C. elegans nuclear hormone receptor, NHR-25, in different cell types. Through a combination of yeast two-hybrid analysis and in vitro biochemistry we identified the single C. elegans SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on at least four lysines. Some of the sumoylation acceptor sites are in common with those of the NHR-25 mammalian orthologs SF-1 and LRH-1, demonstrating that sumoylation has been strongly conserved within the NR5A family. We showed that NHR-25 bound canonical SF-1 binding sequences to regulate transcription, and that NHR-25 activity was enhanced in vivo upon loss of sumoylation. Knockdown of smo-1 mimicked NHR-25 overexpression with respect to maintenance of the 3° cell fate in vulval precursor cells (VPCs) during development. Importantly, however, overexpression of unsumoylatable alleles of NHR-25 revealed that NHR-25 sumoylation is critical for maintaining 3° cell fate. Moreover, SUMO also conferred formation of a developmental time-dependent NHR-25 concentration gradient across the VPCs. That is, accumulation of GFP-tagged NHR-25 was uniform across VPCs at the beginning of development, but as cells began dividing, a smo-1-dependent NHR-25 gradient formed with highest levels in 1° fated VPCs, intermediate levels in 2° fated VPCs, and low levels in 3° fated VPCs. We conclude that sumoylation operates at multiple levels to affect NHR-25 activity in a highly coordinated spatial and temporal manner.Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here, we examine how a single regulatory input, sumoylation, differentially modulates the activity of a conserved  C. elegans, to examine how a single post-translational modification (sumoylation) affected the activity of a conserved TF (NHR-25) in different cell types during animal development. Our work suggests that sumoylation constrains NHR-25 activity in order to maintain proper cell fate during development of the reproductive organ.Animals precisely control when and where genes are expressed; failure to do so can cause severe developmental defects and pathology. Transcription factors must display extraordinary functional flexibility, controlling very different sets of genes in different cell and tissue types. To do so, they integrate information from signaling pathways, chromatin, and cofactors to ensure that the correct ensemble of genes is orchestrated in any given context. The number of regulatory inputs, and the complex physiology and large numbers of cell and tissue types in most experimentally tractable metazoans have rendered combinatorial regulation of transcription nearly impenetrable. We used the powerful genetics and simple biology of the model nematode,  Tissue-specific and cell type-specific transcriptional networks underlie virtually every aspect of metazoan development and homeostasis. Single TFs, operating within gene-specific regulatory complexes, govern distinct gene regulatory networks in different cells and tissues; thus, combinatorial regulation underpins tissue- and cell type-specific transcription. Determining the precise mechanisms whereby such specificity arises and how networks nevertheless remain flexible in responding to environmental and physiological fluctuations is an interesting challenge. TFs integrate signaling information from co-factors, chromatin, post-translational modifications, and, in the case of nuclear hormone receptors, small molecule ligands, to establish transcription networks of remarkable complexity.C. elegans, a simple metazoan with powerful genetic tools, a compact genome, and an invariant cell lineage leading to well-defined tissues. NHRs are DNA-binding TFs characterized by a zinc-finger DNA binding domain (DBD) and a structurally conserved ligand binding domain (LBD) C. elegans encodes 284 NHRs while humans only have 48 NHRs C. elegans ortholog of vertebrate SF-1/NR5A1and LRH-1/NR5A2, and arthropod Ftz-F1 and fulfills many criteria for the study of tissue-specific transcriptional networks nhr-25 highlight the broad roles of the receptor, and its genetic interaction with numerous signaling pathways  Here, we approach this problem by studying a covalent modification of a nuclear hormone receptor (NHR) in S. pombeSUMO  proteins serve as post-translational modifiers and are related to but distinct from ubiquitin The extent of sumoylation of a given target can be regulated by varying the expression, localization, stability or activity of components of the sumoylation machinery in response to external and internal cellular cues C. elegans nuclear hormone receptor NHR-25, and the physiological relevance of this nuclear hormone receptor-SUMO interaction. Using a combination of genetics, cell biology, and in vitro biochemistry we sought to understand how signaling through sumoylation impacts NHR-25's role in animal development, and how sumoylation affects the NHR-25 transcriptional network.Elucidating how a single nematode NHR integrates cellular signals to regulate specific genes in distinct tissues will advance our understanding of metazoan transcription networks. To this end, we examined how sumoylation regulates the C. elegans SUMO homolog (SMO-1) in a genome-wide Y2H screen using the normalized AD-Orfeome library, which contains 11,984 of the predicted 20,800 C. elegans open reading frames We identified an interaction between NHR-25 and the single C. elegans and other eukaryotes regulates TFs and chromatin, thus is well positioned to impact NHR-25 gene regulatory networks. Furthermore, spatial and temporal expression patterns of smo-1 and nhr-25 during development largely overlap smo-1 loss-of-function (lf) mutants display a fully penetrant protruding vulva (Pvl) phenotype, reflecting disconnection of the vulva from the uterus smo-1 RNAi or mutation also cause low penetrance of ectopic induction of vulval cells, which can generate non-functional vulval-like structures known as multivulva (Muv) smo-1 mutants, nhr-25 reduction-of-function leads to a Pvl phenotype, but does not cause Muv nhr-25 Pvl phenotype results from defects in cell cycle progression, aberrant division axes of 1° and 2° cell lineages, and altered vulval cell migration . Because at an earlier stage NHR-25 is also necessary for establishing the anchor cell (AC) nhr-25 depletion on later developmental events.SMO-1 was an enticing NHR-25 interacting partner to pursue. SUMO in smo-1 and nhr-25 were simultaneously inactivated, animals exhibited a fully penetrant vulvaless (Vul) phenotype and an exacerbated Muv phenotype  and smo-1(lf);nhr-25(RNAi) animals. Lineage analyses showed that following simultaneous inactivation of both smo-1 and nhr-25, daughters of all VPCs normally responsible for vulva formation,  failed to undergo the third round of vulval cell division (smo-1(ok359) and smo-1(ok359);nhr-25(RNAi) backgrounds, the expression of the 1° marker, egl-17::YFP exhibited ectopically high expression in P5.p and/or P7.p  animals, the P.p cell, which normally divides only once and fuses into the hypodermal syncytium, kept dividing  the acidic glutamic acid residues within the three consensus sumoylation sites were mutated to alanine. NHR-25 3EA leaves the acceptor lysines available, but is predicted to inhibit sumoylation by impairing interaction with UBC9. While wild type NHR-25 was clearly sumoylated, the 3EA mutation severely impaired sumoylation  or three arginine substitutions (NHR-25 3KR). When only one predicted acceptor lysine was available (2KR), we detected a single dominant sumoylated species, whereas for NHR-25 3KR, sumoylation was abrogated . We perfoylation .in vitro on three lysines and that C. elegans SMO-1 does not readily form polySUMO chains, unlike yeast SMT3 and mammalian SUMO2.When sumoylation reaction times were extended 5–20 fold, additional species of sumoylated NHR-25 were generated . These sC. elegans sumoylation to date have used hE1, hUBC9, and hSUMO proteins E. coli and tested their activity in in vitro sumoylation assays. Our CeE1 preparation was inactive, but was effectively substituted by hE1. Under those conditions, our CeUBC-9 and CeSMO-1 catalyzed sumoylation of the SF-1 hinge-LBD fragment  consensus sites, previously shown to be responsive to NHR-25 To begin to investigate how sumoylation affects NHR-25-dependent transcriptional activity, we employed a HEK293T cell-based assay. We used a luciferase reporter driven by four tandem Ftz-F1  and CYP11A1 (CYP) genes. We assessed NHR-25 binding to these elements using yeast one-hybrid (Y1H) and electrophoretic mobility shift assays (EMSAs). The Y1H assays indicated that NHR-25 bound the MIS and CYP11A1 elements (ku217) mutant, which has impaired DNA binding in vitroE. coli clearly bound MIS and CYP11A1 sites singly , and a third SUMO acceptor lysine (K84) within the DBD region between the second zinc finger and the conserved Ftz-F1 box  background  or by use of nhr-25(ku217), a reduction-of-function allele of nhr-25, abrogated reporter expression even in smo-1 knockdown animals  wild type NHR-25; (ii) NHR-25 3KR; or (iii) SMO-1 alone. Although egl-17 is typically used as a 1° and 2° cell fate marker during vulva development, it is expressed in all VPCs in earlier stages egl-17 promoter rather than commonly used VPC driver, lin-31, because the heterodimeric partner of LIN-31 is sumoylated and directly involved in vulva development To examine functionally the consequences of NHR-25 sumoylation, we returned to the roles of egl-17 promoter) but not in hyp7 or seam cells  drove Muv induction at the P8.p position, mimicking smo-1 RNAi .pxx cells , and Pegl-17::NHR-25(3KR) backgrounds (smo-1(RNAi) animals NHR-25::GFP was re-expressed in the AC at the “bell stage”  in VPCs P4.p and P8.p . However, regulation of lin-3 by NHR-25 in the anchor cell has also been suggested smo-1 RNAi is unlikely to cause Muv induction since, developmentally, this expression occurs much later than VPC fate determination. In wild type animals, NHR-25 levels are therefore downregulated in the AC, which may be required for proper completion of AC invasion and/or fusion. Additionally, the cell division arrest seen in nhr-25 RNAi leading to the Pvl phenotype was enhanced by inactivation of smo-1 , which impairs uterine-vulval connections The role(s) of NHR-25 and SMO-1 in vulval induction are likely pleiotropic. Multiple vulval development factors are sumoylated -25 RNAi . Synthetof smo-1 . For insivisions , Table 1How could unsumo∶sumo NHR-25 balance regulate 3° cell fate? Sumoylation might alter NHR-25 levels or activity in a manner that shifts the unsumo∶sumo NHR-25 ratio, which in turn acts as a switch to determine NHR-25 output. The activities of a mammalian nuclear hormone receptor have been shown to shift dramatically with signal-driven changes in levels of receptor activity The finding that overexpression of NHR-25 strongly provoked a Muv phenotype suggests that sumoylation state of NHR-25 in VPCs is exquisitely regulated. Such regulation might be accomplished by subtle changes in availability of SUMO in different VPCs, not detected by our assays, or by the relative activities of the sumoylation machinery and the SUMO proteases. A similar competition for constant levels of SUMO regulates Epstein-Barr virus infections, where the viral BZLF protein competes with the host PML protein for limiting amounts of SUMO1 C. elegans DAF-12 receptor. Indeed, such expansion of the concept of signaling ligands could “de-orphan” many or all of the 283 C. elegans nuclear hormone receptors for which no traditional ligands have been identified. Detection of noncovalent ligands is very challenging; numerous mammalian NHRs remain “orphans” despite intensive efforts to find candidate ligands and evidence that the ancestral NHR was liganded It is intriguing to consider SMO-1 as an NHR-25 ligand parallel to hormones or metabolites bound noncovalently nuclear hormone receptors in other metazoans, and by the C. elegans, SMO-1 can bind proteins non-covalently C. elegans. As both S. cerevisiae SUMO (SMT3) and vertebrate SUMO2 and SUMO3 form polySUMO chains, it appears that C. elegans has lost the ability to form polySUMO chains.There are three ways in which SUMO can potentially interact with target proteins: i) non-covalent binding, where a protein binds either free SUMO or SUMO conjugated onto another protein; ii) sumoylation, where SUMO associates covalently with a target protein through an isopeptide linkage; and iii) poly-sumoylation, where chains of SUMO are built up from an initially monosumoylated substrate. In D. melanogaster Ftz-F1 and mammalian LRH-1  are sumoylated on two sites within the hinge region of the protein, between the DBD and LBD an LRH-1  [7], -1-propanesulfonate}, and purified by anion-exchange chromatography using a MonoQ column  and eluted with a 1 M ammonium acetate gradient. Peak fractions were pooled, concentrated and 6×His-MBP-NHR-25 was purified by size-exclusion chromatography using an S200 column . Peak fractions containing 6×His-MBP-NHR-25 were pooled, concentrated, dialyzed into 20 mM Tris pH 7.5, 50 mM NaCl, 10% glycerol, flash frozen in liquid nitrogen, and stored at −80°C. Later purifications used only nickel affinity chromatography. Using this preparation in sumoylation assays produced results similar to those obtained using the preparations purified over the three aforementioned columns. 6×His-MBP-NHR-25 (amino acids 1–173) was expressed and purified using a single nickel affinity chromatography step, as described above for the 6×His-MBP-NHR-25 (amino acids 161–541) fragment.Recombinant hE1, hUBC9, hSUMO1, hSUMO2, hSENP1, and murine SF-1 LBD were purified as described et al.in vitro transcribed proteins were used as substrates, 50 µl reactions were generated using a TnT T7 Quick Coupled Transcription/Translation System (Promega). 16 µl of this reaction was then used as a substrate in a 25 µl sumoylation reaction using the same molarities as described above. When SUMO protease was required, 1.25 µg of hSENP1 was added. Reactions were incubated at 37°C for the desired time, and stopped by boiling in protein sample buffer . Proteins were resolved by SDS-PAGE on either 4–12% Bis-Tris gradient gels (Invitrogen) or 3–8% Tris acetate gels (Invitrogen) followed by either Coomassie staining or immunoblotting. For immunoblotting, anti-NHR-25, anti-guinea pig-HRP (Santa Cruz), and anti-guinea pig-IR800 (Li-Cor) antibodies were used. Blots were developed using a LAS500 imager  or an Odyssey laser scanner (Li-Cor).Reactions were performed as described by Campbell et al.2, 10 mM DTT, 10 mM ATP, and a 1 µM concentration of double-stranded oligonucleotide). Sequences of oligonucleotides are provided in Reactions were performed as described by Campbell C. elegans was cultured at 20°C according to standard protocols and the wild type strain is the N2 Bristol strain unc-119(ed4) syIs90 [egl-17::YFP+unc-119(+)], OP33 unc119 (ed3); wgIs33 [nhr-25::TY1::EGFP::3×FLAG(92C12)+unc-119(+)], VC186 smo-1(ok359)/szT1[lon-2(e678)]; +/szT1, MH1955 nhr-25(ku217). The following transgenic strains were generated for this study: HL102 jmEx102[Pegl-17::Myc::NHR-25_mCherry+rol-6(su1006)], HL107, HL108, HL110 are independent lines carrying jmEx107[Pegl-17::Myc::NHR-25(3KR)_mCherry+rol-6(su1006)], HL117 jmEx118 [Pegl-17::Myc::SMO-1_mCherry+rol-6(su1006)], HL111 and HL112 are independent lines carrying jmEx111[Pgrl-21::Myc::NHR-25_mCherry+rol-6(su1006)], HL121 jmEx121[Pgrl-21::Myc::SMO-1_mCherry+rol-6(su1006)], HL113 and HL114 are independent lines carrying jmEx113[Pwrt-2::Myc::NHR-25_mCherry+rol-6(su1006)], HL115 and HL116 are independent lines carrying jmEx115[Pwrt-2::Myc::SMO-1_mCherry+rol-6(su1006)], HL153 jmEx153[8×NR5RE (WT):pes-10Δ:NLS-3×Venus:unc-54 3′-UTR+Pmyo-2::tdTomato], HL155 jmEx155[8×NR5RE (MUT):pes-10Δ:NLS-3×Venus::unc-54 3′-UTR+Pmyo-2::tdTomato], HL170 nhr-25(ku217); jmEx153.[Pegl-17(1914 bp)::Myc::NHR-25_polycistronic_mCherry], pJW774 [Pegl-17(1914 bp)::Myc:: NHR-25(3KR)_polycistronic_mCherry], pJW773 [Pegl-17(1914 bp)::Myc::SMO-1_polycistronic_mCherry], pJW526 [Pgrl-21(746 bp)::Myc::NHR-25_polycistronic_mCherry], pJW775 [Pgrl-21(746 bp)::Myc::SMO-1_polycistronic_mCherry], pJW524[Pwrt-2(1380 bp)::Myc::NHR-25_polycistronic_mCherry], pJW776[Pwrt-2(1380 bp)::Myc::SMO-1_polycistronic_mCherry]. The following Gateway-based constructs were generated in pCFJ150 [8×NR5RE(WT):pes-10Δ:NLS-3×Venus:unc-54 3′-UTR] and pJW1110 [8×NR5RE(MUT):pes-10Δ:NLS-3×Venus::unc-54 3′-UTR]. Plasmids were prepared using a PureYield Plasmid Midiprep System (Promega) followed by ethanol precipitation, or a Qiagen Plasmid Midi kit (Qiagen). Transgenic strains were generated by injecting 50 ng/µl of each plasmid into the C. elegans gonad 8×NR5RE reporter strain generation, N2 animals were injected with 30 ng/µl of the reporter plasmid and 5 ng/µl of co-injection marker Pmyo-2::tdTomatoThe following Gateway-based constructs were generated in pKA921: pJW522nhr-25 RNAi, synchronized L2 larvae (19–20 hours after hatching) were fed on bacteria expressing nhr-25 dsRNA to bypass the anchor cell (AC) defect. smo-1 RNAi was performed on late L4 or young adults. For in vivo reporter assays, sodium hypochlorite-treated eggs were placed on RNAi plates seeded with dsRNA induced bacteria.Feeding RNAi was performed as described, with the indicated alterations to the protocol To score vulva induction, nematodes were anesthetized in 10 mM levamisole, mounted onto 5% agar pads  and the number of daughter cells for each VPC were counted under differential interference contrast (DIC) optics. For lineaging analyses, the division pattern was followed under DIC from the two to eight cell stages PEHQVSSSTTDQNNQINYFDQTKC (24 a.a. 141–163); SLHDYPTYTSNTTNC (15 a.a. 250–263); TSSTTTGRMTEASSC (15 a.a. 283–296) RYLWNLHSNXPTNWEC (16 a.a. 507–521).A peptide-based anti-NHR-25 antibody was raised in guinea pig . Animals were immunized against four short peptides in the hinge and LBD regions: TGAAGGTCA and TCAAGGTCA  Human embryonic kidney (HEK) cell line 293T was maintained in Dulbecco's modified Eagle's medium , supplemented with 10% fetal bovine serum. Transfections were performed with polyethyleneimine . The transcriptional activity of NHR-25 was tested with a luciferase vector carrying a CMV basic promoter driven by two copies of the Ftz-F1 binding consensus sequences Figure S1SMO-1 interaction is specific to NHR-25. (A) Yeast two-hybrid analysis of the indicated proteins fused to the Gal4 activation domain (AD) or DNA binding domain (DB). Empty vector (No insert) controls are shown. β-galactosidase (LacZ) and HIS3  reporters were assayed, and yeast viability was confirmed by growth on a plate lacking leucine and tryptophan (-Leu-Trp). Both NHR-25β and NHR-31 displayed self-activation activity, precluding analysis of their interactions with any of the AD fusions. (B) Due to the size of the matrix, the strains were plated on two plates. To rule out variation between plates, a negative control  and two positive controls (RFS-1 interaction with RAD-51 (ii) and R01H10.5 (iii), respectively) are provided for each plate.(TIF)Click here for additional data file.Figure S2smo-1(lf) and smo-1(lf); nhr-25(RNAi) cause defects in 2° cell fate. (A) Pegl-17::YFP expression in vulval cells at the 4-cell stage (1° cell fate marker) in the animals of the indicated genotypes. Ectopically high expression of Pegl-17::YFP was observed in 2° fated cells in smo-1(ok359) and smo-1(ok359); nhr-25(RNAi) animals. (B) The egl-17::YFP vulva marker is expressed in smo-1 (lf)-induced multivulva. Wild type expression of egl-17::YFP seen in vulD (a) and vulC (b) in late vulva morphogenesis. In smo-1 (ok359) and smo-1 (ok359); nhr-25 (RNAi) backgrounds (c and d), Muv is induced and the 1°/2° vulva marker egl-17::GFP is ectopically expressed. * indicates ectopic vulvae. (C) egl-17 has been reported to be expressed in all Pn.p cells egl-17 promoter for in vivo overexpression ([egl-17::NHR-25(3KR)_polycistronic_mCherry] transgenic animal is provided.pression  from a v(TIF)Click here for additional data file.Figure S3SMO-1 expression is required for NHR-25 to interact with UBC-9. (A) Indicated proteins were fused to the Gal4 activation domain (AD) or DNA binding domain (DB). Empty vector (No insert) controls are shown. (A) Yeast two-hybrid data confirmed that the SMO-1 V31K β-sheet mutation still binds to UBC-9, which indicated that the mutation did not disrupt the protein. The SMO-1 di-glycine deletion (ΔGG) prevented the interaction with UBC-9. (B) Yeast three-hybrid analysis. The indicated AD and DB fusions were expressed along with the pAG416 low copy yeast expression vector carrying either no insert or SMO-1. β-galactosidase staining is provided in A and B.(TIF)Click here for additional data file.Figure S4In vitro sumoylation reactions were resolved by SDS-PAGE and visualized by Coomassie staining  or anti-NHR-25 immunoblotting (C). (A and B) used a recombinant SF-1 partial hinge-LBD fragment as a substrate and (C) used a recombinant 6×His-MBP-NHR-25 (amino acids 161–541) fragment. All reactions used recombinant hE1. In (A), hE2 (UBC9) and hSUMO1 were used. (B and C) used CeUBC-9 and CeSMO-1. Recombinant hSENP1 SUMO protease was included in each experiment to demonstrate that bands reflected sumoylated species. A size standard in kilodaltons (kDa) is provided.Confirmation of activity of sumoylation enzymes. (TIF)Click here for additional data file.Figure S5in vitro on three lysines. In vitro sumoylation reactions were resolved by SDS-PAGE and visualized by anti-NHR-25 immunoblotting (A) or Coomassie staining (B). Both reactions used hE1, hE2, hSUMO1, and a recombinant NHR-25 substrate (6×His-MBP-NHR-25 (amino acids 161–541)). In (A) recombinant hSENP1 SUMO protease was included. In (B), the substrates were wild type NHR-25 (WT) and NHR-25 2KR (K170R K236R) and NHR-25 3KR (K165 K170R K236R) mutants where SUMO acceptor lysines were mutated to arginine. A size standard in kilodaltons (kDa) is provided.The NHR-25 hinge domain is sumoylated (TIF)Click here for additional data file.Figure S6Sumo1 and SMO-1 do not readily form poly-SUMO chains. (A) Anti-NHR-25 immunoblots on sumoylation reactions incubated for the indicated number of hours. E1 enzyme was incubated with the indicated E2 and SUMO combinations. Methyl-hSUMO1 is a modified protein that blocks SUMO chain formation. The asterisk (*) indicates a non-specific band in the NHR-25 substrate control lane (no sumoylation enzymes added). NHR-25 isoforms predicted to contain one, two, and three SUMO proteins covalently attached are indicated . (B–E) Short course sumoylation time courses using hE1, hE2, and hSUMO1  or hE1, CeUBC-9, and CeSMO-1 . The substrate was recombinant 6×His-MBP-NHR-25 (amino acids 161–541). Reaction time in minutes, and a size standard in kilodaltons (kDa) are provided. The final lane is a substrate only control. Coomassie stained polyacrylamide gels  and anti-NHR-25 immunoblots  are shown.(TIF)Click here for additional data file.Figure S75′-YCAAGGYCR-3′  Sumoylation affects NHR-25 binding to canonical SF-1 sites. (A) Sequence of binding sites used in the Y1H and EMSA experiments. The mutation in the MIS binding site (MIS MUT) is underlined. The canonical binding site of the NHR-25 ortholog, SF-1, is (TIF)Click here for additional data file.Table S1Overexpression of NHR-25 in hyp7 or seam cells does not cause Muv induction. Table providing scoring of overall multivulva (Muv) induction in the indicated strains/genotypes, as well as induction in individual VPCs. Number of animals (n) scored for each strain genotype is provided. Use of brackets denotes transgenic genotypes.(DOCX)Click here for additional data file.Table S2NR5 family Response Element. The NR5RE oligos carry an mMIS and hCYP11A1 binding site. SF-1 binding site is highlighted in bold.Sequences of oligonucleotides and gBlocks used in this study. All sequences are displayed in a 5′ to 3′ orientation. (A) Primers used to clone the indicated cDNAs and promoters. Sequences of the attB recombination sites and Myc and FLAG epitopes are indicated as described in the table. (B) Sequences of the primers used to generate the indicated mutations by site-directed mutagenesis. (C) gBlocks used in this study. NR5 binding sites and minimal promoters are indicated as described in the table. (D) Sequences of oligonucleotides from SF-1 target gene promoters used in EMSA assays are shown. m, mouse; h, human; MIS, Mullerian Inhibiting Substance; ; CYP11A1, Cytochrome P450, Family 11, Subfamily A, Polypeptide 1; 2×NR5RE, nuclear receptor (DOCX)Click here for additional data file.Table S3unc-54 3′-UTR marks the tissues where the array is expressed. pCFJ150 is used to generate C. elegans expression vectors through three-fragment Gateway cloning Plasmids generated for this study. The vector backbones used for Gateway cloning are provided, as is a description of each vector. pDONR221 (Invitrogen) and pDONR-P4P1r (Invitrogen) are entry vectors for cDNA and promoter cloning, respectively. pAD and pDB are Y2H vectors for generating N-terminal fusions of the Gal4 activation domain (AD) and DNA binding domain (DB), respectively, to proteins of interest (DOCX)Click here for additional data file.


Juvenile dermatomyositis (JDM) is characterised by chronic inflammation in skeletal muscle, skin, and other target organs. Prognosis has improved in the past 10-20 years, but it is still a disease with high morbidity and considerable sequelae such as muscle weakness, joint contractures, calcinosis and lipodystrophy. There is increasing evidence that single nucleotide polymorphisms (SNPs) are associated with chronic inflammatory diseases. In the present study, we investigated known SNPs within genes that previously have been associated with inflammatory myopathies, including JDM and dermatomyositis.The aim of the study was to investigate known polymorphisms within the genes encoding Toll-like receptor (TLR)1-10; nucleotide binding oligomerisation domain (NOD)1-2; interferon-induced helicase C domain-containing protein 1 (IFIH1); and melanoma differentiation associated gene 5 (MDA5), as well as single nucleotide polymorphisms (SNPs) in tumour necrosis factor (TNF)-alpha and protein tyrosine phosphatase, non-receptor type 22 (PTPN22) and their association with the disease course of Juvenile Dermatomyositis (JDM) in a population-based follow-up study.In 53 JDM patients SNPs in the pattern recognition receptor genes encoding TLR1-10, DDX58 (RIG-I), IFIH1 (MDA5), NOD1 (CARD4), and NOD2 (CARD15) were analysed using an in-house multiplex bead-based assays. The TNF-alpha (rs1800629) and PTPN22 (rs2476601) polymorphisms were genotyped by pyrosequencing.The TLR2-rs1898830 SNP was found to be associated with calcinosis (p=0.006) and lipodystrophy (p=0.0006). Patients with the G/G genotype had a significantly higher risk  of developing lipodystrophy than patients with the A/A or A/G genotypes.No other SNPs were significantly associated with JDM outcome.The present study suggests an association between TLR2-rs1898830 and disease outcome in JDM.


We recently showed from a Swedish cohort that nearly half of boys born post-term (≥42 weeks gestation) were overweight or obese at 16 years of age. We hypothesized that post-term children would display features of insulin resistance and the metabolic syndrome even in their pre-pubertal years.90 healthy pre-pubertal children aged 4–11 years, with birth weight appropriate-for-gestational-age were studied: 36 children born post-term (18 boys and 18 girls) and 54 children (36 boys and 18 girls) born at term (38–40 weeks). Insulin sensitivity was measured using Bergman’s minimal model. Other assessments included fasting lipid and hormonal profiles, body composition using whole-body dual-energy x-ray absorptiometry, and 24-hour ambulatory blood pressure monitoring.-4·min−1·(mU/L); p<0.0001). Post-term children had an adverse lipid profile, with higher total cholesterol  and LDL  concentrations. Further changes suggestive of the metabolic syndrome among post-term children included an increased android to gynoid fat ratio , and a reduction in the normal nocturnal systolic blood pressure dip . Post-term children also had higher serum leptin , lower adiponectin , and lower IGFBP1  concentrations.Insulin sensitivity was reduced in post-term children (8.44±0.74 vs 13.55±0.89 x10Our study shows for the first time that post-term children have early features of the metabolic syndrome, including reduced insulin sensitivity, adverse lipid profile, and increased abdominal adiposity.


The alpha-subunit of cone transducin (TαC) is specifically expressed in cone photoreceptors and is required for colour vision. In order to better understand the molecular genetics controlling the initiation of cone photoreceptor-specific expression in vivo, we have utilised zebrafish to identify cis-regulatory elements in the upstream promoter region of the TαC gene.Appropriate transcriptional regulation is required for cone photoreceptor development and integrity. To date, only a few -like sequences, with potential functional redundancy, are located in this 2.5 kb promoter. PRE-1-rho which has the highest sequence and structural homology to PRE-1 is located in the rhodopsin promoter. Surprisingly, PRE-1 and PRE-1-rho are functionally distinct. We demonstrate that PRE-1, but not PRE-1-rho, is sufficient to enhance expression from a heterologous UV cone promoter. PRE-1 is also sufficient to enhance expression from a heterologous rhodopsin promoter without altering its rod photoreceptor specificity. Finally, mutations in consensus E-box and Otx sites prevent PRE-1 from forming complexes with eye nuclear protein and enhancing photoreceptor expression.A 0.5 kb TαC promoter fragment is sufficient to direct cone-specific expression in transgenic larvae. Within this minimal promoter, we identify photoreceptor regulatory element-1 (PRE-1), a unique 41 bp sequence. PRE-1 specifically binds nuclear factors expressed in ocular tissue. PRE-1 is not required for cone-specific expression directed from a 2.5 kb TαC promoter. However, PRE-1cis-regulatory module that is sufficient to enhance the initiation of photoreceptor-specific gene expression in differentiating rod and cone photoreceptors.PRE-1 is a novel  Underlining these molecular differences, these photoreceptor-specific isoforms associate with distinct forms of blindness. Mutations in the gene encoding rod transducin alpha (TαR) cause inherited night blindness whereas mutations in the gene encoding cone transducin alpha (TαC) cause achromatopsia, or total colour blindness  and appropriate unlabelled competitor for 10 minutes at 4°C prior to incubation with the labelled probe for 30 minutes with increasing concentrations of nuclear protein. For competition assays, cold competitor was added prior to the addition of labelled probe in 50, 100, 150 or 200 fold molar excess. The reactions were electrophoresed on 4-8% non-denaturing polacrylamide gels followed by autoradiography.Nuclear protein extracts were prepared from adult zebrafish eyes and mid body in the presence of protease inhibitors as previously described . Probes gnat2 ENSDARG00000042529, opn1sw1 ENSDARG00000045677, opn1sw2 ENSDARG00000017274, rho ENSDARG00000002193, gnat1 ENSDARG00000044199 and pde6a ENSDARG00000075911) and housekeeping genes (Bactin1 ENSDARG00000037746 and gapdh ENSDARG00000043457), and gnat2 orthologues  were obtained from the ENSEMBL database. These 10 kb sequences were aligned with the 41 bp PRE-1 sequence in both orientations using Clustal W and of the top 20 alignments identified by Clustal W [10 kb 5' promoter sequences of zebrafish photoreceptor phototransduction genes (lustal W , only seCBE: Crx-binding element; CPRE-1: cone photoreceptor regulatory element 2; dpf: days post fertilization; EGFP: enhanced green fluorescent protein; IRBP: interphotoreceptor retinoid binding protein; LCR: locus control region; NRE: Nrl response element; PRE-1: photoreceptor regulatory element 1; PCE: photoreceptor conserved element; TBS: Tris-buffered saline; TαC: cone transducin α subunit; TαR: rod transducin α subunit; ZOP: zebrafish rod opsin promoter; zUVOP: zebrafish UV opsin promoterin vivo activity levels of constructs. BNK and SS performed the electrophoretic mobility shift assays. BNK, SB and JH conceived, designed and supervised the study, and contributed to writing the manuscript. All authors read and approved the final manuscript.MEM drafted the manuscript and tested the Figure S1 - Alignment of PRE-1 Sequence to Known Photoreceptor Regulatory Elements. Schematic showing regions of homology of PRE-1 to known photoreceptor cis-elements.Click here for fileFigure S2 - Location of PRE-1-Like Sequences in GNAT2 Orthologues. Schematic showing the location of PRE-1-like sequences in 10 kb promoter fragments of gnat2 orthologues from human, gorilla, cow, rat, mouse, cat, dog and fugu.Click here for file


We present a patient with tenofovir disoproxil fumarate (TDF)-induced Fanconi syndrome, osteomalacia and concurrent nucleos(t)ide reverse transcriptase inhibitor related CD4 cell decline. Sequentially measured intracellular (ic) tenofovir-diphosphate (TFV-DP) levels were extremely high, with plasma TFV just slightly elevated.3 by December 2007, but then slowly decreased to 181 despite a persistently undetectable HIV-1 RNA. There was 13 kg weight loss when he developed diabetes mellitus in April 2008. In May 2008 blood and urinalysis were compatible with Fanconi syndrome. Creatinine clearance (CCl) had decreased from 76 (2003) to 44 mL/min (GC). Serum 1,25(OH)-vitamin D was low (20 pmol/L), but 25(OH)-vitamin D and PTH were normal. MRI of feet and knees showed patchy bone marrow oedema without fractures, DXA demonstrated osteopenia and bone biopsy confirmed the diagnosis osteomalacia. While CCl decreased, plasma TFV increased only slightly (from 0.23 in July 2004 to 0.36 mg/mL in September 2008), but ic TFV-DP levels were found to be extremely high, 3630 fmol/106 cells (mean TFV-DP in patients on LPV-r 233.1 fmol/106 cells [6 cells), but plasma TFV was undetectable, illustrating the long ic half-life. Ic dideoxy adenosine-triphosphate (ddATP) levels were also high, 123 fmol/106 cells (n 5.06 fmol/106 cells) as were ddI plasma levels (max. 0.304 mg/L). TDF and ddI were replaced by raltegravir/nevirapine and phosphate, calcium and 1,25(OH)2-VitD temporarily supplemented. Two months later symptoms disappeared, CCl improved to 62 mL/min, CD4s increased (286 cells/mm3) and oral antidiabetics could be stopped.In June 2008 a 53-year-old Caucasian male complained of severe pain in his lower extremities. He was diagnosed with HIV in 1995 and developed some polyneuropathy and severe lipoatrophy after initiation of ART in 1996. In September 2003 TDF 300 mg qd, didanosine (ddI) 250 mg qd and lopinavir-ritonavir (LPV-r) 400/100 mg bid were started. CD4 cells had increased from 136 to 489/mmThis case illustrates the severe clinical impact of protracted unrecognised TDF-related toxicity and is suggestive of a causal relationship between TDF use, concomitant factors and ic TFV-DP accumulation fig . A signi


Antagonist anti-CD28 antibodies prevent T-cell costimulation and functionally differentiate from CTLA4Ig since they cannot block CTLA-4 and PDL-1-mediated coinhibitory signals. They demonstrated efficacy in suppressing effector T cells while enhancing regulatory T cells function after organ transplantation.In vitro, FR104 and CTLA4-Ig (LEA29Y) dose-dependently prevented human T cell proliferation to a similar extent in mixed lymphocytes reactions with EC50 at 0.16 and 0.18 micrograms/ml, respectively. IL-2 secretion after stimulation with SEE superantigens was also inhibited in a similar manner.Here we evaluated FR104, a novel antagonist pegylated Fab’ anti-CD28 monovalent antibody, in xenogeneic graft-versus-host disease (GVHD) induced in NOD/SCID mice infused with human PBMC and compared efficacy with CTLA4Ig molecules. NOD/SCID mice adoptively transferred with human PBMC presented > 80% engraftment after a week and developed a florid GVHD in the third week due to the proliferation of xenogeneic human T cells and their infiltration into the liver, gut, lung and skin, eventually leading to weight loss and sacrifice. Treatment of recipient mice with 5 mg/Kg FR104 biweekly from day 0 to day 25 reduced target organs infiltration by human T cells and completely prevented weight loss and mortality (p < 0.0001). Mice continued gaining weight and did not succumb even a month after treatment withdrawal. Flow cytometry analysis 2 month post engraftment revealed that FR104 treated mice displayed low level of human cells engraftment (<10%), indicating that targeting the CD28/B7 pathway protected from acute GVHD by inhibiting donor T cell expansion. The therapeutic effect of FR104 was dependent on CTLA-4, since co-administration of FR104 with blocking anti-CTLA4 antibodies completely abrogated FR104-mediated protection. This might be explained by the fact that CTLA-4 signals attenuate T cell responses independently of CD28. In Contrast, biweekly administrations of CTLA4-Ig (LEA29Y or Abatacept), which reduces CD28-CD80/86 costimulation as well as CTLA-4-CD80/86 coinhibition, was ineffective to protect mice from severe GVHD, whereas a less intensive protocol of administration (once a week) was partially effective.Our data suggests that antagonist anti-CD28 monovalent antibodies might lead to higher therapeutic indexes, by sparing CTLA-4, as compared to CD80/CD86 antagonists, to dampen T cell activation in transplantation.


Arabidopsis thaliana, one of the natural hosts for CMV, with Myzus persicae . Virus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of Infection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0. Inhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins  appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between the effects of Fny-CMV on this plant and those seen previously in tobacco (inhibition of resistance to aphids) may have important epidemiological consequences. Viruses induce extensive biochemical changes in plants . These cMacrosiphum euphorbiae was inhibited (consistent with encouragement of transmission), whereas feeding by Myzus persicae was enhanced, which is less likely to encourage PVY transmission by members of this aphid species  and incubated at 37°C overnight. Tissue was washed several times in 70% ethanol before observation of the indigo staining  revealed that in plants with disrupted miRNA utilization (35S:2b-expressing lines 2.30F and 3.13F and ago1-25), basal levels of camalexin were significantly higher  than in non-transformed (NT) plants and pad3-1 mutant plants. Interestingly, the double transgenic plants expressing the 2b and 1a proteins had levels of camalexin similar to NT plants. Aphid infestation resulted in significantly increased levels of camalexin relative to the mock-treatment in the 35S:2b-expressing line 2.30F and in the mutant ago1-25 . (C) The mean relative growth rate of individual aphids feeding on Fny-CMV-infected wild-type and pad3-1 mutants  was lower compared to aphids feeding on mock-inoculated plants, n≥24. The decrease in aphid growth rate on Fny-CMV-infected pad3-1 mutants was slightly less severe than in virus-infected wild-type plants  indicating that camalexin can make only a minor contribution at best to aphid resistance induced by Fny-CMV. Error bars represent standard error of the mean. Different letters are assigned to significantly different groups.(TIF)Click here for additional data file.Figure S8Symptoms induced by CMV reassortants in wild-type Arabidopsis plants. Viral reassortants generated using combinations of CMV genomic RNAs 1, 2 or 3 (subscript) from either Fny-CMV (F) or LS-CMV (L) were used to inoculate wild-type Arabidopsis plants (Col-0 ecotype). Mock indicates a mock-inoculated plant. Plants were photographed at 14 days post-inoculation. Scale bars represent 1 cm.(TIF)Click here for additional data file.Figure S9dcl1-9 mutant plants.Aphid performance on virus-infected wild-type plants and  Mean relative growth rate of individual aphids feeding on dcl1-9 mutant and wild type (WT) Arabidopsis plants, n≥24. Error bars represent standard error of the mean. Different letters are assigned to significantly different groups . (TIF)Click here for additional data file.Figure S10Percentage occurrence of waveforms produced in electrical penetration graph (EPG) analysis of feeding aphids over 12-hour recordings. (A) Ingestion from the phloem  was significantly increased for aphids feeding on Fny2b-transgenic plants in the second half of recordings. (B) Ingestion from the phloem  was significantly increased for aphids feeding on ago1-25 transgenic plants across the whole recording. Statistical significance was tested by Student’s t-test compared to percentage occurrence of phloem ingestion on mock-inoculated plants for the first and second halves of the recording and for the whole recording. (TIF)Click here for additional data file.Figure S11ago1-25 mutants.Glucosinolate accumulation in wild-type plants and  Glucosinolate accumulation of Fny-CMV-infected wild-type plants and ago1-25 mutants was analyzed using high performance liquid chromatography, n=3. Error bars represent standard error of the mean. Abbreviations: 4MI3M, 4-methoxy-indol3yl-methylglucosinolate, and I3M, indol-3-yl-methylglucosinolate. Different letters are assigned to significantly different results (ANOVA with post-hoc Tukey’s tests P<0.05).(TIF)Click here for additional data file.Figure S12Phenotypes of transgenic Arabidopsis plants constitutively expressing various Fny-CMV proteins. Appearance of plants (from independent transformed lines) expressing transgenes encoding the CMV proteins 1a, 2a, 2b, movement protein (MP), and coat protein (CP) under the control of the constitutive cauliflower mosaic virus 35S promoter. Vector control is a plant from a line transformed with ‘empty’ pBI121.1. Plants were five weeks old when photographed. The 2b-transgenic lines 2.30F and 3.13F were constructed by Lewsey and colleagues [lleagues . Scale b(TIF)Click here for additional data file.Figure S13CMV proteins transgenically expressed in Arabidopsis are biologically active. (A) The transgenic plants expressing the 1a [1a (c)] or 2b and 1a [2b/1a (a) and 2b/1a (b)] ORFs of Fny-CMV were inoculated with a mixture of synthetic RNAs 2 and 3 (RNA 2+3) generated by invitro transcription of the plasmids pFny209 and pFny309, respectively. Three weeks post-inoculation, CMV coat protein (CP) was detected by DAS-ELISA in the non-inoculated leaves indicating that virus replication had occurred . (B) Transgenic plants expressing the 2a (2a) and truncated 2a (2a_Tr) ORFs were inoculated with synthetic RNAs 1 and 3 (RNA 1+3) generated by invitro transcription of the clones pFny109 and pFny309, respectively. Three weeks post inoculation CMV CP was detected by DAS-ELISA in the non-inoculated leaves indicating that virus replication had occurred and therefore that the 2a-derived transgenes were expressing enzymatically active 2a (RNA-dependent RNA polymerase) protein and complementing efficient viral replication and spread. (C) Transgenic plants expressing the MP gene [independent lines MP (a) and MP (c)] were inoculated with RNA 1 and 2 and a modified RNA 3 containing the GFP gene in place of the MP ORF (RNA 1+2+CP), where CP represents the synthetic RNA transcribed from pF:GFP/CP as described previously . Three weeks post-inoculation, CMV CP was detected by DAS-ELISA in the non-inoculated leaves indicating that the transgenically expressed MP had complemented cell-to-cell movement and facilitated systemic movement of RNAs 1, 2 and the modified RNA 3. (D) Expression of CP was directly detected by DAS-ELISA in leaf tissue from transgenic plants expressing the CP ORF. Additional transgenic plants in (A-C) have been inoculated with a mixture of RNAs 1, 2, and 3 (RNA1+2+3), which constitutes the whole Fny-CMV genome, as a positive control. Non-transgenic (NT) plants and mock-inoculated (Mock) transgenic plants gave a background signal in DAS-ELISA.(TIF)Click here for additional data file.Figure S14dcl2/4 double mutant plants.Relative accumulation of Fny-CMV and Fny-CMVΔ2b in wild-type and  Viral RNA accumulation was measured (by RT-Q-PCR) relative to the accumulation of Fny-CMV RNA in wild-type plants at 14 days post-inoculation, n=3. Error bars represent standard error of the mean. the experiments was repeated three times with similar results. Different letters are assigned to statistically different groups .(TIF)Click here for additional data file.Figure S15dcl2/4 mutant plants: Percentage occurrence of waveforms produced in electrical penetration graph (EPG) analysis of feeding aphids over 12-hour recordings on virus infected dcl2/4 mutants.Aphid feeding behavior on  Ingestion from the phloem  was significantly increased in the second half of the recording for aphids feeding on Fny-CMV infected dcl2/4 mutant plants. Ingestion from the phloem was significantly decreased for aphids feeding on Fny-CMVΔ2b-infected dcl2/4 mutant plants. Statistical significance was tested by Student’s t-test compared to percentage occurrence of phloem ingestion on mock-inoculated plants for the first and second halves of the recording and for the whole recording. (TIF)Click here for additional data file.Figure S16A truncated form of the Fny2a protein had the capacity to induce anti-aphid resistance. Growth rate of individual aphids feeding on transgenic plants expressing a truncated form of the Fny2a protein (35S:2a_Tr) and non-transgenic (NT) plants, n≥24. Error bars represent standard error of the mean. Asterisks indicate significant differences (Student’s t-test): *, P<0.05; **, P<0.01; ***, P<0.001.(TIF)Click here for additional data file.Figure S17dcl2/4 double mutant plants.High performance liquid chromatography analysis of glucosinolate accumulation in Fny-CMV-infected (Fny-CMV) and Fny-CMVΔ2b-infected (Fny-CMVΔ2b)  Statistical analysis did not reveal any significant differences compared to mock-inoculated plants (Mock) , except for the increased accumulation of 4-methoxy-indol-3-yl-methylglucosinolate in Fny-CMVΔ2b-infected dcl2/4 double mutants . Error bars represent standard error of the mean. Data presented represents the mean accumulation of each glucosinolate extracted from tissue from at least three plants per treatment grouped and repeated independently three times.(TIF)Click here for additional data file.Spreadsheet S1(XLS)Click here for additional data file.Spreadsheet S2(XLS)Click here for additional data file.Spreadsheet S3(XLS)Click here for additional data file.Table S1Primers used in generation of transgenic plants expressing proteins derived from Fny-CMV.(DOC)Click here for additional data file.Table S2Primers used in RT-Q-PCR analyses.ELONGATION FACTOR 1α (EF1α) and GAPDH were used as stable reference transcripts to control for loading.(DOC)Click here for additional data file.


MicroRNA-22 (miR-22) is a muscle-enriched miRNA required for optimum cardiac gene transcription and adaptation to hemodynamic stress by pressure overload in mice. Recent evidence also suggests that miR-22 induces hypertrophic growth and it is oftentimes upregulated in end stage heart failure. However the scope of mRNA targets and networks of miR-22 in the heart failure remained unclear. We analyzed transgenic mice with enhanced levels of miR-22 expression in adult cardiomyocytes to identify important pathophysiologic targets of miR-22. Our data shows that forced expression of miR-22 induces a pro-hypertrophic gene expression program, and it elicits contractile dysfunction leading to cardiac dilation and heart failure. Increased expression of miR-22 impairs the Ca2+ transient, Ca2+ loading into the sarcoplasmic reticulum plus it interferes with transcription of estrogen related receptor (ERR) and PPAR downstream genes. Mechanistically, miR-22 postranscriptionally inhibits peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), PPARα and sirtuin 1 (SIRT1) expression via a synergistic circuit, which may account for deleterious actions of unchecked miR-22 expression on the heart.Increasing evidence suggests that microRNAs are intimately involved in the pathophysiology of heart failure.  Heart failure (HF) is a complex disorder defined by impaired myocardial contractility, deprivation of cardiac bioenergetics, increased myocyte cell death, and collagen scar formation. A major risk factor for HF comes from maladaptive cardiac hypertrophy, which occurs as a result of sustained hemodynamic overload due to chronic hypertension and/or cardiac injury ,2.At the cellular level pathological hypertrophy is characterized by an increase in cardiac myocyte cell size, increased protein synthesis, sarcomeric remodeling, and a switch to the hypertrophic molecular gene program that mimics that observed in fetal heart development . Patholoin vivo studies in mice suggest that the heart enriched, microRNA-22 (miR-22) is involved in the pathogenesis of HF  and NFAT activation [i.e. regulator of calcineurin 1 splice variant 4 (Rcan1-4)] in ventricles of TG-M and TG-H mice between 5- and 12-weeks of age , Caveolin-3 (Cav3), Homer-1, and Pgc1a were confirmed as repressed in miR-22 transgenic hearts by quantitative PCR and/or immunoblot . In agreement with a previous report, transfection of a miR-22 mimic lead to potent hypertrophy of NRVC characterized by increased protein synthesis, cell size, and increased Nppa expression  were decreased in 5-week old miR-22 transgenic mice. Secondly, we also scrutinized transcript levels of Iroquois related homeobox 4 (Irx4), a proximal transcriptional brake on hypertrophic traits acting within the PGC-1α/ERR-regulatory axis  in 12-week old TG-H hearts are known transcriptional targets of the PGC-1α/ERR complex  involved in fatty acid uptake, transport, esterification and/or oxidation . To explore this question, we isolated cardiac myocytes from 16-week old TG-M and WT mice and measured calcium transients. When paced at 1Hz, the Ca2+ transient amplitude was lower in TG-M compared to WT myocytes  activity in TG-M mice compared to WT mice  of miR-22 LOF and GOF mediated cardiomyopathy are especially complex because miR-22 operates upstream of many critical transcription factors with a high level of connectivity and therefore the phenotype(s) cannot be easily be assigned to a single gene. In the future it will be important to determine if miR-22 is inversely correlated with expression levels of SIRT1, PGC-1α, and PPARα in human diseased hearts.We have previously shown that absence of vocation . Our mol disease –28. ThisFigure S1Representative Doppler Lead II surface electrocardiogram trace from 5-week old TG-H and WT mice.(TIF)Click here for additional data file.Figure S2miR-22 transgenic mice.Apoptosis analysis in Histomorphometric quantification of TUNEL-positive cells in cardiac sections obtained from 12-week old TG-H and control mice. (n = 3-4.) Student t test. **, P<0.01.(TIF)Click here for additional data file.Figure S3miR-22 targeting effects.Transcriptome microarray detection of (A) Sylamer analysis of microarray shows specific enrichment of miR-22 heptamer ‘seed’ matches among downregulated genes in TG-H hearts. miR-22 seed heptamers are shown in shades of blue while all other mouse miRNA seeds are shown in grey. (B) Pie chart shows the number and distribution of downregulated transcripts in TG-H hearts with 6-9-mer seed matches in 3’ UTR. (C and E) Relative cardiac expression levels of miR-22 target genes were evaluated by qPCR in (E) 5- or (C) 12-week old WT, TG-M, and TG-H mice. (D) CAV3 protein levels were determined by immunoblot in hearts of 12-week old TG-H and WT mice. (F) Schematic shows conserved motifs potentially bound by miR-22 in the 3’ UTRs of Camta2 and Homer1. (G) Quantitative PCR detection of sarcomeric genes in 12-week old TG-M versus WT hearts. Bars represent expression normalized with WT set equal to 1.0. Student t test, (C and G); or 1-way ANOVA with the Dunnett post hoc test, (E). *, P<0.05; **, P<0.01; ***, P<0.001.(TIF)Click here for additional data file.Figure S4miR-22 expression in primary cultures of neonatal rat ventricular cardiac myocytes (NRVC).Cellular and molecular effects of increased (A) Representative α-actinin immunostaining and cardiomyocyte cell size of NRVC transfected with either a miR-22 or control (Crtl) miRNA mimic in the absence  or presence of phenylephrine (PE); n = 3-5 experiments, >40,000 cells per group. (B) Immunoblot detection of PGC-1α, PPARα, and SIRT1 levels in NRVC cells transfected with either a miR-22 or control miRNA mimic. GAPDH was used as a loading control. Repression: protein levels in miR-22 over-expressing cells relative to control miRNA.  Real time quantitative PCR detection of indicated genes in NRVC cells transfected with miR-22 or control miR mimic (n = 3 experiments). Student t test, ; or 2-way ANOVA with the Tukey’ post hoc test, (A) *, P<0.05; **, P<0.01.(TIF)Click here for additional data file.Figure S5Transcriptome microarray Gene Set Enrichment Analysis.Gene set enrichment analysis (GSEA) tool was applied on the transcriptome microarray to identify significantly repressed or induced Gene Ontology, Biological Process, Molecular Function, or Cellular Compartment categories in miR-22 transgenic hearts. *, P<0.05; **, P<0.005.(TIF)Click here for additional data file.Table S1Hemodynamic Analysis of TG-H mice and WT controls.(PDF)Click here for additional data file.Table S2Genes Dysregulated in miR-22 transgenic hearts.(PDF)Click here for additional data file.Table S3Potential Targets of miR-22 in the heart.(PDF)Click here for additional data file.


Blood platelets undergo a carefully regulated change in shape to serve as the primary mediators of hemostasis and thrombosis. These processes manifest through platelet spreading and aggregation and are dependent on platelet actin cytoskeletal changes orchestrated by the Rho GTPase family member Rac1. To elucidate how Rac1 is regulated in platelets, we captured Rac1-interacting proteins from platelets and identified Rac1-associated proteins by mass spectrometry.-/-P-Rex1-deficient mice were treated with platelet agonists or exposed to platelet activating surfaces of fibrinogen, collagen and thrombin. Platelets from -/- P-Rex1mice responded to platelet agonists and activating surfaces similarly to wild type platelets.Here, we demonstrate that Rac1 captures the Rac guanine nucleotide exchange factor P-Rex1 from platelet lysates. Western blotting experiments confirmed that P-Rex1 is expressed in platelets and associated with Rac1. To investigate the functional role of platelet P-Rex1, platelets from These findings suggest that P-Rex1 is not required for Rac1-mediated platelet activation and that the GEF activities of P-Rex1 may be more specific to GPCR chemokine receptor mediated processes in immune cells and tumor cells. Upon exposure to agonist signals of vascular injury, platelets spread out on sites of vessel damage to form thrombotic plugs ,2. DurinRho family GTPases are regulated in a cyclical manner by different classes of Rho-GTPase binding proteins. When platelets are stimulated to form a plug over the site of vascular injury, guanine nucleotide exchange factors (GEFs) such as Vav1 bind the Rac1 GTPase in its GDP conjugated form and catalyze a nucleotide exchange reaction to form Rac1-GTP . Rac1-GT9/ml. Lysates were prepared from resting platelets or platelets activated with 5 U/ml thrombin for 5 minutes. Immobilized Rac1-GST or GDP and GTP-loaded Rac1-GST were added to precleared lysates and incubated for 1 hour at 4°C. Rac1-associated proteins were eluted into Laemelli sample buffer and separated by PAGE. Silver-stained gel slabs from thrombin stimulated Rac1-GST eluates corresponding to 70 - 250 kD  from wild type (+/+P-Rex1) or -/- P-Rex1mice were placed on 100 μg/ml fibrinogen-coated coverglass in the presence of vehicle, the ADP scavenger apyrase (2 U/ml), or the GPCR agonists thrombin (1 U/ml) or ADP (10 μM) for 45 minutes at 37°C and were examined using differential interference contrast (DIC) microscopy. Platelets from wild type and -/- P-Rex1mice attached to fibrinogen surfaces at the same level -mediated events in immune cells  and tumoADP: adenosine 5'-diphosphate; CXCR: chemokine receptor; GEF: guanine nucleotide exchange factor; GST: glutathione S-transferase; GTP: guanosine-5'-triphosphate; GPCR: G-protein coupled receptor; MPER: Mammalian Protein Extraction Reagent; mTOR: mammalian target of rapamycin; PAR: protease activated receptor; PIP3: phosphoinositol-3,4,5 phosphate; P-Rex1: phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1.The authors declare that they have no competing interests.-/- -Rex1mice. All authors read and approved the final manuscript.All authors designed and carried out experiments. JEA wrote the manuscript. HCW supplied P


Natronomonas pharaonis halorhodopsin 3.0), we selectively inhibited D1-MSNs during cocaine exposure, which resulted in a behavioral blockade of cocaine-induced locomotor sensitization. Moreover, inhibiting these NAc D1-MSNs during cocaine exposure reversed the down-regulation of Tiam1 gene expression and protein levels. These data demonstrate that altering activity in specific neural circuits with optogenetics can impact the underlying molecular substrates of psychostimulant-mediated behavior and function.Exposure to psychostimulants results in structural and synaptic plasticity in striatal medium spiny neurons (MSNs). These cellular adaptations arise from alterations in genes that are highly implicated in the rearrangement of the actin-cytoskeleton, such as T-lymphoma invasion and metastasis 1 (Tiam1). Previous studies have demonstrated a crucial role for dopamine receptor 1 (D1)-containing striatal MSNs in mediating psychostimulant induced plasticity changes. These D1-MSNs in the nucleus accumbens (NAc) positively regulate drug seeking, reward, and locomotor behavioral effects as well as the morphological adaptations of psychostimulant drugs. Here, we demonstrate that rats that actively self-administer cocaine display reduced levels of Tiam1 in the NAc. To further examine the cell type-specific contribution to these changes in Tiam1 we used optogenetics to selectively manipulate NAc D1-MSNs or dopamine receptor 2 (D2) expressing MSNs. We find that repeated channelrhodopsin-2 activation of D1-MSNs but not D2-MSNs caused a down-regulation of Tiam1 levels similar to the effects of cocaine. Further, activation of D2-MSNs, which caused a late blunted cocaine-mediated locomotor behavioral response, did not alter Tiam1 levels. We then examined the contribution of D1-MSNs to the cocaine-mediated decrease of Tiam1. Using the light activated chloride pump, eNpHR3.0 (enhanced  Striatal medium spiny neurons (MSNs) play a crucial role in the molecular, cellular, and behavioral responses to psychostimulants . PreviouOne such set of molecules altered by cocaine are cytoskeletal-associated proteins that can directly alter structural plasticity, including spine density and dendritic branching in MSNs . The actNatronomonas pharaonis halorhodopsin 3.0 (eNpHR3.0), the yellow/green light activated chloride pump, during cocaine-induced locomotor sensitization and then examined Tiam1 gene expression and protein levels. Overall our studies demonstrate the regulation of Tiam1 following behavioral sensitization and cocaine self-administration. Moreover, we now begin to examine how cell type specificity, particularly the D1-MSNs, contributes to the underlying molecular mechanisms of psychostimulant induced structural plasticity and behavior. Furthermore, we examine whether altering activity in specific circuits with optogenetics can influence these underlying molecular mechanisms.Our previous study examined Tiam1 levels in the NAc after non-contingent home cage cocaine i.p. injections  but did ad libitum food and water. Sprague-Dawley rats, used in the self-administration experiment, were maintained on a 12-h reverse light/dark cycle ad libitum food and water. All studies were conducted in accordance with the guidelines set up by the Institutional Animal Care and Use Committee’s at The University of Maryland School of Medicine, The University at Buffalo, The State University of New York, and Mount Sinai Medical School.Mice used for optogenetic experiments were D1-Cre hemizygote (line FK150) or D2-Cre hemizygote (line ER44) bacterial artificial chromosome (BAC) transgenic mice on a C57Bl/6 background . Acute brain slices containing NAc were cut using a microslicer (Ted Pella) in cold sucrose-aCSF, which was derived by fully replacing NaCl with 254 mM sucrose and saturated by 95% O2 and 5% CO2. Slices were maintained in holding chamber with aCSF for 1 h at 37°C. Patch pipettes (3–5 MΩ), for whole-cell current-clamp and voltage-clamp recordings, were filled with internal solution containing the following: 115 mM potassium gluconate, 20 mM KCl, 1.5 mM MgCl2, 10 mM phosphocreatine, 10 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 2 mM magnesium ATP, and 0.5 mM GTP . Whole-cell recordings were carried out using aCSF at 34°C (flow rate = 2.5 ml/min). For current-clamp recordings to measure green (532 nm) light inhibition of action potentials we measured the effect of green light eNpHR3.0 activation on current (50 pA) injection-induced spiking. eNpHR3.0 activation-induced outward current was measured in voltage-clamp. Current- and voltage-clamp experiments were performed using the Multiclamp 700B amplifier and data acquisition was carried out in pClamp 10 (Molecular Devices). Series resistance was monitored during the experiments, and membrane currents and voltages were filtered at 3 KHz (Bessel filter). For light delivery to NAc slices, a 200 μm optical fiber  was connected via a FC (fiber-optic)/PC  adaptor to a 532 nm green laser diode (OEM Laser Systems).Whole-cell recordings were obtained from NAc MSNs in acute brain slices from D1-Cre mice that were injected with adeno-associated virus (AAV)-double-floxed inverted orientation (DIO)-eNpHR3.0-enhanced yellow fluorescent protein  followed by 4% paraformaldehyde (PFA). Brains were immersed in PFA overnight and then cryopreserved in 30% sucrose. Brains were cryosectioned (Leica) at 35 μm into 0.1 M PBS. Brain sections were blocked in 3% normal donkey serum with 0.3% Triton-X for 1 h at room temperature. Sections were then incubated overnight at room temperature in primary antibodies, 1:5,000 chicken anti-GFP (Aves) and 1:1,000 mouse anti-Cre (Millipore) diluted in the above blocking solution. On the second day, tissue sections were rinsed in 0.1 M PBS followed by a 1-h incubation at room temperature in secondary antibodies, 1:1,000 donkey anti-chicken-Alexa488 and 1:1,000 donkey anti-mouse-Cy3 (Jackson ImmunoResearch). Immunofluorescence was imaged on an Olympus Bx61 confocal microscope.For self-administration (SA) studies, male Sprague-Dawley rats weighing approximately 300 g at the start of the experiments were used. Rats were implanted with chronic indwelling jugular catheters and singly housed for the rest of the SA phase of the experiment in order to protect the catheter/harness assembly. The catheter implantations, maintenance, and patency testing procedures have been previously described elsewhere . Only ran = 6) or 1.0 mg/kg/infusion cocaine (n = 6). Cocaine hydrochloride  was dissolved in sterile saline. Two-hour daily testing occurred during the animals” dark cycle in Med-Associates experimental test chambers fitted with two snout poke ports, using a fixed ratio (FR) 1 schedule for 10 test sessions . After injections, implantable fibers  bilaterally to the NAc through a 62.5-μm split fiber patch cord coupled to a green (532 nm) or blue (473 nm) laser diode (OEM) by FC/PC connection. Locomotion was video tracked with Topscan tracking software . The animals were then sacrificed 24 h after the final session and NAc punches were quickly collected and stored at -80°C.2 PCR Profiler Array experiments, blue light stimulation was performed in the home cage. Mice received 15 min of 10 Hz blue (473 nm) light pulses (for 30 s every minute) for 5 days. The animals were then sacrificed 24 h after the final session and NAc punches were quickly collected and stored at -80°C.For ChR2 stimulation to D1-MSNs and D2-MSNs for RTCT method described previously (All NAc tissue punches were collected 24 h after the last cocaine administration and stored at -80°C. RNA was extracted using Trizol (Invitrogen) and the RNeasy Micro Kit (Qiagen) with a DNase step. RNA concentration was measured by Nanodrop Spectrophotometer and 400 ng cDNA was then synthesized using reverse transcriptase iScript cDNA synthesis kit (Bio-Rad). mRNA expression changes were measured using quantitative polymerase chain reaction (qPCR) with SsoAdvanced SYBR Green Supermix kit (Bio-Rad) or IQ SYBR Green Supermix (Bio-Rad). Quantification of mRNA changes was performed using the delta delta eviously , using gTiam1 mouse, forward: CGGAACAGATGGAAACACCT, reverse: AGAGACGACCACCCTTTCCT; Tiam1 rat, forward: GGTGCTGACACACGCCCCAA, reverse: AGGAAGCATGCCGCGTCCTC; caspase 1 mouse, forward: ATGCCTGGTCTTGTGACTTGG, reverse: AATGTCCCGGGAAGAGGTAGA; caspase 3 mouse, forward: AGCTGTCAGGGAGACTCTCAT, reverse: TTGAGGTAGCTGCACTGTGG; caspase 8 mouse, forward: GCGTGAACTATGACGTGAGC, reverse: AAGCCATGTGAACTGTGGAGA; caspase 9 mouse, forward: CGAAGCTCTCATGGCTTGGA, reverse: ACTGCTCCACATTGCCCTAC; neuronal nuclei (NeuN) mouse, forward: CAGACACTGGGGAAGACCTG, reverse: CCTAGGAACCCCCTGTCTGG; GAPDH mouse, forward: AGGTCGGTGTGAACGGATTTG, reverse: TGTAGACCATGTAGTTGAGGTCA; GAPDH rat, forward AACGACCCCTTCATTGAC, reverse TCCACGACATACTCAGCA.2 First Strand Kit, then run on a RT2 PCR Profiler Array , according to manufacture’s protocols and as previously described , phosphatase inhibitor cocktails I and II  and protease inhibitors (Roche) using a ultrasonic processor . Protein concentrations were determined using DC protein assay (Bio-Rad) and then 20 μg samples of total protein were loaded onto a 7.5% precast Tris–HCl polyacrylamide gel (Bio-Rad). The samples were transferred to a nitrocellulose membrane and blocked for 1 h in blocking buffer, 5% non-fat dry milk in Tris buffered saline (pH 7.6) with 0.1% Tween. Blocked membranes were incubated overnight at 4°C in blocking buffer with primary antibodies using either 1:1,000 rabbit-anti-Tiam1  or 1:2,000 β-Tubulin . Membranes were then incubated with goat anti-rabbit peroxidase-labeled secondary antibodies  in blocking buffer. The bands were visualized using SuperSignal West Dura Extended Duration substrate (Pierce). Bands were quantified with ImageJ Software and normalized to β-Tubulin to control for equal loading.post hoc test were used to analyze the self-administration and cocaine-mediated locomotor activity data. Student’s t-test or two-way ANOVAs followed by a Tukey’s post hoc test were used for mRNA, RT2 PCR Profiler Array, and protein analysis.Statistical analysis was performed with Graphpad Prism software. Repeated measures. Two-way analysis of variance (ANOVA) followed by with Bonferroni Figure 1A) for RNA and protein isolation. We find Tiam1 gene expression and protein levels are decreased in the NAc of rats that self-administer cocaine .Previously, we demonstrated that Tiam1 protein levels were down-regulated after repeated home cage cocaine exposure  and alsoin vivo in the NAc could alter cytoskeleton regulated genes. We expressed ChR2 or EYFP in the NAc of D1-MSNs and D2-MSNs by stereotaxic injection of the Cre-inducible adeno-associated virus AAV-DIO-ChR2-EYFP or AAV-DIO-EYFP to the NAc in D1-Cre and D2-Cre BAC transgenic mice. Mice received 5 days of 15 min blue light (473 nm) pulses to the NAc and gene expression analysis was performed on NAc tissue . Similarly, we see no change in Tiam1 mRNA levels after optogenetic activation of D2-MSNs during cocaine exposure, compared to EYFP cocaine controls despite observing decreased cocaine-mediated locomotor activity on days 4 and 5 under these conditions .Since altering activity in D1-MSNs vs. D2-MSNs oppositely regulates psychostimulant-mediated behaviors and reinforcement  we next c tissue  using thFigure 3A). We first validated the utility of green light (532 nm) to photo-inhibit NAc D1-MSNs expressing eNpHR3.0 and observed hyperpolarization of the membrane potential as well as an outward current during green light illumination of eNpHR3.0 expressing D1-MSNs . As expected, green light delivered to NAc D1-MSNs expressing eNpHR3.0 caused a decrease in D1-MSN firing . Finally, we found that 5 days of 60-min green light illumination to the NAc in vivo does not alter markers of cell death or NeuN gene expression  in NAc of D1-Cre mice expressing EYFP or eNpHR3.0-EYFP in D1-MSNs.We next investigated the contribution of D1-MSNs to the cocaine-mediated decrease in Tiam1, since many studies have demonstrated a positive role for D1-MSNs in the dorsal striatum and NAc in mediating the molecular, cellular, and behavioral responses to drugs of abuse . To assein vivo with green light during repeated saline or cocaine  injections over a 5-day period. As expected, cocaine treated D1-Cre mice expressing EYFP displayed enhanced cocaine-induced locomotion when compared to saline treated EYFP or eNpHR3.0 mice . In contrast, inhibition of D1-MSNs, expressing eNpHR3.0, with green light significantly blocked cocaine-induced locomotion . We observed significant differences on days 2–4 in cocaine treated D1-Cre eNpHR3.0 mice relative to D1-Cre EYFP cocaine treated mice . We next tested whether blocking cocaine-induced locomotor activity by silencing D1-MSNs can alter Tiam1 gene expression and protein levels in the NAc. As expected, we found that D1-Cre mice expressing EYFP had reduced levels of Tiam1 gene expression and protein levels in the NAc after 5 days of repeated cocaine  when compared to the saline EYFP or saline eNpHR3.0 groups . Consistent with our findings that inhibiting D1-MSNs, expressing eNpHR3.0, with green light attenuates cocaine-induced behavioral responses; we find that silencing D1-MSNs also reverses the cocaine-mediated negative regulation of Tiam1 gene and protein expression levels .We then illuminated NAc D1-MSNs, expressing eNpHR3.0 or EYFP, Repetitive cocaine exposure alters structural plasticity changes that are tightly correlated with the induction of behavioral sensitization . These cThe mechanism by which actin-cytoskeleton molecules are regulated after cocaine behavioral plasticity is unknown. We investigated the role for NAc MSN subtypes in regulating the actin-cytoskeleton molecules, including Tiam1, by using optogenetics to control activity in MSN subtypes. We find that increasing neuronal activity repeatedly over 5 days in D1-MSNs resulted in the down-regulation of Tiam1 mRNA, similar to the effects of cocaine. We observe no change in Tiam1 levels when activating D2-MSNs. We then investigated the role for NAc MSN subtypes in regulating the cocaine-mediated decrease in Tiam1 by activating D2-MSNs or inhibiting D1-MSNs during cocaine locomotor behavioral sensitization. Studies from our group and other groups using optogenetics or designer receptor activated by designer drugs (DREADDs) demonstrate a role for dorsal striatum and NAc D1-MSN activity in positively mediating motivational and motor behaviors . ConsistAlthough the precise molecular mechanisms by which Tiam1 is regulated in NAc MSNs after cocaine exposure is unclear, one intriguing mechanism is through an increase in the transcription factor deltaFosB which may mediate the expression of Tiam1. Indeed, deltaFosB, the long lasting isoform of the FosB gene, is stably increased selectively in D1-MSNs by cocaine  and has An important caveat to this study is the lack of cell type-specific Tiam1 gene expression or protein expression. Thus, we do not yet know whether the Tiam1 changes are occurring in D1-MSNs or the other MSN subtype or interneurons. Future studies combining these optogenetic manipulations of cocaine-mediated behaviors with cell type selective gene expression profiling  of Tiam1Collectively, our study associates a role for D1-MSN activity in regulating actin-cytoskeleton dynamics in cocaine dendritic and behavioral plasticity. We furthermore, show the utility of using optogenetics to manipulate psychostimulant-mediated behaviors to define the molecular mechanisms associated with drugs of abuse. Future studies to manipulate activity in specific cell subtypes during drug abuse behavioral paradigms coupled with molecular profiling could potentially lead to novel therapeutic targets to treat drug addiction.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.