RESEARCH ARTICLE Open Access Regulation of GAD65

Research Article Open Access Regulation Of Gad65-Free PDF

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Singh et al BMC Molecular Biology 2012 13 28 Page 2 of 12. http www biomedcentral com 1471 2199 13 28, cytokines like IL 1 beta have been shown to act as potent tumor suppressor protein and significantly down. negative regulators 15 GAD is an enzyme found in regulated in higher grades of breast cancer 44. high concentration in the pancreas and brain where it The present study delineates a novel mechanism of. catalyzes the conversion of glutamic acid to gamma regulation of GAD65 expression by two tumor suppressor. amino butyric acid GABA GABA is an inhibitory proteins SMAR1 and p53 We show that both the pro. neurotransmitter that is important in the pancreas as a teins can individually and synergistically upregulate. messenger between neurons and pancreatic cells GAD65 expression We demonstrate that SMAR1 binds. 16 17 Specific experiments in animal models have to GAD65 promoter in vitro and in vivo to upregulate its. shown that GAD expression is necessary for the auto mRNA expression Our results show that STZ treatment. immune destruction of cells One study in particular leads to upregulation of SMAR1 and p53 expression On. performed on NOD mice which are close to humans the other hand MDM2 expression is downregulated lead. in their autoimmune manifestation of IDDM incorpo ing to increased stability of p53 The stabilized p53 in turn. rated an antisense GAD transgene into a subset of mice binds to SMAR1 leading to an increased expression of. and found that IDDM development was significantly GAD65 We also show a temporal increase of SMAR1 and. reduced 18 p53 proteins on GAD65 promoter upon STZ treatment. Streptozotocin STZ derived from a fermentation Further studies using mouse islets confirmed our find. broth of Streptomyces achromogenes has been widely ings regarding the synergy between p53 and SMAR1 in. used to study the beta cell destruction both in vitro and GAD65 expression Taken together our results reveal a. in vivo models 19 20 The drug Streptozotocin STZ is novel mechanism of regulation of major auto antigen. a glucose analogue N methylnitrosocarbamoyl D glu GAD65 by SMAR1 and p53. cosamine that is rapidly transported into the cells via. the glucose transporter Glut2 21 and is known to be Results. metabolized readily upon entry into the cell The exact SMAR1 binds to multiple DNA sequences in genome. mechanism of STZ s toxicity is not fully understood but SMAR1 is documented to be a transcriptional activator. it has been suggested that its primary effect on beta cells repressor in a context dependent manner based on its. is DNA damage by alkylation unscheduled DNA synthe DNA binding abilities In vivo SMAR1 binds to the pro. sis DNA adducts chromosomal aberrations and DNA moters of Cyclin D1 and 5 UTR of cytokeratin 8. strand breaks induced by free radical generation 22 23 36 37 To gain a better understanding of the transcrip. The tumor suppressor protein p53 can cause cell cycle tion regulatory activities of SMAR1 we wanted to delin. arrest upon DNA damage induced activation 24 30 In eate various genomic DNA sequences bound by. doing so it facilitates the repair of damaged DNA or SMAR1 Therefore we resorted to a novel pull down. eliminates irreversibly damaged or abnormally growing assay Briefly mouse genomic DNA was sheared to ob. cells to prevent potential transformation Mdm2 helps to tain small fragments of approximately 500 bp These. maintain steady state levels of p53 under normal condi fragments were incubated with glutathione bead bound. tion through ubiquitination pathway 31 33 STZ has GST SMAR1 recombinant protein and the bound DNA. also been shown to induce p53 protein in MIN6 cells fragments were subsequently sequenced Glutathione. 34 but the exact mechanism is largely unknown One beads as well as bead bound GST were the controls used. study also reported p53 antibodies circulating in patients to negate non specific binders The resulting fragments. suffering from type 1 diabetes 35 were sequenced and then aligned using MEME software. Gene regulation is one of the most complex processes and a 50 mer consensus was derived Figure 1A Fur. involving cross talk between a variety of proteins nu ther manual alignments of the derived sequences lead to. clear matrix DNA and many other DNA binding fac identification of a hexamer TAATPu Py Pu consensus. tors The function of nuclear matrix is to provide a solid SMAR1 binding sequences where first four nucleotides. platform for efficient transcription A number of matrix are conserved while fifth one can be either pyrimidine. associated DNA region binding proteins MARBPs are or purine and the last nucleotide is either of the purines. known to be involved in regulation of transcription Figure 1A sequence shown in red Further we were. SMAR1 is one such MARBP which was earlier shown to able to validate our method by identification of some of. be involved in the regulation of Cyclin D1 and CK8 ex the already known promoters bound by SMAR1 includ. pression 36 37 SMAR1 specifically binds to putative ing cyclin D1 36 Bax 45 5 UTR of cytokeratin 8. MAR MAR of the transcriptional enhancer E at 37 One of the unique promoters found to be bound. the T cell receptor locus 38 39 This protein is by SMAR1 was GAD65 while the other isoform of GAD. known to cause cell cycle arrest and activates p53 i e GAD67 promoter did not show any binding In order. through its serine 15 phosphorylation as well as through to confirm that SMAR1 indeed binds to the GAD65 pro. disruption of its interaction with MDM2 40 43 It is a moter we performed CNBr pull down assay The. Singh et al BMC Molecular Biology 2012 13 28 Page 3 of 12. http www biomedcentral com 1471 2199 13 28, Figure 1 Isolation of DNA fragments bound by SMAR1 A MEME software based alignment of the SMAR1 binding DNA sequences for the. consensus for SMAR1 binding B CNBr pull down assay by consensus and by GAD65 promoter DNA samples were processed for western blot. analysis using SMAR1 specific antibody PI represents pre immune and Imm represents the immune complexes C D GST p53 GST SMAR1. GST Rxn5 DNA binding domain of SMAR1 GST Rxn6 Protein binding domain of SMAR1 along with GST and bead only controls was used for. in vitro pull down assays for DNA protein interactions The resulting DNA fragments were amplified by PCR using GAD65 C and GAD67 D. specific primers, consensus sequence from GAD65 promoter and the start site We found a strong p300 consensus element. general consensus oligonucleotide were individually 820 bp upstream and a p53 binding site 560 bp. coupled to CNBr beads and Rin 5f cell lysate was passed juxtaposed to SMAR1 binding sites A detailed map of. through the column The protein samples were eluted various binding sites is shown in Additional file 1. from the CNBr columns and processed for Western, blotting using SMAR1 specific antibody Our result SMAR1 binds to GAD65 promoter and upregulates its. showed that SMAR1 bound with equal propensity to expression. both GAD65 promoter sequence and the general con We further verified the binding of SMAR1 to GAD65 pro. sensus oligo Figure 1B lanes 4 and 5 In vitro pull moter using mobility shift assays A 120 bp probe from. down assays followed by PCR amplification showed that GAD65 promoter which harbors the potential MAR and. GST p53 350 548 aa DNA binding domain of SMAR1 SMAR1 consensus binding site was radiolabelled and used. 36 and the full length SMAR1 were able to precipitate for the assays EMSA using radiolabelled GAD65 pro. GAD65 promoter Figure 1C lanes 2 3 4 The pro moter probe showed a SMAR1 DNA complex formation. tein binding domain of SMAR1 i e GST SMAR1 160 Figure 2A lane 2 and a cold competitor reduced this. 350 aa GST bound beads and glutathione beads alone complex formation Figure 2A lane 3 showing the speci. did not show any amplification Figure 1C lanes 5 7 ficity of binding GAD67 and Actin Figure 2B lanes 1 3. showing the specificity of the interaction All these and 4 6 respectively promoter specific probes did not. samples were further analyzed for GAD67 promoter show any complex formation with SMAR1 recombinant. binding While we detected GAD67 promoter bound protein Also competition with cyclin D1 promoter oligo. to GST p53 neither the full length nor the DNA bind greatly reduced the complex formation on GAD65 oligo. ing domain of SMAR1 showed any binding to GAD67 and reflected the specificity of the complex formation. promoter Figure 1D lane 2 It is well known that Figure 2C lane 3 Similarly super shift assays with. p53 binds to GAD67 promoter 34 From the above SMAR1 specific antibody on using Rin cell lysate helped. mentioned results it is clear that SMAR1 specifically document SMAR1 complex formation on GAD65 pro. binds to the consensus sequence in GAD65 promoter moter oligo Figure 2D lane 2 and 3 The use of cold. GAD65 has a TATA less promoter and various other competitor in this experiment significantly reduced the. factors are known to regulate it Therefore we analyzed specific complex formation Figure 2D lane 4. GAD65 promoter for other transcription factor binding After confirming that SMAR1 binds to GAD65 pro. sites in silico A careful analysis of the sequence showed moter we proceeded to check the in vivo effect of. that SMAR1 binds 870 bp upstream of transcription SMAR1 binding on the promoter It is known that. Singh et al BMC Molecular Biology 2012 13 28 Page 4 of 12. http www biomedcentral com 1471 2199 13 28, Figure 2 SMAR1 binds to GAD65 promoter A Electro mobility Shift assay EMSA was done using GAD65 promoter DNA fragment which is.
bound by GST SMAR1 in EMSA lane 2 while cold competitor C C lane 3 reduced the binding showing the specificity of the interaction B. Promoter fragments of GAD67 and actin was used as negative controls and did not show any binding with GST SMAR1 proteins C Competitive. EMSA shows SMAR1 interaction with GAD65 promoter lane 3 is greatly reduced in presence of a competitor of cyclin D1 promoter oligo. compared to no competition lane lane 1 D Supershift assay using Rin5f cell lysate without lane 2 or with SMAR1 antibody lane 3 showing. shift in the GAD65 promoter band The upper band in lane 3 represents GAD65 SMAR1 SMAR1 ab complex while lower band shows SMAR1. GAD65 promoter complex only E Luciferase assay showing that SMAR1 and p53 together leads to up regulation of GAD65 promoter activity. Coexpression and knock down of SMAR1 and p53 in various combinations was used to look of luciferase activity of GAD65 promoter F Rin 5f. cells were treated with FLAG SMAR1 as well as siRNA of SMAR1 and western blot was carried out to test their effect over SMAR1 expression. upper panel Similarly cells were treated with FLAG p53 as well as siRNA of p53 and western blot using p53 antibody was done to check its. expression levels lower panel, GAD65 is the predominant form in rat while in mouse one of them is indispensible for activation of GAD65. both the forms are expressed Rat insuloma cell line promoter It has been reported that phosphorylation of. Rin 5f cells were co transfected with a luciferase re SMAR1 at serine 370 residue reduces its DNA binding. porter construct driven by GAD65 promoter and ex activity 30 unpublished data Transfection of S370A. pression plasmids siRNAs of SMAR1 and p53 The mutant SMAR1 led to a reduced GAD65 promoter ac. results show that GAD65 promoter drives the expres tivity compared to the wild type SMAR1 This was not. sion of reporter gene upon over expression of SMAR1 overcome by ectopic expression of p53 Figure 2E. or p53 witnessed by an increase of 4 and 4 5 folds lanes 8 9 respectively This result clearly indicates. respectively Figure 2E On the other hand knock that direct binding of SMAR1 is essential for GAD65. down of either of these proteins leads to a decreased promoter activation and that the effect of SMAR1 is. luciferase activity driven by GAD65 promoter Over not through stabilization activation of p53 In order to. expression of SMAR1 and p53 together lead to the verify our results we performed western blot analysis. highest luciferase counts 6 folds increase indicating to confirm over expression as well as siRNA mediated. their additive effect on GAD65 promoter On the knockdown of SMAR1 and p53 Figure 2F shows the. other hand the knockdown of both lead to negligible expression levels of SMAR1 upper panel as well as. promoter activity Knock down of p53 and over ex p53 lower panel in Rin5f cells. pression of SMAR1 partially rescued 1 5 folds the, luciferase activity These results indicate that although SMAR1 leads to upregulation of GAD65 expression. SMAR1 or p53 individually can up regulate GAD65 Next we verified the expression of GAD65 upon over ex. promoter activity their synergistic activity is required pression of SMAR1 RT PCR results showed that upon. for maximal promoter activity that in turn reflects the SMAR1 over expression GAD65 and p53 mRNA levels. transcriptional activation On the other hand either are elevated in a dose dependent manner Figure 3A. Singh et al BMC Molecular Biology 2012 13 28 Page 5 of 12. http www biomedcentral com 1471 2199 13 28, Figure 3 SMAR1 upregulates GAD65 expression A RT PCT analysis of GAD65 and p53 upon dose dependent SMAR1 over expression Actin. was used as loading control B Densitometric analysis of the RT PCR showing 4 fold increase in p53 and 7 fold increase in GAD65 mRNA levels. upon SMAR1 over expression C SMAR1 was over expressed in Rin5f cells and samples were processed for western blot analysis 48 hrs post. transfection Figure shows western blot analysis of t. RESEARCH ARTICLE Open Access Regulation of GAD65 expression by SMAR1 and p53 upon Streptozotocin treatment Sandeep Singh2 Varsheish Raina1 Pavithra

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