPT, FPT, MPT→FPT, and FPT→MPT. They were treated with Proteinase K and bisulfite and purified according to the manufacture's protocol. Purified gDNA were amplified using Advantage 2 Polymerase Mix with Arom Set5F primer, 5′- GGY GTT AGG TTT GAT GAA TTT ATG GTT TGG-3′ and Arom Set5R primer, 5′- ACA CCT TTA ATC RAT The ratio of methylated to unmethylated CpG at each position were compared between the treatment groups  by Fisher's exact test using R . A p-value of less than 0.05 was considered to be statistically significant.Figure S1Nucleotide position of gonad-specific transcription start sites (TSSs) of aromatase in the red-eared slider turtle. TSSs were examined in total RNA from pooled gonads at embryonic stage 16, 19, and 21 at FPT (n = 6/stage) using (A) RNA ligase-mediated rapid amplification of cDNA ends (RLM-5′RACE) and (B) A conventional 5′RACE using SMARTTM RACE cDNA Amplification kit (Clontech). X-axis represents the distance in base-pairs from the translation start codon (ATG), and the y-axis represents the number of clones examined.(TIF)Click here for additional data file.Figure S2Comparative analysis of the transcription factor binding sites (TFBSs) of the aromatase gene among species. TFBSs at approximately 700 bp of the 5′-flanking regions including the translation initiation codon (ATG) and transcription start site (TSS) of the red-eared slider turtle and four other species were predicted using the MatInspector (Genomatix) and aligned relative to the gonad- or brain-specific TSSs. GenBank accession numbers of these species are as follows: alligator (AY029233), chicken (D50335), quail (D50336), and zebra finch (AH008871). Dashed lines indicate missing sequence information of the alligator aromatase in the GenBank. Colored marks represent TFBSs with a 5′ to 3′ (above the line) or a 3′ to 5′ (below the line) orientation relative to the TSS. Color codes for the transcription factors are as follows: pink: SF1 (vertebrate steroidogenic factor 1), light green: ERE (estrogen response elements), ocher: FOX (fork head domain factors), dark green: DM (DM domain-containing transcription factor), purple: SOX (Sox/SRY-sex/testis determining and related HMG box factors), and dark blue: HEAT (heat shock factors). TATA: TATA box. Horizontal black arrow: TSS. Reverse triangle: ATG. Solid vertical arrows: conserved SF1/ERE and Sox binding sites. Dashed vertical arrows: conserved SOX binding sites.(TIF)Click here for additional data file.Figure S3Relative positions of the designed primers in . Black line: known aromatase cDNA sequence of the red-eared slider turtle (GenBank accession no. AF178949). Orange line: unknown 5′-flanking region. Black arrows: aromatase gene-specific reverse primer (GSP) positions. A translation start codon, ATG was previously identified in the red-eared slider turtle (TIF)Click here for additional data file.


MEN1 gene predisposing to the multiple endocrine neoplasia type 1 (MEN1) syndrome can also cause sporadic pancreatic endocrine tumors. MEN1 encodes menin, a subunit of MLL1/MLL2-containing histone methyltransferase complexes that trimethylate histone H3 at lysine 4 (H3K4me3). The importance of menin-dependent H3K4me3 in normal and transformed pancreatic endocrine cells is unclear. To study the role of menin-dependent H3K4me3, we performed in vitro differentiation of wild-type as well as menin-null mouse embryonic stem cells (mESCs) into pancreatic islet-like endocrine cells (PILECs). Gene expression analysis and genome-wide H3K4me3 ChIP-Seq profiling in wild-type and menin-null mESCs and PILECs revealed menin-dependent H3K4me3 at the imprinted Dlk1-Meg3 locus in mESCs, and all four Hox loci in differentiated PILECs. Specific and significant loss of H3K4me3 and gene expression was observed for genes within the imprinted Dlk1-Meg3 locus in menin-null mESCs and the Hox loci in menin-null PILECs. Given that the reduced expression of genes within the DLK1-MEG3 locus and the HOX loci is associated with MEN1-like sporadic tumors, our data suggests a possible role for menin-dependent H3K4me3 at these genes in the initiation and progression of sporadic pancreatic endocrine tumors. Furthermore, our investigation also demonstrates that menin-null mESCs can be differentiated in vitro into islet-like endocrine cells, underscoring the utility of menin-null mESC-derived specialized cell types for genome-wide high-throughput studies.Inactivating mutations in the  MEN1 encoding menin, a component of histone methyltransferase complexes, in 27–44% of tumors DAXX or ATRX that encode subunits of a chromatin-remodeling complex Whole exome sequencing of different tumor types has identified mutations in various genes whose products are associated with epigenetic processes that are involved in chromatin modification Men1-knockout (Men1-ko) mouse embryos Men1 loss in mouse models driven by RIP-Cre, GLU-Cre, or PDX1-Cre show islet endocrine cell-type restricted tumorigenesis, implicating an essential role for menin in islet endocrine cell homeostasis Menin acts as a tumor suppressor in the autosomal dominant multiple endocrine neoplasia type 1 (MEN1) syndrome characterized by tumors of hormone producing cells of the parathyroids, enteropancreatic endocrine tissues, and anterior pituitary HOXA7, HOXA9, and the HOX cofactor MEIS1HOX genes and MEIS1Surprisingly, menin's association with MLL is pro-oncogenic in MLL-associated leukemia cells. About 50–60 different translocations involving the MLL1 gene are known to cause acute lymphoid and myeloid leukemias with increased expression of specific homeobox (HOX) genes such as Men1-ko mouse embryos have been established. ESCs can undergo multi-lineage differentiation in vitro producing specialized cell types retaining an intact normal diploid karyotype (unlike cell lines and tumors that are aneuploid) Men1-ko (menin-null) mouse ESCs (mESCs) cannot complete hematopoietic differentiation in vitro due to reduced Hoxa9 expression in vitro.The direct role of H3K4 trimethylation (H3K4me3) catalyzed by menin-containing MLL complexes in pancreatic islet endocrine cells is unclear, and the functional relevance of H3K4me3 catalyzed by the MLL1 and MLL2 complexes with or without menin in islet cells has not been elucidated. Pancreatic endocrine cells are found in the islets that comprise only ∼1% of the pancreas. Large quantities of live cells are generally required for most genome-wide occupancy analysis assays. Menin-null human islets are rarely available, which makes islets from MEN1 mouse models  an attractive alternative source. However, to obtain large quantities of live cells, one would need to isolate and pool islets from a large number of mice. Embryonic stem cells (ESCs) from in vitro differentiation of wild-type as well as menin-null mESCs into pancreatic islet-like endocrine cells (PILECs) in order to obtain a source of cells with a homogenous and diploid genetic background for global menin-dependent H3K4me3 and gene expression analyses. We used ChIP-Seq and microarray analysis to profile genome-wide H3K4me3 and gene expression, respectively, in wild-type and menin-null mESCs and PILECs. Specific and significant loss of menin-dependent H3K4me3 was observed at imprinted Dlk1-Meg3 locus in menin-null mESCs, and at all four Hox loci in menin-null PILECs. These H3K4me3 losses were accompanied by reductions in gene expression. Meg3  is an imprinted long non-coding RNA that acts as a tumor suppressor DLK1-MEG3 and HOX loci is associated with sporadic pituitary tumors and parathyroid tumors, respectively We performed Men1-ko (menin-null) mouse embryonic stem cells (mESCs) were deficient in completing hematopoietic differentiation in vitroin vitro differentiation of menin-null mESCs into pancreatic islet-like endocrine cells (PILECs). Menin-null mESCs used in our study showed no growth defects, and were not compromised for embryoid body (EB) formation .Histone methyltransferase complexes contain menin, specifically the MLL1- and MLL2-containing COMPASS-like MLL complexes that are known to deposit the histone H3 lysine 4 trimethyl mark (H3K4me3). Homozygous FigureS2, and TablesS2). Examination of H3K4me3 levels at genes that were at least 2-fold upregulated or 2-fold downregulated in menin-null mESCs compared to wt mESCs revealed that expression changes in menin-null vs wt mESCs cells did not accompany changes in H3K4me3 levels  in menin-null mESCs and menin-null PILECs, respectively (TableS3), suggesting that the role of menin-dependent H3K4me3 in mESCs is limited and specific. Since a vast majority of these regions were intergenic, we focused on those that were near genes. Among the most significant differentially H3K4 trimethylated regions between wt and menin-null mESCs were the promoters of imprinted Meg3 and Mest (mesoderm specific transcript) genes that experienced complete loss of H3K4me3 in menin-null mESCs compared to wt mESCs . Meg3 and Mest were expressed 14-fold and 6-fold less (p<0.005) in menin-null mESCs compared to wt mESCs.To determine the connection, if any, between the loss of menin-dependent H3K4me3 and transcription, we examined the microarray gene expression data in wt and menin-null mESCs. Since H3K4me3 is generally associated with active and poised gene promoters TableS1 and S3). Among them were five transcripts encoded from genes within the ∼300 Kb Dlk1-Meg3 locus  that had ∼4–13 fold reduced expression in menin-null mESCs compared to wt mESCs . The Hox clusters, each containing 9 to 11 genes, are located on chromosomes 6, 11, 15, and 2 in the mouse genome. The 39 HOX genes are a highly conserved family of transcription factors essential for body axis patterning, development and differentiation. Although HOX genes are essential during embryogenesis, their regulation and target genes are not well defined. HOX genes are epigenetically silenced in undifferentiated ESCs, and their activation is associated with differentiation. Hox genes in mESCs are known to be marked by bivalent histone modifications of active H3K4me3 and repressive H3K27me3 Hox loci in both wt and menin-null mESCs . RT-PCR assay confirmed the loss  of expression for many Hox genes , we found that menin loss results in a significant loss of H3K4me3 only at a limited number of loci in mESCs and PILECs, thus defining a specific role for menin in modulating gene expression at these loci (Dlk1-Meg3 region on mouse chromosome 12 (human chromosome 14q32) is an imprinted locus consisting of multiple maternally expressed noncoding RNA genes and paternally expressed protein-coding genes  of the endocrine tumor types seen in familial MEN1, except in sporadic pituitary tumors (only 1–5% with MEN1 mutation) Meg3 as a menin target gene provides insights into the role of menin-regulated genes as potential candidates for pituitary tumorigenesis. Meg3 is essential for embryonic development indicated by the premature lethality of Meg3 knockout mice; therefore, the role of Meg3 in tumor development could not be assessed in mouse models Meg3 silencing did not affect the viability or proliferation of menin-null mESCs, its silencing in mature differentiated cells in vivo upon menin loss or from other causes could contribute to tumorigenesis. Our data suggests that perhaps other menin-independent mechanisms, activated during the differentiation of mESCs into PILECs, induce the expression of transcripts from the Dlk1-Meg3 locus, which would mean that the Meg3 locus is essential for PILEC differentiation. Identification of such mechanisms could lead to a better understanding of Meg3's role in early differentiation and in tumorigenesis.The HOX gene expression in mature adult cells is essential for the maintenance of cellular identity; for instance, in cells with high turnover such as in the proliferation and differentiation of blood cells. Abnormal HOX gene expression has been observed not only in acute leukemias but has also been associated with oncogenesis in breast, cervical, lung, ovarian, prostate, and thyroid cancers HOX gene activation; without menin, specific HOX gene expression is reduced thus preventing leukemogenesis HOX genes by an earlier study showed upregulation of 23 HOX genes among familial MEN1 parathyroid tumors with biallelic MEN1 loss, and downregulation of 5 HOX genes among sporadic parathyroid tumors without MEN1 loss HOX genes was noted Hox gene expression is not essential for differentiation of mESCs into islet endocrine lineage in vitro, it could be causative for endocrine tumorigenesis in mature cells. However, the role of HOX downregulation in pancreatic endocrine tumors from menin loss or from other causes needs to be determined.HOX and DLK1-MEG3 genes) during the initiation and progression of tumors found in MEN1 or MEN1-like sporadic endocrine tumors will be instrumental in correlating these events as biomarkers and/or causes of endocrine neoplasia.Further investigations analyzing the regulation of menin targets , rabbit anti-menin , rabbit anti-NeuroD1 , rabbit anti-Oct3/4 , mouse anti-Tubulin , normal rabbit IgG , HRP-conjugated rabbit and mouse secondary antibodies .Men1-ko (menin-null) mESCs (3.2N) Wild-type mESCs (TC-1) and 6 mESCs in each 100 mm dish.Wild-type mESCs and menin-null mESCs were differentiated into pancreatic islet-like endocrine cells (PILECs) Table S5.Total RNA was isolated using Trizol (Invitrogen) and further purified using the RNeasy kit (Qiagen) from 2 independent cultures of wt or menin-null mESCs and 2 independent cultures of differentiated wt or menin-null PILECs. RNA quality was assessed on the Agilent Bioanalyzer. Each sample was processed and analyzed at the NIDDK microarray core facility using an Affymetrix microarray platform. Labeled RNA samples were hybridized to Affymetrix Genechip mouse genome 430, 2.0 array. Microarray data were normalized and analyzed using the Affymetrix Genechip software, Microarray Analysis Suite 5.0. For RT-PCR, DNase I (Ambion) treated RNA samples were reverse transcribed using oligo-dT and SuperScript III (Invitrogen), and the first strand cDNA was used for PCR in standard PCR reactions with Taq Gold (Applied Biosystems). PCR products were analyzed by agarose gel electrophoresis. The primers sequences are listed in Whole cell protein extract (WCE) from wt or menin-null mESCs, and wt or menin-null mESCs differentiated into PILECs was prepared in buffer containing 1X TBS, 0.1% Igepal, and protease inhibitors (Roche). Protein concentrations were determined with a detergent-compatible protein assay (Bio-Rad). Equal amount of proteins (50 µg) were separated by SDS-PAGE, electro-blotted onto nitrocellulose membranes, and detected with appropriate antibodies and ECL (Millipore).mESCs differentiated into PILECs in gelatinized chamber slides were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, and stained with pro-insulin C-peptide mouse monoclonal antibody  and anti-mouse secondary antibody conjugated to Texas Red (Jackson ImmunoResearch Laboratories). Microscopy and photomicrography was performed with an epifluorescence microscope (Zeiss).6 mESCs undifferentiated or differentiated was used for each ChIP with 5 µg antibody . About 20 ng of DNA was obtained from each H3K4me3 ChIP of chromatin from 2×106 cells. ChIP DNA was used for ChIP-PCR assays or for preparation of ChIP-Seq libraries. ChIP-PCR was performed in standard reaction conditions with Taq Gold (Applied Biosystems), and the products were analyzed by agarose gel electrophoresis. The primers sequences are listed in Cells in culture dishes were cross-linked with 1% formaldehyde, and processed for chromatin lysate preparation, and chromatin immunoprecipitation (ChIP) using the ChIP assay kit (Millipore). Chromatin lysate was sonicated with a Bioruptor system (Diagenode) to yield a DNA smear averaging 250 bp. Sonicated chromatin lysate from 2×10Sequenced 36-bp reads were aligned to the mouse reference genome (mm9 assembly), and only those reads/tags that mapped to unique genomic locations with at most two mismatches were retained for further analysis. The mapped tags for each sample were converted to a browser extensible data (BED) file, detailing the genomic coordinate of each tag. Summary files, displaying the normalized number of tags in 200-bp windows, in BED format were used for viewing in the UCSC Genome Browser, and to generate screenshots. For generating tag density plots, data across samples were normalized by the total number of reads within each sample.All the ChIP-Seq and Gene expression microarray data generated for this study were deposited in the NCBI GEO repository under the accession number GSE37776.Figure S1Data quality of genome-wide H3K4me3 ChIP-Seq from mESCs. (A) UCSC genome browser images of H3K4me3 profiles (top two tracks) at a randomly selected ∼600 Kb region on chromosome 17 in wild-type mESCs. The top track shows H3K4me3 data from a previously published report (17). The middle and bottom track shows the data  generated for this study. Genes within the locus are shown at the bottom. (B) Normalized average tag density across a gene unit (left) and 10 Kb surrounding the transcription start site (TSS) (right) is shown. All genes in the mouse genome were used to calculate the average tag density.(TIF)Click here for additional data file.Figure S2Differentially expressed genes in menin-null cells. Number of genes that were at least 2-fold differentially expressed (p<0.005) in menin-null vs wild-type (WT) mESCs and menin-null vs WT pancreatic islet-like endocrine cells (PILECs) are shown. Overlaps between the differentially expressed subsets are represented as venn diagrams.(TIF)Click here for additional data file.Table S1Genes differentially expressed in menin-null vs wild-type mESCs.(XLSX)Click here for additional data file.Table S2Genes differentially expressed in menin-null vs wild-type pancreatic islet-like endocrine cells.(XLSX)Click here for additional data file.Table S3Genomic regions with differential H3K4me3 in menin-null vs wild-type mESCs.(XLSX)Click here for additional data file.Table S4Genomic regions with differential H3K4me3 in menin-null vs wild-type pancreatic islet-like endocrine cells.(XLSX)Click here for additional data file.Table S5PCR Primers.(XLSX)Click here for additional data file.


Epac2 locus that are essential for its transcriptional control.Epac 1 and Epac 2  are multidomain proteins that mediate cellular responses upon activation by the signaling molecule cAMP. Epac1 is ubiquitously expressed, whereas Epac2 exhibits a restricted expression pattern. The gene encoding Epac2 gives rise to at least three protein isoforms  that exhibit confined tissue and cell specific expression profiles. Here, we describe alternative promoter usage for the different isoforms of Epac2, and demonstrate that the activity of these promoters depend on the DNA methylation status. Bisulfite sequencing demonstrated that the level of methylation of the promoters in different tissues correlates with Epac2 isoform expression. The presented data indicate that the tissue-specific expression of the Epac2 isoforms is epigenetically regulated, and identify tissue-specific differentially methylated promoter regions within the  Epac 1 and Epac 2  are multidomain proteins that mediate responses via the intracellular signaling molecule cAMP. Binding of cAMP to the cAMP-binding domain activates the Epac proteins, which then act as exchange factors for the small G proteins Rap1 and Rap2 by catalyzing the exchange of bound GDP for GTP  Two separate genes encode Epac1 and Epac2, and alternative promoter usage and differential splicing of Epac2 we identified a potential promoter region for Epac2A, and determined that all three promoters exhibited transcriptional activity in a heterologous cell system. Moreover, we established that promoters of Epac2 are targets for DNA methylation. DNA methylation is an epigenetic mechanism that plays critical roles in differentiation, development and disease. In somatic tissues of mammals, DNA methylation occurs nearly exclusively on cytosines (C) that are followed by guanines (G) (CpG sites). Bisulfite sequencing revealed that CpG sites within the intragenic promoter regions of Epac2 were subjected to DNA methylation and that the methylation status correlated with promoter activity, such that they were demethylated in the tissue where they are active, but methylated in other tissues.We have very limited knowledge about the mechanisms that control the confined expression of the different Epac2 isoforms. Potential promoters for Epac2B and Epac2C have been described in exon 1b uniprot.org (A2ASW3 protein entry number) (Three Epac2 protein isoforms are described in the literature  number) . This la number) 1. In thi number) . This is number) . Transcr number) .Due to the low expression levels of Epac2 in most tissues and the lack of well functioning antibodies, the expression profile of Epac2 has mainly been determined by Northern blotting and reverse transcriptase (RT)-PCR. The applied RT-PCR primers have not, however, been designed to distinguish between the different isoforms. To examine the potential presence of more than one Epac2 isoform within the known Epac2 expressing tissues, primers that specifically amplify each isoform were designed see . Epac2A2Epac2, genomic sequences corresponding to regions around the alternative translational start sites were analyzed by the FlyBase eukaryotic promoter prediction computational tool (http://flybase.org) and the rVISTA evolutionary conservation analysis tool To identify novel potential promoter regions in Epac2 promoter regions, the sequence corresponding to the Epac2 gene region  was analyzed with the cpgplot software by employing default CGI prediction settings of; length >200 bp, GC content >50% and observed CpG/expected CpG >0,60 Epac2. This CGI contains a total of 60 CpG-sites and coincides with the Epac2A-promoter region  , which make up the majority of the CpG-sites in the CGI  A-B, in To establish the methylation pattern of the Epac2B-promoter, primers were designed to amplify 6 CpG sites across a region of 438 bp  comparedi.e. adrenal cortex, endocrine pancreas, brain and pituitary , with the exception of the Epac2A promoter region, which was nearly completely demethylated in all cells and tissues examined  Epac2, T-DMRs are frequently found in promoters located in intragenic regions, and are also enriched in the proximity of alternative TSSs, suggesting that DNA methylation influences tissue specific alternative promoter usage vs. pancreatic endocrine cells.Whole genome analyses indicate that approximately 70% of annotated gene promoters are associated with a CGI, and moreover that CGI promoters generally are kept hypomethylated regardless of the transcriptional status of the gene In the Epac2A promoter, CpG sites overlap with binding sites for transcription factors such as Sp1 and E2F that are enriched at CGI promoters In the Epac2C promoter, three CpG sites are located at corresponding positions in the murine and human promoters, but none of these overlap with predicted transcription factor binding sites. However, of potential interest is that a CpG site in the murine promoter is part of a recognition sequence for the nuclear receptor LXR that is known to have several functions in this organ As described in the introduction, the Epac2 isoforms exhibit a strict tissue specific expression pattern. The results presented in At present little is known about the functional differences of the Epac2 isoforms and about the physiological significance for their restricted expression patterns. The three domains that differ between Epac2A, Epac2B and Epac2C . Mice were kept under a 12h light/dark cycle with food and water 2 (2M) and collagenase (300 μg/ml) for 25 minutes at 3,8 ml/min. The liver was excised and incubated in Williams E Medium I (VME I) containing 100 U/ml penicillin, 100 μg/ml streptomycin and FBS (10%) to release the hepatocytes. All subsequent steps were performed on ice or at 4°C. The solution was filtered through a 40 μm grid, pelleted at 50×g for 5 min, and resuspended in WME I (20 ml). The cell suspension was transferred to a tube containing 1∶1 WME I and Percoll (20 ml) and centrifuged at 750×g for 5 min. The cells were washed twice in WME I (40 ml) at 50×g, 5 minutes. Finally the cells were resuspended in WME I (20 ml). Cells were seeded on 6- well plates pre-coated with collagen. 3×105 cells were seeded into WME I (2 ml)/well, after one wash with PBS. After 4 hours, the medium was changed to WME II .Primary hepatocytes were isolated from C57BL/6 mice. A venous cannula perforated the hepatic portal vein to give access to the mouse’s blood supply. A tube was connected to the cannula, and EBSS  was infused into the vein at 3,8 ml/min. To avoid accumulation of fluid the inferior vena cava was cut immediately after the infusion started. After 20 min the perfusion solution was changed to EBSS (100 ml) containing CaClGenomic DNA from mouse tissues, paraffin sections or primary cells was isolated employing the QIAGEN DNeasy kit  following the manufacturer’s instructions.RNA was isolated from materials pre-stored at RNA-later buffer, using the RNeasy Protect Mini Kit . One μg of total RNA was employed for the cDNA synthesis, performed with the iScript cDNA synthesis kit from BioRad . RT-PCR was performed with 20 ng cDNA template and GoFlexi DNA polymerase  on a MJ Research PTC-200 under the following conditions: initialization at 96°C for 2,5 min and 33 cycles of 30s at 96°C, 25s at 59°C and 30s at 72°C, and a final elongation step for 10 min at 72°C , 3 mM EDTA and 10% glycerol, supplemented with complete protease inhibitor cocktail EDTA-free, Roche Applied Science). As positive controls for the Epac2 isoforms in the immunoblotting experiments, Cos-1 cells were transfected with flag-tagged Epac2A/Epac2B/Epac2C expression vectors  using Fugene 6 (Roche Applied Science). Whole-cell extracts were prepared by lysis in RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM phe-nylmethylsulfonyl fluoride, 2 mM sodium orthovanadate, and 2 µg/ml aprotinin and leupeptin).2 anesthesia; the pancreas was dissected out and kept on ice in buffer . The tissue was then injected with collagenase P and cut into 3–4 pieces before digestion in a shaking water bath (37°C) for 7 minutes. The collagenase digestion was terminated by addition of BSA (0.1 g/mL), and the remaining tissue was thoroughly washed with ice-cold buffer. Islets of Langerhans were then separated from exocrine tissue by handpicking with a pipette under a stereomicroscope. Isolated islets and exocrine tissue, were immediately put on RNA-later (Sigma) for storage before RNA or DNA isolation, or frozen at −80°C for protein extraction.C57BL/6J mice were killed by decapitation in deep COProtein extracts  were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with I-block and incubated overnight with primary antibodies and subsequently with anti-mouse antibodies for 1h at RT. Antibody dilutions were 1∶100 for the anti-Epac2 antibody , whereas anti-actin  and anti-mouse horseradish peroxidase (HRP)-conjugated antibodies (Santa Cruz Biotechnology) were used at 1∶10000. Chemiluminescence signals were developed by the SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology).in vitro by using the SssI CpG methylase . Plasmid DNA was incubated at 37°C for four hours with SssI (2 U/μg DNA) in presence of S-adenosylmethionine (SAM) (160 μM), with an addition of extra SssI (0.3 U/μg DNA) and SAM (160 μM) after two hours. The unmethylated control plasmids were treated as above, but in the absence of SssI.Fragments corresponding to the putative Epac2 promoter regions were amplified by PCR with primers as shown in Transformed African Green Monkey Kidney Fibroblast Cells (Cos-1) were plated in 24-well plates and transiently transfected the following day using FuGENE HD . The cells were transfected with reporter plasmids (200 ng/well), and 48 h later, the cells were lysed in luciferase buffer . Luciferase activities were measured in total cell extract using the Luciferase Assay Kit A  according to manufacturer’s protocol on a LUCY-3 luminometer .Laser dissections were performed with a system comprised by an inverted microscope  and an air-cooled nitrogen laser (model VSL-37 ND-S) from P.A.L.M. Microlaser Technologies GmbH . Micro-dissection was performed on 7-μm-thick paraffin sections of mouse adrenal glands. Paraffin embedding and sectioning were performed according to standard protocols.Genomic DNA (5–500 ng) was subjected to sodium bisulfite modification with the EZ DNA Methylation-Gold kit  according to the producer’s instruction. PCR amplification was carried out using primers see  specificFigure S1Alignment of mouse Epac2 protein isoforms. Full amino acid sequence (aa) is shown for each of the Epac2 isoforms; Epac2A1 , Epac2A2, Epac2B and Epac2C. Functional domains are shown colored; cAMP-A and B; cAMP-binding domain A (red) and B (green), DEP; Dishevelled/Egl–10/Plekstrin domain (yellow), REM; Ras exchange motif domain (blue), RA; Ras association domain (orange), RasGEF; Guanine nucleotide exchange factor (GEF) for Ras-like small GTPases (pink). Stars below sequence alignment indicate full aa identity (100%) among all four Epac2 isoforms. Note the dissimilarity at AA 145/147  between Epac2A and Epac2B due to translation from Exon1b for the Epac2B isoform (grey box). Also, note the lack of the 18 aa peptide in Epac2A2, compared to Epac2A1/Epac2B . Alignment was produced by ClustalX (1.83), and modified in GeneDoc by employing the following mouse protein sequences; Epac2A1; A2ASW4, Epac2A2; A2ASW3, Epac2B; A2ASW8 and Epac2C; Q9EQZ6-2.(TIF)Click here for additional data file.Figure S2rVISTA evolutionary conserved genome alignment. rVISTA genome alignment of evolutionary conserved regions (ECR) between the human hg19-genome and the mouse mm10 base genome was performed on the three Epac2 promoter regions: A; Epac2A promoter , C; Epac2C promoter (chr2 72179738–72180207).(TIF)Click here for additional data file.Figure S3In silico prediction of CpG islands in Epac2. The sequence corresponding to Epac2, expanded by 3 kb upstream and downstream of the gene, was submitted to EMBOSS for CpG-Island (CGI) prediction (http://www.ebi.ac.uk/Tools/emboss/cpgplot/). Only one CGI, containing 60 CpG-sites over 548bp, was predicted in this sequence. The CGI overlaps with the Epac2A promoter region. For clarity, a region of 1000bp covering the CGI is shown. Default settings were employed in analysis. A; Calculated observed/expected ratio of CpG patterns . B; Calculated percent G’s+percent C’s in the region (Percent C+G content); C; The resulting CGI-region identified, based on A and B. For further details see website documentation and original publication by Gardiner-Garden & Frommer (TIF)Click here for additional data file.