CFTR is tightly controlled by regulators including CCAAT/enhancer binding proteins (C/EBPs). We previously reported that the transcription factors YY1 and USF2 affect CFTR expression. We can now demonstrate that C/EBPβ, a member of the CCAAT family, binds to the CFTR promoter and contributes to its transcriptional activity. Our data reveal that C/EBPβ cooperates with USF2 and acts antagonistically to YY1 in the control of CFTR expression. Interestingly, YY1, a strong repressor, fails to repress the CFTR activation induced by USF2 through DNA binding competition. Collectively, the data strongly suggest a model by which USF2 functionally interacts with YY1 blocking its inhibitory activity, in favour of C/EBPβ transactivation. Further investigation into the interactions between these three proteins revealed that phosphorylation of C/EBPβ influences the DNA occupancy of YY1 and favours the interaction between USF2 and YY1. This phosphorylation process has several implications in the CFTR transcriptional process, thus evoking an additional layer of complexity to the mechanisms influencing CFTR gene regulation.The promoter of the cystic fibrosis transmembrane conductance regulator gene  CFTR expression is functional in maturation CFTR promoter et al. CFTR. Through the functional analysis of sequence variations within the CFTR promoter, our laboratory identified other trans-acting proteins including YY1 and USF2 with repressor and activator activities, respectively CFTR gene sequence between −226 and +135 bp upstream of the major transcription initiation site The CCAAT/enhancer binding proteins (C/EBPs) encompass a family of structurally similar yet functionally and genetically distinct transcription factors with functional roles in a number of physiological activities C/EBPβ encodes the different polypeptide isoforms liver-enriched activatory protein (LAP) Through the differential usage of AUG codons within the same transcript, et al. HIV-1 LTRDetermining the function of C/EBP transcription factors may be achieved in part through protein-protein interactions Alternatively, multiple phosphorylation events have been shown to be critical in the regulation of members of the C/EBP family CFTR gene expression and attempt to unravel the molecular mechanisms involved. Considering that C/EBPβ phosphorylation has already been demonstrated to modulate its DNA binding capacity and the transcriptional activities of other genes CFTR transcription.In this report, we investigate the role of C/EBPβ in the control of For transfection assays the following expression vectors were used: pBS-C/EBPβ , pCMV-LIP and pCMV-LAP , pCMV-Flag-A-C/EBP , pCDNA3-hLAP-T235A , pCR3-USF2  and pCDNA3-YY1 .cis-acting elements, mutations within the core CAAT were generated from the WT-pGL3 plasmid using an oligonucleotide-directed mutagenesis system  according to the manufacturer’s instructions. Mutagenesis primers are available on demand. The presence of mutations and the sequences fidelity were verified by dideoxynucleotide sequencing.To study the importance of the C/EBPβ CFTR, were transiently transfected as previously described CFTR promoter plasmid, 0.008 µg of internal control pRL-SV40 containing Renilla luciferase and 0.004 to 0.08 µg of each expression vector. Transfections with siRNA were performed using Fugene6® reagent (Roche diagnostics) with 1.2 µl of either control  or C/EBPβ siRNA  for three wells. When indicated, medium containing sodium fluoride  was added to cells. All luciferase activities represent at least three independent experiments with each construct tested in triplicate per experiment.Human bronchial epithelial cells, Beas2B, expressing endogenous Protein extracts, resuspended directly in 1X Laemmli sample buffer, were resolved on a 10% SDS-polyacrylamide gel as previously described CFTR primers . Reverse transcription was performed as previously described  primers  using th6 cell equivalents of lysate were used. Following shearing of the crosslinked chromatin by sonication, chromatin was incubated at 4°C on a rotating stand overnight with either 3 µg of anti-C/EBPβ, anti-USF2, anti-YY1 or non-specific antibodies (anti-HA). ChIP samples (immunoprecipitated or input DNA) were analyzed by either classical PCR or quantitative PCR. For both analyses, primers used for CFTR promoter amplification are reported in CFTR gene which lacks both C/EBPβ and YY1 motifs: 5′-TGTGGGGAGGGAAATAGATG-3′ and 5′-GCAGAGTTTGCAGTGAGCTG-3′. The fold change in the specific binding was normalized to mock input values. For quantitative ChIP, all PCR reactions were performed in triplicate in a final volume of 10 µl containing 2 µl of sample, 5 µM of each primer (ChIP assays were performed using the Chromatin Immunoprecipitation Assay Kit (Upstate) following Upstate ChIP protocols, with minor modifications. As indicated, Beas2B cells were transfected with either expression vectors or C/EBPβ siRNA and/or treated with 10 mM of NAF for 4 hours. Prior to harvesting, cells were incubated with 5 mM of DTBP for 30 min at RT, followed by treatment with formaldehyde 1% for 5 min. For each immunoprecipitation, 2×10h primer , 0.1 µl Nuclear extracts were prepared from Beas2B cells as previously described t-test. Data were considered statistically significant at p<0.05. All graphical data and statistical analyses were generated with GraphPAD Prism software (Version 3.0). Densitometric analyses were performed using computerized densitometry and Quantity One® software (Biorad). Densitometric values for both protein band (immunoblot) and promoter CFTR amplicons (ChIP) were determined in areas of equal size and were reported in normalized arbitrary units relative to either expression of LaminA/C protein or input (non-immunoprecipitated chromatin), respectively.Transfection data are expressed as the mean ± S.E. Paired comparisons were made using student’s CFTR promoter, we performed chromatin immunoprecipitation (ChIP) assays using crosslinked protein-DNA complexes prepared from Beas2B cells. The immunoprecipitates obtained with antibodies directed against the C-terminus of C/EBPβ and a non-specific control protein were used as templates to amplify a 360 bp DNA fragment corresponding to the minimal CFTR promoter. Quantitative PCR (qPCR) amplifications of DNA immunoprecipitated by anti-C/EBPβ revealed that endogenous C/EBPβ binds to the CFTR promoter in Beas2B cells (CFTR gene devoid of any C/EBPβ binding motif (data not shown).To evaluate whether C/EBPβ binds to the minimal 2B cells . This bi2B cells  was enfo2B cells . As a neCFTR promoter fused to a luciferase-coding cDNA in Beas2B cells. Overexpression of the C/EBPβ-LAP construct resulted in a 3.7 fold increase promoter activity (CFTR promoter and the observed decrease in promoter activity supported a dominant negative influence of the LIP isoform as previously reported CFTR gene promoter activation following transient co-transfections of the dominant-negative (A-C/EBP), reported to dimerize with C/EBPβ thus blocking its DNA binding  were performed to identify putative binding sites for the transcription factor C/EBPβ. The results indicated that the CFTR minimal promoter region contains different binding motifs for C/EBPβ as depicted in CFTR promoter directly or exerts its effects through an indirect activation of other proteins that do directly influence CFTR, mutagenesis reactions of the core sequence (TTNNNCAATNNN) of each C/EBPβ binding site in the reporter construct were performed. As shown in CFTR transcription, this would suggest the implication of additional transcription factors.CFTR in proximity to the C/EBPb3 motif. In a first set of experiments, transient co-transfection assays were carried out to evaluate the putative cooperative role of C/EBPβ with USF2, Sp1 and SRF positive factors (binding motifs depicted on CFTR promoter is activated by the concerted action of C/EBPβ and USF transcription factors.Based on the knowledge that C/EBPβ heterodimerization is important for its DNA binding CFTR promoter CFTR promoter activity. This hypothesis was investigated by transient co-transfections with both C/EBPβ and YY1 expression vectors. As observed in CFTR gene devoid of any YY1 motif was performed (denoted negative control). C/EBPβ overexpression significantly reduced the YY1 DNA binding activity  or vanadate  treatment at different concentrations on the CFTR transcription. While vanadate did not modify the CFTR expression level (data not shown), treatment with either 5 mM or 10 mM NAF significantly increased the luciferase activity to over 1.5- and 2.5-fold, respectively . Western blot assays were carried out to assess the protein expression levels under the indicated treatments. Immunoblotting with a specific anti-phospho-C/EBPβ antibody showed that treatment with NAF increased the level of phosphorylated C/EBPβ (CFTR expression was observed after A-C/EBP overexpression following NAF treatment (data not shown). These results indicate that NAF-induced C/EBPβ phosphorylation is involved in CFTR activation. To assess whether the increased CFTR expression could be an indirect consequence of the general effects of NAF on promoter efficiency, we performed transfection assays using mutant hLAPT235A vector (denoted T235A), in which the conserved Thr235 is mutated to Ala. As presented in CFTR. The results confirmed the data obtained by gene reporter assays . As depicted in CFTR promoter but the level of binding was much lower by comparison with the phosphorylated C/EBPβ. These results were confirmed by ChIP-qPCR showing that LAPT235A overexpression induced a considerable decrease in C/EBPβ binding (nearly 3-fold) that was associated with a modest increase in YY1 binding activity (1.5-fold) when compared to endogenous DNA binding activity for either the C/EBPβ or YY1 transcription factors (set as 1), respectively . The DNA binding activity for USF2 was not altered following C/EBPβ or LAPT235A overexpression (data not shown). These data show that phosphorylation of C/EBPβ increases its DNA binding ability and disrupts the YY1 occupancy of its DNA cognition site.Considering that C/EBPβ appears to undergo phosphorylation in correlation with the acquisition of DNA-binding activity in vivo  and YY1 in vivo  occupancConsequently, we investigated whether this latter effect is mediated by interactions between YY1 and USF2. Co-immunoprecipitation assays demonstrated that NAF treatment does indeed favour the interaction between YY1 and USF2 . As a potrans-acting factors play pivotal roles in airway epithelial cell differentiation during lung organogenesis SP-A and CFTRtrans-acting factors including SRF, YY1, USF2, Sp1 and Nrf2 that contribute to CFTR transcriptional activity CFTR expression pattern, we investigated the functional role of C/EBPβ alongside some previously reported transcription factors.C/EBPβ and both related polypetides LAP (liver-enriched activatory protein) and LIP (liver-enriched inhibitory protein) belong to the CCAAT/Enhancer binding proteins (C/EBP) family of transcription factors. These nuclear CFTR promoter activity in bronchial epithelial cells. Based on knowledge of the CFTR promoter being quite weak, this trans-acting factor may be considered as a strong activator. The results obtained with the C/EBPβ-specific siRNA and with mutants for the C/EBPβ binding site demonstrate the importance of C/EBPb3 and its binding motif located within a region previously documented to contain two mutations identified in CF patients CFTR mRNA level was never completely abolished in our assessments is evidence of the C/EBPβ transcription factor controlling CFTR transcription in a coordinated fashion with other partners. We have illustrated here its cooperation with USF2. Moreover, considering the repressor role of YY1 in CFTR transcriptional regulation CFTR transcriptional activation elicited by C/EBPβ is antagonized by YY1 overexpression. We also showed that this functional antagonism between YY1 and C/EBPβ might be partially due to mutually exclusive DNA-binding activities. In addition, although we previously reported that USF cooperates with Sp1 to transactivate the CFTR gene and that YY1 antagonizes SRF, we failed to demonstrate any cooperativity between C/EBPβ and either SRF or Sp1 in our promoter context. Indeed, USFs and Sp1 may act as negative regulators when they interact with transcription factors bound simultaneously to the same site or at other sites, suggesting a dual mode of regulation of genes by these factors . Interestingly, the results of this study confirm the complex link between the multiple transcription factors that regulate CFTR transcription.The data presented in this report show that C/EBPβ positively influences CFTR activation induced by USF2. Several concordant results strongly argue in favour of USF contributing towards C/EBPβ-mediated CFTR activation through a functional USF/YY1 interaction. This interaction could at least partially explain the cooperation between C/EBPβ and USF2 and the DNA competition observed between C/EBPβ and YY1. Based on these data, we propose a model of interaction between USF and YY1 by which USF decreases the inhibitory activity of YY1, either through interacting with the domain mediating the YY1 repressive activity or by impeding access of co-regulators. Indeed, p300 which has been shown to bind YY1, may be essential for YY1 repressor activity via physical interaction To further explore the relationship between the C/EBPβ, USF2 and YY1 factors, transient co-transfections were performed. Enforced expression of YY1, a powerful repressor, was unable to inhibit CFTR transcriptional activity. By using the Ser/thr phosphatase inhibitor NAF, we demonstrated an increase in C/EBPβ phosphorylation, DNA occupancy and endogenous CFTR expression in bronchial epithelial cells. To confirm these results, we performed transient transfection, RT-qPCR and qChIP assays by using a LAPT235A expression vector, mutated on threonine 235. We found that this mutant protein, unable to be phosphorylated on its Thr residue, could only induce a modest activation of CFTR transcription compared to the increased CFTR expression induced by C/EBPβ overexpression and significantly altered the CFTR mRNA level. In addition, the overexpression of the LAPT235A protein induced a considerable decrease in the C/EBPβ DNA binding activity  and an increase in the YY1 DNA occupancy. The ChIP analyses were consistent with the results obtained with the NAF treatment. However, other sites of phosphorylation, not studied in our system, might be implicated in C/EBPβ activity as was previously reported (13). These data are in agreement with a recent study showing that phosphorylation of the CREB transcription factor influences CFTR expression CFTR transcriptional activation (CFTR regulated expression observed during the development of certain tissues including lung formation In addition to their acquisition of transcriptional activity through selective interactions, transcription factors also require posttranslational modifications in order to function. This highlights the need to study other levels of regulation. Interestingly, it has been shown that C/EBPβ is mainly activated by serine/threonine phosphorylation, which results in its heterodimerization and an increase in its DNA binding tivation . This fiA crucial point raised is which factors help determine between antagonism and cooperation within a given cell? The answer to this question is very complex and to date not yet elucidated. One important consideration is the expression level of each transcription factor which depends on the specific cell type. For instance, it has been reported that the LIP/LAP ratio may dictate the C/EBPβ transcriptional activity and depend on the cell type CFTR gene.This work has highlighted that such a mode of regulation involving cooperative and antagonistic interaction coupled with posttranslational modification, is an interesting avenue that we will pursue in the future to further unravel the complex regulation of the


Phosphatase and TENsin homologue deleted from the human chromosome 10), TPTE (Transmembrane Phosphatase with TEnsin homology) and TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) belong to a family of dual-specific lipid and protein phosphatases. PTEN is a well characterized tumor suppressor, which plays crucial role in cell survival, cell cycle regulation, cell proliferation as well as adhesion, motility and migration of cells. The C2-domain of PTEN is essential for PTEN-functions. We have isolated a novel 1019 bp human TPIP cDNA (TPIP-C2) from a human testis cDNA library. In silico analysis of the cDNA revealed that it is produced from the TPIP-locus on the human chromosome 13 by alternative RNA-splicing. It has a unique 5′-Alu sequence, a LINE sequence followed by a 582 bp Open Reading Frame (ORF) encoding a 193 aa polypeptide with a partial phosphatase domain and a C2-domain. TPIP-C2 mRNA is expressed in human testis and in mouse tissues. Mouse testis and brain showed higher levels of TPIP-C2 mRNA in comparison to the heart, liver and kidney under normal physiological conditions. TPIP-C2 mRNAs from human and mouse testes show extensive sequence identity. Over-expression of TPIP-C2 cDNA in HeLa cells strongly (up to 85%) inhibited cell growth/proliferation and caused apoptosis in a caspase 3-dependent manner. These findings suggest for the first time that a TPIP splice-variant mRNA with a partial phosphatase domain and a C2-domain is expressed in cells and tissues of human and murine origins under normal physiological conditions. Inhibition of cell growth/proliferation and induction of apoptosis by overexpression of TPIP-C2 mRNA in HeLa cells suggest that it may be involved in negative regulation of cell growth/proliferation.Alternative splicing of mRNAs is known to involve a major regulation of gene expression at RNA level in mammalian cells. The PTEN ( TP53 and is mutated or deleted in a wide variety of cancers. In addition to genetic mutations, somatic, germ-line and promoter mutations of PTEN are responsible for Cowden syndrome (CS), Bannayan-Riley-Ruvalcaba Syndrome (BRRS), Proteus and proteus-like syndrome etc 383 phosphorylation/dephosphorylation Mammalian genomes contain relatively less numbers of genes, which encode large numbers of proteins. This is effective by alternative splicing of the primary transcripts to generate splice-variants (SVs) of mRNAs, which code for isoforms of proteins with variable functions. The PTEN/MMAC1 (Mutated in Multiple Advanced Cancers-1)/TEP1 (TEnsin-like Phosphatase-1) was identified as a tumour suppressor gene from the human chromosome 10q23.3 Other phosphatases of PTEN-family include TPTE and TPIP. TPTE is a testis-specific gene expressed from the human chromosome 21, while TPIP is expressed from the human chromosome 13. Both TPTE and TPIP have multiple splice-variants i.e., TPTEα, β, γ, δ In the present study, we have isolated a novel TPIP splice-variant (TPIP-C2) mRNA from a human testis cDNA library. TPIP-C2 mRNA can code for a 193 aa putative C2-domain-like protein. It is produced from the human TPIP-locus on chromosome 13 by alternative RNA-splicing and expressed in human testis and mouse tissues. The TPIP-locus of mouse genome is expressed as TPIP-C2 mRNA in the tissues. Functional assay showed that ectopic expression of TPIP-C2 cDNA caused up to 85% suppression of cell growth/proliferation and induced apoptosis in human cervical carcinoma (HeLa) cells. Therefore, the alternatively spliced TPIP-C2 mRNA encoding an isolated C2-domain-like protein may exist in mammalian cells and tissues and negatively regulate signaling events involved in regulation of cell growth/proliferation.AluSx-class, a 61 nt. (268–328) LINE sequence belonging to L3CR1 class followed by a 582 nt (340–921) encoding a 193 aa ORF  DNA probe identified genomic DNA fragments of expected sizes based on in silico analysis of the genomic organization of the locus + transcript of 2.5 kb was specifically detected in the human testis RNA but not in the spleen, thymus, prostate, ovary, small intestine, colon and peripheral blood leukocytes RNA  blot and ytes RNA . A similytes RNA  was eithytes RNA . The ful control . These rWe checked TPIP-C2 mRNA expression in mouse tissues. RT-PCR analysis was carried out to check whether TPIP-C2-like mRNA is expressed in mouse tissues or not, although no TPIP RNA/cDNA has been reported from mouse till date. We developed a specific RT-PCR assay to distinguish TPIP-C2 transcript from RNAs produced from other TPIP-isoforms. Based on the sequence homology, the ORF-primers (P1 and P2)  are expeWe checked possible cellular function of TPIP-C2 mRNA in human cells. TPIP-C2 cDNA was expressed from pCDNA-TPIP-C2 plasmid in HeLa cells by stable transfection followed by G418-selection resulting into G418-resistant colonies expressing TPIP-C2 mRNA. Overexpression of TPIP-C2 mRNA caused up to 85% decrease in the number of G418-resistant colony formation. This is shown by two independent experiments with two different DNA concentrations (2.5–10 µg DNA per 10 cm plate and 1–5 µg DNA per well of 6 well plate). The suppression of cell growth/proliferation increased from 12% to 85% with increasing amount of cDNA transfected into the cells showing a DNA-dose-dependent effect . The magFrom these results it is clear that TPIP-C2 mRNA is a splice variant (SV) from the human TPIP-locus and the TPIP-C2 protein product is completely identical with the C2-domain of other TPIP isoforms . Therefore, we inserted a HA-tag to make pCDNA-TPIP-C2-HA expression plasmid so that upon transfection, the protein can be detected by western blot using anti-HA antibody. Expression of HA-tagged TPIP-C2 protein is shown in HeLa cells transfected with the HA-tagged plasmid . AntibodTo further validate the growth/proliferation suppressive effect of TPIP-C2 in HeLa cells, cell proliferation was measured by a cell viability assay (MTT) in TPIP-C2 transfected HeLa cells. The transfection efficiency was ∼40% to 50% as determined by transfection of 1 µg of pEGFP-N2 plasmid in HeLa cells . As showIn this study, we report a new isoform of TPIP mRNA, which was isolated from the human testis cDNA library and we named it as TPIP-C2 mRNA, this is in addition to the previously described TPIPα, β, γ and δ isoforms. Mammalian genome maintains the genomic DNA as the blue print of the organism with high degree of consistency and transcribes it into large numbers of RNAs with high degree of fidelity and variability. Primary transcripts from many human genes are processed by different posttranscriptional mechanisms to produce isoforms of RNAs and proteins for carrying out various cellular functions The TPIP-C2 SINE is also present in 3′-UTR of many other human transcripts indicating its functional significance. Since Alu sequences are abundant and polymorphic in the human genome and most of the human genome is transcriptionally active, we expect presence of Alu sequences in several RNAs. This may have functional consequences and evolutionary significance. SINE and LINE elements located in promoters of some human genes have been reported to contain high-affinity binding sites for hormone receptors, their presence in enhancers suggest role of these mobile DNA elements in regulation of gene expression TPIP-C2 mRNA is strongly expressed in human testis as a 2.5 kb mRNA and weakly expressed in other tissues . It is d2+ ligands, and thus it is similar to the C2 domains of the Ca2+-independent protein kinase C (PKC)-isotypes 2+-dependent manner as shown for C2A-domain of synaptotagmin I in vitro via its C2-domain and mutation of basic residues in this region reduces PTEN's membrane affinity and its ability to suppress growth of glioblastoma tumor cells The TPIP-C2 protein belongs to the PTEN-C2 superfamily of phosphatases. The three-dimensional structure of PTEN provides a deep insight into potential mechanisms by which the PTEN phosphatase can recognize and dephosphorylate its 3-phosphate-containing phospholipid substrates. The PTEN-C2 domain lacks the canonical CaInterestingly, over-expression of TPIP-C2 mRNA in HeLa cells also showed similar effects like cell cycle-arrest, suppression of cell growth/proliferation and induction of apoptosis like PTEN  and 5. T2+-dependent membrane binding 2+-independent PTEN-like C2-domain protein, probably membrane localized, and it induces apoptosis and negatively regulates cell growth/proliferation possibly as a dominant negative molecule by interfering with proteins involved in the interactions of extracellular matrix (ECM) and cell surface associated as well as intracellular proteins. The detailed molecular mechanism of the cell growth/proliferation inhibition by TPIP-C2 mRNA/protein needs further investigation.It has been reported that isolated PTEN C2-domain can inhibit cell migration in U373 cells The 1.019 kb TPIP-C2 cDNA was sequenced and analyzed online using BLAST, ORF finder and RepeatMasker for genomic localization, possible ORF and repeats. CLUSTAL-W amino acid sequence alignment and Kyte and Dolittle hydropathy score were carried out, genomic organization of TPIP-C2 was found by comparing with the 119.33 kb human genomic sequence on chromosome 13 (NT_024524.13) by BioEdit online. The 5′ splice site was identified by comparing TPIP-C2 cDNA with the genomic contig (NT_024524.13). Prediction and comparison of domains was carried out by ELM, SMART through ExPASy Proteomics tools.32P]dATP (specific activity = 4000 Ci/mmole) was from BRIT, India. Anti-β-actin monoclonal antibody (A5316), anti-HA-antibody (H9658), anti-caspase 3 active (C8487), MTT (M2128), Propidium iodide (P4170) were from Sigma-Aldrich.Molecular biology and tissue culture grade materials and reagents were purchased from Sigma-Aldrich (U S A), New England Biolabs and Promega (U S A), synthetic oligonucleotides were from Life Tech. (U S A) and Microsynth (Switzerland), Lipofectamine™ 2000 (Invitrogen), the human testis λgt11 cDNA library, the human RNA-blots for expression analysis and pEGFP-N2 plasmid were from Clontech (U S A). The radioisotope,  primer, 0.5 mM dNTPs, 20 U RNasin and 100 U of M-MLV Reverse Transriptase in 1× M-MLV RT-reaction buffer in 25 µl. First strand cDNA mixture was used as template and pCDNA-TPIP-C2 plasmid was used as positive control (Pc) for PCR. The reaction contained 1× PCR buffer 2SO4, 0.2 mM dNTPs, 2.0 mM MgCl2, 25 pmole primers (12.5 pmole each), 1.0–5.0 µl 1st strand cDNA templates and 1.0 U Taq DNA polymerase. The PCR cycle parameters were initial denaturation at 94°C for 4 min followed by 20–35 cycles of denaturation at 94°C for 45 sec, annealing at 68°C (TPIP-ORF) or 51°C (TPIP-C2 and β-actin), 60°C (GAPDH) for 45 sec-1 min, polymerization at 72°C for 1 min and a final extension of 10 min at 72°C. TPIP-ORF primers amplified a 603 bp C2-ORF from human TPIPα, γ and δ, TPTEα, β, γ mRNAs, whereas, TPIP-C2 primers are specific for the TPIP-C2 transcript and amplified a 733 bp amplicon. The 311 bp β-actin and 452 bp GAPDH products were used as control and normalization signals. The PCR products  were electrophoresed in 1.5% agarose-TBE gels, photographed by an AlphaImager 3400 gel-documentation system and densitometry of specific DNA bands was carried out  by the AlphaImager software.RNA was isolated from mouse tissues and human cells. First strand cDNAs were synthesized from 1.0 µg of total RNA with 0.5 µg of oligo dT . The introns are depicted as dotted line and exons as black boxes. The number below the exons represents length of respective exon. The exon sequences corresponding to the chromosome 13 genomic regions are mentioned (upper panel). TPIP-C2 exons and introns are with respect to human DNA sequence from the clone RP11-408K19 on chromosome 13 [AL590076] (lower panel).(A) TPIP-C2 cDNA (1.019 kb inserted DNA) is cloned at EcoR I site of pBSKII(PDF)Click here for additional data file.Figure S232P] labeled 1.019 kb full length TPIP-C2 cDNA probe showing homologous RNA expressions due to presence of SINE/LINE sequences present in the full length TPIP-C2 cDNA. Almost all samples show the signals. A number of controls are indicated at the right hand side of the MTE blot.(A) Sources of human cells and tissues for the mRNAs used in the dot blots. (B) Hybridization of the MTE-blot with [(PDF)Click here for additional data file.Figure S34–101 copies of the plasmid template (bottom panel) showing the sensitivity of PCR reaction at 101 copy number. (B) Chromatograms of DNA sequencing of one each type of representative RT-PCR products cloned from the mouse tissues showing single nucleotide changes from the expected sequence. (C) The amino acid changes with respect to the single nucleotide changes in TPIP-C2 193 aa. ORF are shown. M: Marker, Pc: positive control, Lc: 20 ng of 0.74 kb DNA as a loading control for densitometric measurements, B: brain, H: Heart, T: testis, L: liver and K: kidney of mouse.Expression of TPIP-C2 mRNA in mouse tissues. (A) TPIP-ORF amplicon is 605 bp (339–921 nt) and TPIP-C2 amplicon is 733 bp (285–2017 nt) as shown by PCR/RT-PCR products amplified by using P1+P2 and P3+P4 primers, respectively. TPIP-ORF primers contain extra flanking sequences and Hind III site at their 5′-ends, thus, instead of 582 bp amplicon, it amplifies 605 bp DNA. Establishment of TPIP-C2-specific PCR assay (top panel), Lane 1 and 2: pCDNA-TPIP-C2 plasmid control for TPIP-ORF and TPIP-C2 PCR, lane 3 and 5: TPIP-ORF PCR with ORF 339–921 and ORF 285–1087 templates, lane 4: ORF 339–921 and β-actin templates were mixed and PCR-amplified for TPIP-C2 and β-actin (311 bp) products with respective primers, lane 6: TPIP-C2 PCR from ORF 285–1087 template, TPIP-C2 plasmid PCR with 10(PDF)Click here for additional data file.Table S1Homology of TPIP-C2 CDNA with human genome.(DOC)Click here for additional data file.Table S2Presence of TPIP-C2 SINE and LINE in human transcripts.(DOC)Click here for additional data file.


Giardia lamblia is a protozoan parasite that is found worldwide and has both medical and veterinary importance. We applied the transcription start sequence (TSS-seq) and RNA sequence (RNA-seq) techniques to study the transcriptome of the assemblage A WB strain trophozoite. We identified 8000 transcription regions (TR) with significant transcription. Of these regions, 1881 TRs were more than 500 nucleotides upstream of an annotated ORF. Combining both techniques helped us to identify 24 ORFs that should be re-annotated and 60 new ORFs. From the 8000 TRs, we were able to identify an AT-rich consensus that includes the transcription initiation site. It is possible that transcription that was previously thought to be bidirectional is actually unidirectional. Giardia lamblia , a member of the family Hexamitidae, is a diplomonad parasitic protozoan that infects humans and that was discovered by Leeuwenhoek (1681). Giardia has a worldwide distribution and infects a wide range of animals in addition to humans. It is a common cause of diarrhea in both developed and developing countries. For example, Giardia is the most commonly detected human intestinal parasite in the United States Giardia is simple, containing only the trophozoite and cyst stages. The trophozoites are pear shaped, measuring 12–15 µm in length and 4–9 µm in width, with a ventral sucking disk, four pairs of flagella and two identical nuclei. The cysts are oval in shape, measuring 5×7–10 µm, and they contain four nuclei The life cycle of Giardia occurs when the parasite escapes the immune response of the host due to the expression of variant surface proteins (VSP). Giardia has approximately 228 VSP genes, and all of them seem to be transcribed; however, only one is expressed at the cell membrane. The mechanism of selective expression seems to be related to RNA interference and post-transcription regulation. Switching between VSPs leads to changes in the cell wall antigens, allowing the parasite to escape the host’s immunity Recurrent infection with GiardiaGiardia lamblia assemblage A was sequenced and published. The assemblage A genome is ∼12 Mb distributed over 5 chromosomes with nearly ∼ 77% representing open reading frames (ORF) with multiple repeats, short intergenic regions, few introns and short untranslated 5′ and 3′ regions (UTRs) Giardia has the ability to translate mRNAs with very short 5′-UTRs, which may be as short as one nucleotide (nt) GiardiaGiardia might have some split genes There are seven different genetic assemblages known for Giardia, improving our understanding of the unique features of these parasites and allowing us to identify expressed genes and verify annotated ones. Combining the oligo-capping method The availability of second-generation sequencers has made it easier and less expensive to study the gene expression profile of Giardia has a small genome and only two stages and it is an important human and animal pathogen with a widespread distribution of infection, it is a good target for such analysis. Determining which genes are transcribed and making these data available to the research community is important to understanding the encystation process and the mechanisms by which Giardia escapes immunity. Here, we applied both TSS-seq and RNA-seq methods to Giardia lamblia assemblage A trophozoites cultured in vitro. We aimed to study the relationship between transcription start sites and annotated genes, to confirm known data about Giardia, to identify possible new ORFs and to determine whether bidirectional transcription is due to conserved motifs.Another method is RNA sequencing (RNA-seq), whereby cDNA is directly sequenced, allowing for correct identification and quantitation of gene expression and detection of introns Giardia lamblia trophozoites  of the same strain that was used to obtain the genome sequence were maintained in modified TYI-S-33 medium 6/ml by centrifugation for at 1500 rpm for 4 minutes. Five volumes of Trizol  were added to the parasite pellet, mixed by pipetting and then drop-frozen in liquid nitrogen. A pestle and a mortar were used to grind the Trizol pellets to ensure complete destruction of the cell walls and improve the RNA extraction yield. The RNA was then purified.5′-AATGATACGGCGACCACCGAG-3′) (5′-CAAGCAGAAGACGGCATACGA-3′) under the following reaction conditions: 15 cycles of 94°C for 1 min, 56°C for 1 min and 72°C for 2 min. The PCR fragments were size-fractionated and used for the sequencing reactions with the Illumina GA. In total, 36 cycles of the sequencing reactions were performed according to the manufacturer’s instructions.A sample containing 200 µg of the obtained total RNA was subjected to oligo-capping by treatment of the RNA with bacterial alkaline phosphatase  and tobacco acid pyrophosphatase , followed by ligation with RNA-oligo (5′-AAUGAUACGGCGACCACCGAGAUCUACACUCUUUCCCUACACGACGCUCUUCCGAUCUGG-3′) using RNA ligase (TaKaRa) Giardia lamblia ATCC50803 genomic sequencing  with the sequence alignment program Eland. Unmapped or redundantly mapped reads were removed from the data set. Reads with more than two mismatches were also removed. The reads were sorted into TSS sites, and the TSS sites were then clustered. Due to the compactness of the genome and the small intergenic distance, a size window of 10 nts was used to count the TSS sites. Overlapping transcription windows were merged and designated as transcription regions (TRs). TSS reads were considered to be in different TRs if they were separated by more than 10 bases without any TSS reads in between. The start position of a TR or the position of a TSS site with the highest sequence read number was used to evaluate the correlation between TRs and ORFs. For further details, see the supplementary material and methods, The obtained sequences were mapped onto the To calculate the background distribution, the Poisson distribution was used. The background distribution was estimated to be ≈1.3 reads for a 10-nt window. In this study, a TR was considered significant if it contained a TSS site with 5 or more reads.To verify the mapping results for the TSS reads, 20 TRs 100 nt downstream from the start site that were more than 500 nt away from any ORF and 10 TRs of 200 nt that were within 500 nt upstream an ORF were chosen to be amplified using RT-PCR. Primer-BLAST was used to design the specific primers st strand cDNA, which was followed by synthesis of the 2nd strand. The ends were repaired, and the 3′ ends were adenylated. The fragments were ligated to adapters and amplified for 15 cycles.To verify the TSS reads and confirm their relationships to ORFs, high throughput RNA-seq was performed using TruSeq™ (Illumina) according to the manufacturer’s protocol. Briefly, 1 µg of total RNA was purified and fragmented and then used to synthesise the 1The RNA-seq tags were mapped to the genome. Due to the presence of multiple repeats and the small genome size, Integrative Genomics Viewer (IGV) The metagenomic ORF finding tool Orphelia was used to read multiple ORFs in a 700 base pair model to identify ORFs when no ORFs were found within the 500-nt downstream TR WebLogo, a web-based application that can be used to draw logos for conserved sequences in relation to their positions, was used to detect and draw a sequence consensus Giardia lamblia, WB clone, ATCC50803. A total of 6,343,253 34-base sequences was generated and mapped to the same strain as that used for genome version 1.1 (http://Giardiadb.org/common/downloads/release-1.1/Glamblia), allowing a 2-base mismatch. Reads were deposited at DDBJ Sequence Read Archive, accession number DRA001089.We obtained 2,600,245 uniquely mapped reads that were sorted into 404,331 TSS sites , which contains 9747 ORFs. Of these, 3766 ORFs are deprecated. According to the distance from the first ATG, the TRs were categorised into four categories, A, B, C and D  TRs were located more than 100 nt upstream of the 1st ATG, and 1881 (23.5%) of these were located more than 500 nt upstream (position D category). The transcripts from these further upstream TRs could correspond to transcripts with long 5′-UTRs or sterile polyadenylated transcripts. Elmendorf et al. We used RT-PCR to verify some of the TSS data. Twenty targets were chosen from TRs located more than 500 nt upstream of annotated ORFs (Position D), and 10 targets were selected from the TRs that were within 500 nt of ORFs . In total, 17 out of the 20 position D category TRs and 5 out of 10 of the position A, B or C category TRs were amplified, as shown in We used Orphelia Giardia genome contains 9747 ORFs, of which 3766 ORFs are deprecated. We found 4187 ORFs with TRs located from 500 nt upstream of the start codon to the end of ORF. Among them, 554 are deprecated ORFs. A total of 111 of the 575 deprecated ORFs had TRs within 100 nt upstream of the start codon. Thus, these 554 ORFs may be re-considered, or only the 369 ORFs that do not completely overlap with non-deprecated genes. From the RNA-seq data, we could evaluate the expression of 363 of the 575 deprecated ORFs. An example is the deprecated gene GL50803_23497 shown in The Giardia strains that have been sequenced  family; 5 are re-annotated ORFs and 8 are new ORFs that were expressed according to both TSS and RNA-seq. Another 6 ORFs with Ankyrin-like conserved domains were also found. Using BLAST®, we identified similar genes in either the same 50803 strain or in other equenced .Teodorovic et al. We evaluated our results to detect the bidirectional relationships of the 8000 significantly expressed TRs to the entire set of 63795 TRs. With up to 300 nt difference, we found total of 3686 bidirectional pairs of TRs. Of those pairs, 1175 pairs  had significant expression in both directions, while 2521 pairs had significant expression in one direction and insignificant expression to the opposite direction. We attempted to determine whether there is any conserved sequence (consensus) for the highly expressed bidirectional TRs by examining the sequences 150 nt upstream and 150 nt downstream from the nucleotide positioned midway between the start sites of each pair. However, we were not able to find any conserved sequences for a promoter region.Giardia lack motifs patterns seen in other eukaryotes so we decide to use alignment tools to find any conserved sequences. Using the start position as a land mark, we failed to find any consensus within 100 nt upstream of the start position of the TR. We thought that the transcription start site with highest copy reads might be the most effective point of reference. We examined 50 nt up stream and 50 nt downstream from that position and identified an AT-rich consensus, which is shown in Giardia. This finding demonstrates the importance of precise mapping of TSS.As the 5`-UTR is very short, this result raised the possibility that there is no real bidirectional transcriptional consensus, but that unidirectional transcription events occur near each other. Thus, we decided to examined the presence of a consensus for the entire 8000 TRs. The use of motif search tools was unsuccessful as Giardia. A variant of this consensus was predicted by Holberton and Marshall ATTTTAAAAT) was among the sequences suggested by Yee et al., We created a weight matrix for this consensus and evaluated the TR regions again. We found that only 565 TRs lack this consensus. Although many researchers have predicted some sequences such as (CAAT) or (AG) as conserved motifs within 40-100 nt upstream of the transcription initiation site, we did not find any other conserved consensus within 150 nt upstream or downstream of the TSSs with highest reads. Furthermore, we examined up to 300 nt upstream of the TSSs with the highest reads for 565 TRs that lack the transcription initiation consensus. We did not detect any conserved consensus for these 565 TRs. We did find this consensus variant among the sequences reported by Teodorovic et al., As the consensus is somewhat symmetrical, we investigated the possibility of true bidirectional transcription, allowing a ±5-nt difference. We found that 928 pairs of TRs  were bi-directionally significantly expressed, while the antisense transcripts of 1195 TRs were insignificantly expressed. The occurrence of bidirectional transcription was not related to the symmetry of the consensus, as it is in some cases  A; the sGiardia. We failed to identify any other motifs in Giardia. This raises the question of how transcription starts in other places that lack the transcription initiation consensus. Further work is needed to address this question. The presence of the transcription initiation consensus for the majority of the genes shows how simple yet efficient the transcription mechanism of Giardia is.Combining the TSS-seq and RNA-seq techniques was a powerful approach for identifying new genes, confirming or re-annotating known genes and identifying unusually long 5`-UTRs. TSS-seq allowed us to identify the correct transcription sites, which helped us to find the transcription initiation consensus in Figure S1Mapping of TSS sequence copy and TR clustering.(TIF)Click here for additional data file.Figure S2How to measure Distance between Transcription regions (TR) and open reading frames (ORFs).(TIF)Click here for additional data file.Figure S3Relation between TR and ORFs if the TR overlap an ORF. *If ORF1 and ORF2 is in the same orientation: TR3 is Upstream-TR if ATG of ORF1 is nearer than ATG of ORF2. **If ORF1 and ORF2 is in the opposite orientation: TR4 is Always Upstream-TR irrespective nearness of ATG to ORF1 or ORF2(TIF)Click here for additional data file.Figure S4How to evaluate positions of TRs in relation to ORFs.(TIF)Click here for additional data file.Table S1List of targets and primers used in RT-PCR.(DOCX)Click here for additional data file.Table S2Statistics of TSS reads.(DOCX)Click here for additional data file.Table S3Details of transcription regions (TRs) position in relation to annotated open reading frames(ORFs).(DOCX)Click here for additional data file.


We find that nucleosome arrangement at the hox promoters is a progressive process that takes place over several stages. At stages immediately after fertilization, nucleosomes appear to be largely disordered at hox promoter regions. At stages after activation of the embryonic genome, nucleosomes are detectable at hox promoters, with positions becoming more uniform and more highly occupied. Since the genomic sequence is invariant during embryogenesis, this progressive change in nucleosome arrangement suggests that trans-factors play an important role in organizing nucleosomes during embryogenesis. Separating hox genes into expressed and non-expressed groups shows that expressed promoters have better positioned and occupied nucleosomes, as well as distinct NDRs, than non-expressed promoters. Finally, by blocking the retinoic acid-signaling pathway, we disrupt early hox gene transcription, but observe no effect on nucleosome positions, suggesting that active hox transcription is not a driving force behind the arrangement of nucleosomes at the promoters of hox genes during early development.Nucleosome organization at promoter regions plays an important role in regulating gene activity. Genome-wide studies in yeast, flies, worms, mammalian embryonic stem cells and transformed cell lines have found well-positioned nucleosomes flanking a nucleosome depleted region (NDR) at transcription start sites. This nucleosome arrangement depends on DNA sequence (cis-elements) as well as DNA binding factors and ATP-dependent chromatin modifiers (trans-factors). However, little is understood about how the nascent embryonic genome positions nucleosomes during development. This is particularly intriguing since the embryonic genome must undergo a broad reprogramming event upon fusion of sperm and oocyte. Using four stages of early embryonic zebrafish development, we map nucleosome positions at the promoter region of 37 zebrafish  The nucleosome is comprised of an octamer histone core wrapped nearly 1.7 times by approximately 147 bp of DNA that represents the basic unit of eukaryotic chromatin de novo. These models are reasonably successful at predicting nucleosome positions in yeast C. elegansNucleosome positioning is partially encoded by the DNA sequence and experimental studies have identified sequences that favor  to treat murine ES cells hox clusters have also been observed during mouse embryogenesis using 4C technology hox promoters in human cell lines Nucleosome organization has been analyzed in blastula stage hox genes under different conditions. We first examined nucleosome arrangements at the TSS of all 37 genes at various stages of embryogenesis and find relatively poorly positioned and weakly occupied nucleosomes at 2 hpf and 4 hpf. Notably, no hox genes are expressed at these stages of development and we do not observe NDRs at these time points. At the 6 hpf and 9 hpf time points nucleosomes become better organized. The progressive nature of nucleosome positioning on the invariant sequence of hox promoters through early development suggests an important role for trans-factors in positioning nucleosomes at hox promoters. More detailed analyses revealed that promoters of genes expressed at these stages have better nucleosome organization and occupancy with an NDR immediately upstream of the TSS. Non-expressed promoters have nucleosomes that are less organized and lack an NDR at early stages, suggesting that NDR formation correlates with gene expression. However, blocking hox gene transcription by disruption of the RA signaling pathway results in no change in nucleosome positioning or NDR formation, indicating that transcription does not drive nucleosome organization at hox promoters. Our data therefore indicate that trans-factors act at hox promoters during embryogenesis to dynamically rearrange nucleosomes independently of hox gene transcription.We have mapped nucleosomes near the TSS (herein referred to as ‘promoter’) of 37 zebrafish This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Massachusetts (A-1565).Ekkwill (EK) embryos were collected through natural matings and staged using morphological criteria for two, four, six, and nine hours post fertilization (hpf) as defined by Kimmel et al 33mm CaCl2, 0.33 mM MgSO4, and 0.004% methylene blue). Embryos remained in RA-treated water until they were harvested . Diethylaminobenzaldehyde (DEAB): 4–8 cell embryos (∼1–1.25 hours post fertilization) were treated with 10 uM DEAB diluted in fish-water. Embryos remained in DEAB-water until the developmental stage harvested. Drug concentrations were chosen based on embryonic survival to limit embryonic death.Retinoic acid (RA): 2 cell embryos (∼45 minutes post-fertilization) were treated with 100 nM RA diluted in fish-water  and mechanically dissociated by pipetting. Cells were washed once with PBS, resuspended in 1% formaldehyde in PBS and incubated for 10 minutes at 27°C. The reaction was quenched with equal volume of 1M glycine and cells were spun down at 5000 g.Embryos were collected and the chorion was removed using 10 mg/ml Pronase. Embryos were then washed with Fish ringers  and incubated for 30 minutes on ice. Cells were pipetted vigorously again and diluted 1∶1 with GB buffer . Nuclei were purified by layering on an equal volume of GB and spun at 1000 g for 10 minutes at 4°C.Protocol was adapted from Dennis et al 2007 2, 1 mM CaCl2, 1 mM β-mercaptoethanol, 500 uM spermidine and 500 uM DTT) followed by resuspension in reaction buffer with a titrated amount of MNase  and incubated at 37°C for 10 minutes. Reactions were terminated with 50 mM EDTA and placed on ice. Samples were then diluted in water and treated with 1× RNase cocktail (Ambion: AM2286) and 200 mM NaCl (to remove RNA and reverse crosslinks) and incubated at 55°C for 2 hours. 2 ul proteinase K (20 mg/ml) was added and samples were placed at 65°C overnight. Chromatin was extracted using phenol:chloroform followed by ethanol precipitation. Samples were visualized by gel electrophoresis and samples containing a 80–90% mono-nucleosome DNA (faint tri-nucleosome band visible) were used for tiling array hybridization. Mono-nucleosome sized fragments were gel extracted using the Qiagen Gel Extraction kit (28706).Protocol was adapted from Yuan et al 2005 hox clusters was masked for repetitive sequence using the Sanger Institute’s Zebrafish RepeatMasker (http://www.sanger.ac.uk/Projects/D_rerio/fishmask.shtml). The resulting sequences were used to construct a 144 k feature array of 50 bp probes positioned every 20 bp designed using Agilent eArray web software (https://earray.chem.agilent.com/earray/GEO: GPL16536). Isolated mono-nucleosome sized fragments were hybridized to the hox array using protocols adapted from Agilent protocols substituting COT DNA for salmon sperm DNA . Arrays were scanned using either an Axon 4000B or Agilent’s High-Resolution C Scanner.Zebrafish genome v7 sequence of the seven hox gene was calculated. Log2 ratios were calculated based on normalized r-processed and g-processed signals from the Agilent chip for each probe. Mean signal from two replicates for each sample was assigned to each probe location. Signals were tallied using a 30 bp sliding window with a step of 10 bp for each window. A Lowess fitting line (f = 0.05) was plotted to show the trend of the aggregated signals. Nucleosome spacing was calculated based on the predicted di- and mono-nucleosome sized fragments identified from gel images, represented in Probe sequences were remapped to Zv9 and the distance from the center of a probe to the TSS of the nearest hox gene expression was determined using both Agilent and Affymetrix Zebrafish expression arrays. Only genes found to be expressed by both platforms were included in the RA and WT expression groups. Agilent Arrays: RNA was isolated from retinoic acid treated and untreated WT zebrafish embryos at 2 hpf, 4 hpf, 6 hpf, and 9 hpf embryos using Trizol (Invitrogen#15596-026) following standard procedures. RNA was processed and hybridized to Agilent Zebrafish (V3) Gene Expression Microarrays (G2519F-026437) essentially as outlined in Agilent protocols. Since no hox genes are reported to be expressed maternally, the 2 hpf WT embryonic sample was taken to represent baseline and signal above this baseline was taken to represent expression (GEO: GSE43756 ). Affymetrix Arrays: RNA was isolated from retinoic acid treated embryos at 4 hpf, 6 hpf, and 9 hpf while RNA from untreated embryos was collected at 9 hpf. RNA was processed and hybridized to Zebrafish Genechip Arrays (900487) by the UMass Genomic Core facility using standard Affymetrix protocols. CEL files from Affymetrix were normalized using invariantset probe set and background corrected by mas5 using expresso from the R affy package. Present/absent calls were calculated using mas5 call from R affy package with default parameters (GEO: GSE43755).hox gene cDNA was quantified by QPCR using the Qiagen QuantiFast SYBR Green PCR kit (204054) on an ABI 7300 thermocycler. hox expression was normalized to a beta-actin control. Data represents 3 technical replicates.DEAB-treated embryos were collected at 9 hpf and RNA was extracted using Trizol. cDNA was synthesized using the Superscript III RT First strand cDNA synthesis kit priming with oligo dT (18080-051). hoxb1a: FWD-5′-ACC TAC GCT GAC TTA TCG GCC TCT CAA GGAAG TGT GGC AGC AAT CTC CAC ACGRVS-5′-CTC hoxb7a: FWD-5′-CCA TCC GAA TCT ACC CAT GGT GAG CGCCGA TAC GCC GCC GTC TTG AAA GGRVS-5′-TCT hoxb1b: FWD-5′-GGT TCG TTC AGC AAG TAT CAG GTC TCC CCCAA GTT CCG TGA GCT GCT TGG TGGRVS-5′-TCT hoxb5b: FWD-5′-CCT AAC CCA GGA CCA GTG CAA GAC GGTCC GTC AAA CAC AGA GCG TGC GRVS-5′-CGT hoxb6b: FWD-5′-AGT GCA AGA CGG ACT GCA CAG AAC AGGTCC GTC AAA CAC AGA GCG TGC GRVS-5′-CGT hoxc8a: FWD-5′-AGC AAG AGG CCA CCT TAG CGC AAT ACCCAA TAC GGC GCT TGC GTG TGA GGRVS-5′-CTT hoxc9a: FWD-5′-CGG AGA CTG TTT GGG CTC GAA CGG ATCA TAT CGC CGG TCT CTT GTG AGG TRVS-5′-ACC Beta-Actin: FWD-5′-ATA CAC AGC CAT GGA TGA GGA AAT CCCGT CCA ACA ATG GAG GGG AAA ARVS-5′-GGT hox genes  embryos from 2, 4, 6, and 9 hours post fertilization (hpf). These time points were chosen since zygotic gene expression is initiated at 3–4 hpf in the zebrafish hox genes are not transcribed, while in 6 hpf and 9 hpf embryos some cell populations have begun to differentiate and hox gene transcription is being initiated. Nucleosome densities were determined by micrococcal nuclease (MNase) digestion of cross-linked chromatin isolated from staged embryos (adapted from hox clusters. Randomly fragmented mononucleosome sized genomic DNA (gDNA) was co-hybridized as a control. The nucleosomal signal was expressed as a ratio of the MNase digested fragments to the random gDNA fragments. Nucleosome densities were averaged for 37 zebrafish hox genes  regardless of their expression state Several groups have reported a nucleosome-depleted region (NDR) flanked by −1 and +1 nucleosomes upstream of the TSS in many metazoan genes  relative to 4 hpf (when hox genes are not expressed). This is particularly clear when the amplitude of the −1 nucleosome peak is compared to the amplitudes of the adjacent peaks. Expressing the amplitude of the −1 nucleosomes as a ratio to the +1 nucleosomes reveals that the −1 nucleosome in expressed promoters at 6 hpf and 9 hpf is reduced by 35% and 43%,respectively  signaling pathway is an activator of blockade . Plottin embryos . When ho embryos . Overlay embryos . Hence, hox promoters. Embryos were treated with RA starting at the 2-cell stage and collected at 2 hpf, 4 hpf, 6 hpf and 9 hpf. We initially examined average nucleosome organization at all 37 hox promoters. We find the nucleosome profiles of RA-treated embryos to be similar to the profiles of untreated embryos, although there are some minor differences when overlayed , and from factors that interact with DNA, such as transcription factors and ATP-dependent chromatin modifiers (trans-factors). However, the relative contribution of each mechanism remains unclear. A recent study addressed how cis-elements and trans-factors influence nucleosome positioning in yeast. By using YACs to transfer large DNA fragments between divergent yeast strains, analysis of nucleosome organization in the native strain could be compared to nucleosome organization on the YAC in the new host strain hox promoters in human cell lines identified an NDR upstream of the TSS hox genes, we observe an NDR as soon as −1 and +1 nucleosomes are resolved at the TSS  were initially identified at promoters in yeast, but have subsequently been identified in other cell types. In most cases, NDRs are readily observed in bulk analyses of promoters regardless of whether the promoters are active or not. Indeed, previous analyses of bulk S 9 hpf, . The NDRhox promoters, which was reported to be ∼500 bp hox gene might have a nucleosome positioned upstream of the TSS, thereby reducing the size of the NDR observed when signals from all cells in the embryo are averaged. Indeed, a previous study found nucleosomes to be differentially positioned at the serum albumin enhancer in a tissue specific manner in mouse hox promoters in human cell lines. Second, the difference in NDR length may be due to differences between fish and humans. For instance, divergence of regulatory sequences in the promoters as well as divergence in the trans-factors responsible for nucleosome positioning may lead to different sized NDRs. Support for this possibility comes from the analysis of NDRs in evolutionary divergent yeast species, which were found to have different sized NDRs at orthologous promoters Though the NDRs observed in our study are similar to other bulk studies, they are smaller than the NDR previously observed at human hox promoters would create more uniform nucleosome positions as well as increased amplitude of nucleosome peaks, while the lack of trans-factor binding at non-expressed genes would lead to lower occupancy and less well-positioned nucleosomes. Such competition has been observed at the CLN2 promoter in yeast where binding sites for three sequence specific transcription factors are needed for NDR formation. In the absence of these binding sites, the CLN2 promoter has increased nucleosome occupancy hox promoters and are involved in the regulation of hox transcription, have been suggested to act as pioneer transcription factors capable of binding nucleosome-occupied DNA hox promoters. Since RA-receptors may be bound to DNA even in the absence of RA-signaling hox promoters. Second, NDR formation could be an active process mediated throughout embryogenesis by ATP-dependent remodelers. ATP-dependent SWI2/SNF2 complexes, which slide nucleosomes through DNA sequence, have been previously shown to regulate hox genes hox promoters by DNA binding factors such as the Meis and Pbx factors mentioned above.Our data do not address how NDRs form, but we consider several possibilities. First, NDRs could form in a competitive process. Evidence exists for competition between nucleosomes and trans-factors for binding to specific sequences hox promoters are bivalently marked with both H3K4me3 and H3K27me3 at this stage hox gene expression and homeotic transformations in Drosophilahox promoters must be rapidly modified in order to ensure proper regulation of hox genes.The accessibility of genomic DNA is regulated not only by nucleosome positioning, but also by post-translational modifications made to the N-termini of histone tails, that in turn affect chromatin structure. For instance, histone H3 lysine 4 tri-methylation (H3K4me3) by trithorax group proteins and histone H3 lysine 27 tri-methylation (H3K27me3) by polycomb group proteins, associate with active and inactive promoters, respectively File S1File with Figures S1 and S2. Figure S1Comparison of biological replicates used for calculation of nucleosome densities. Data from two biological replicates were plotted against each other for untreated embryos at 2 hpf (A), 4 hpf (B), 6 hpf (C), 9 hpf (D), as well as for RA-treated embryos at 2 hpf (E), 4 hpf (F), 6 hpf (G), 9 hpf (H) and for DEAB-treated embryos at 9 hpf (I). R2 values are indicated in the top right quadrant of each panel. Figure S2Representative MNase digestion. Cross-linked genomic DNA from 4 hpf embryo was left untreated (lane 2) or treated for 10 minutes at 37°C with serially diluted concentrations of micrococcal nuclease (MNase) increasing from 0.5 units/ml −8 units/ml (lanes 3–6) and separated by agarose gel electrophoresis. Lanes 1 and 7 contain size ladders.(PDF)Click here for additional data file.


An RNA-Seq snapshot of neonatal heart transcriptome showed 1,536 of 20,000 total transcripts were altered (p < 0.05) - 10 up- and 1,526 downregulated. Pathway enrichment analysis with MetaCore™ showed Hltf’s regulation of the G2/M transition (p=9.726E-15) of the cell cycle in heart is nearly identical to its role in brain. In addition, Brca1 and 12 members of the Brca1 associated genome surveillance complex are also downregulated. Activation of caspase 3 coincides with transcriptional repression of Bcl-2. Hltf loss caused downregulation of Wt1/Gata4/Hif-1a signaling cascades as well as Myh7b/miR499 transcription. Hltf-specific binding to promoters and/or regulatory regions of these genes was authenticated by ChIP-PCR. Hif-1a targets for prolyl  and lysyl (Plod2) collagen hydroxylation, PPIase enzymes  for collagen trimerization, and lysyl oxidase (Loxl2) for collagen-elastin crosslinking were downregulated. However, transcription of genes for collagens, fibronectin, Mmps and their inhibitors (Timps) was unaffected. The collective downregulation of genes whose protein products control collagen biogenesis caused disorganization of the interstitial and perivascular myocardial collagen fibrillar network as viewed with picrosirius red-staining, and authenticated with spectral imaging. Wavy collagen bundles in control hearts contrasted with collagen fibers that were thin, short and disorganized in Hltf null hearts. Collagen bundles in Hltf null hearts were tangled and fragmented. Thus, silencing Hltf during heart organogenesis compromised DNA double-strand break repair, and caused aberrant collagen biogenesis altering the structural network that transmits cardiomyocyte force into muscle contraction.HLTF/Hltf regulates transcription, remodels chromatin, and coordinates DNA damage repair. Hltf is expressed in mouse brain and heart during embryonic and postnatal development. Silencing Hltf is semilethal. Seventy-four percent of congenic C57BL/6J Hltf knockout mice died, 75% within 12-24 hours of birth. Previous studies in neonatal (6-8 hour  Congenital heart defects (CHDs) - 1 in 100 newborns - result from errors in the development of heart valves, chambers and blood vessels . Growth postpartum) hearts and brains revealed the Hltf null phenotype derives from defects in the G2/M transition (p=3.360E-10). This finding is consistent with Hltf’s role in the maintenance of genomic stability. Additional findings unique to Hltf null heart – decreased transcripts for Wt1 and Gata4 and their downstream targets, downregulation of transcripts for Brca1/BASC complex in DNA damage repair, disorganization of the collagen fibrillar network via decreased transcription of hypoxia-inducible factor (Hif-1a) and its downstream targets, and decreased transcripts for Myh7b/miR499 – demonstrate a critical role for Hltf in heart function.HLTF/Hltf was first described as a DNA-binding protein -12 and sSanta Cruz Biotechnology, Inc.  was the source of antibodies to N-terminal residues of HLTF (sc-27542X), and Protein A/G PLUS-Agarose (sc-2003) beads. Abcam  was the source of antibodies to Hltf residues 600-700 (ab17984) and the Caspase 3 Assay Kit (ab39401). GE HealthCare  was the source of donkey anti-rabbit IgG HRP (NA934V). Ready Gel Tris-HCl precast polyacrylamide gels , Kaleidoscope Prestained Standards (161-0324) and Immuno-Star Western Kit (170-5070) were purchased from Bio-Rad . Magic Mark XP (LC5602) western protein standards were purchased from the Invitrogen division of Life Technologies . PerkinElmer Life Science Products  was the source of Kodak Film (NEF596). OneTouch Ultra Mini and OneTouch Ultra Mini Blue test strips were purchased from LifeScan , a Johnson & Johnson Company, for blood glucose monitoring. For collagen detection, ScyTek Laboratories, Inc.  was the source of the Picrosirius red stain kit (PSR-2), and Chondrex, Inc,  was the source of the Hydroxyproline assay kit (#6017). Mouse heart extract was prepared with the Active Motif  kit (#40010). DNeasy Blood & Tissue Kit (69506) was purchased from Qiagen  for DNA purification. Expand Long Template PCR System Buffer (11681842001) and PCR nucleotide mix (11814362001) were purchased from Roche Applied Science . NucleoSpin RNA II, a complete kit for isolation and purification of total RNA was purchased from Clontech . SuperScript III first-strand synthesis system for RT-PCR was purchased from the Invitrogen division of Life Technologies . PCR primers  were synHltf knockout mice were developed in collaboration with genOway  as previously documented . ConventThe RT component of RT-PCR was performed according to the manufacturer’s instructions for the combined use of oligo-dT and random hexamer priming. Each 50µl PCR reaction consisted of template DNA, primer pairs  were performed as previously detailed ,20,28,29Differential interference contrast (DIC) images (63x oil) for picrosirius red with excitation and barrier filters for FITC (intrinsic cytoplasmic and elastic fiber green fluorescence) were captured with a Zeiss AxioVert 200 inverted fluorescence microscope, AxioVision software, and an AxioCam-camera. OD values 530-560 nm) for the hydroxyproline content of standards and samples were read with the Infinite M100 PRO Quadruple monochromator microplate reader (Tecan). Intrinsic fluorescence of tissue, including elastic and collagen fibers, were obtained using spectral imaging  were flash frozen and sent to Otogenetics Corp.  for RNA-seq assays. Total RNA was isolated; its integrity and purity were assessed using Agilent Bioanalyzer and OD260/280 . As prevC/A C T/A T A/T/G T/G) binding site [We previously identified a consensus Hltf  enabling direct comparisons of expression levels among transcripts and across experimental conditions. RPKM and total read counts were reported for each gene.Otogenetics via DNAnexus provided an unbiased gene expression analysis report of RNA-seq; alternative splicing analysis of Hltf; mutation/RNA-editing analysis and parallel comparison of expression profiles between null and control samples. FPKM (fragments per kilobase of transcript per million mapped reads) were mapped against mm9 with Tophat (V2.0.5) and cufflinks.cuffdiff (V 2.0.2) for parallel analysis of six mouse RNA-seq samples. Differences between samples were considered statistically significant at q < 0.05. Using a free trial, data were imported to MetaCore™ for pathway analysis . Standard enrichment parameters  were used. Analyses from brain and heart were merged with MetaCore™. p < 0.05 significance level). The p value for the difference between elevations caused by the presence or absence of Hltf expression was p = 0.0001. Regression analyses of expression level were measured by RPKM for control vs. Hltf null values. Hydroxyproline assays were performed on HCl (12N) hydrolyzed whole hearts according to the manufacturer’s protocol. A standard curve with hydroxyproline standards was evaluated by linear regression (r=0.99941). Replicate values of serially diluted samples from null  and control  mice were compared by Student’s t-test (p < 0.05 significance level). Blood glucose levels were compared by the Mann-Whitney U-test (p < 0.05 significance level). The p value was p=0.0084.All statistical tests were conducted with GraphPad Prism V.6.0b (GraphPad Software). Caspase 3 assays were performed on whole heart extract according to the manufacturer’s protocol. Triplicate values were evaluated by ANCOVA  and lysyl (Plod2) hydrolyases that are essential for collagen hydroxylation and subsequent folding  as well These studies provide the first opportunity to explore primary and secondary regulatory effects of Hltf silencing. First, the 250-bp region in the Gata4 promoter  that is Hltf deletion also results in reduced expression of Myh7b/miR499. A common promoter  and co-t These studies extend our understanding of Hltf’s regulatory role in the maintenance of genomic stability in brain  to inclu< 1 event/sample) exon-skip events in both organs. In brain, Hltf proteins from both isoforms were detectable by Western blot in controls [HLTF/Hltf is a prototypical model for alternative splicing. Most human tissues express two HLTF transcripts from alternative splicing of intron 25 in the 3′-UTR . Two procontrols . In hearcontrols ,41. It aHltf is a phosphonuclear protein ,29 engagAs summarized in Transcription of the glucose transporters Glut1 and Glut9 is downregulated in Hltf null heart and brain , respectXenopus, chick and mouse [MiRNA genes are either independent transcriptional units or embedded in introns of host genes. One promoter  and co-tnd mouse . postpartum) heart provides conclusive evidence that the functional impact of Hltf silencing derives from defects in the G2/M transition. These findings are consistent with our previous report on the Hltf null brain [In conclusion, genome-wide transcriptome profiling of neonatal  compared with the Mann-Whitney U-test are significantly different (p=0.0084).(TIF)Click here for additional data file.Table S1DNAnexus alternative splicing analyses quantified the usage of each exon by mapping read counts to the splicesome. Mapping coordinates from the splicesome were converted to mapping coordinates to the genome. Mappings of the same read to the same genomic location were combined, and their posterior probabilities summed. Exon quantification was performed to show the relative expression of all known Hltf exons in heart.(XLSX)Click here for additional data file.Table S2DNAnexus alternative splicing analyses quantified the usage for each possible splice junction in control RNA-seq samples. This resulted in the comparison of different known splice products in addition to the identification of new splice products shown here as Hltf exon-skip events in heart.(XLSX)Click here for additional data file.Table S3RPKM values were used to identify differentially expressed genes. Genes whose expression was decreased in Hltf null mouse heart compared with controls are identified here.(XLSX)Click here for additional data file.Table S4RPKM values were used to identify differentially expressed genes. Genes whose expression was increased in Hltf null mouse heart compared with controls are identified here.(XLSX)Click here for additional data file.Video S1Coronary artery fistula in one Hltf knockout (KO) embryo visualized by VisualSonic cardiac echocardiography.(MP4)Click here for additional data file.


Background: Most genetic alterations in MODY affect transcription factors.Results: We functionally characterize a conserved domain in KLF11, which is disrupted in MODY7 patients.Conclusion: Defects in KLF11 not only affect insulin biosynthesis but also diabetes-associated metabolic gene networks.Significance: Disruption of this KLF11 regulatory domain as in MODY7 has wider effects on β cell gene expression than anticipated, helping to clarify disease mechanisms. 2), we investigated the functional consequences of KLF11 coupling to a TRD3 binding partner. Combined immunoprecipitation and biomolecular fluorescence complementation assays confirmed that activation of three different metabolic G protein-coupled receptors  induces translocation of Gβ2 to the nucleus where it directly binds KLF11 in a manner that is disrupted by the MODY7 A347S variant. Using genome-wide expression profiles, we identified metabolic gene networks impacted upon TRD3 disruption. Furthermore, A347S disrupted KLF11-mediated increases in basal insulin levels and promoter activity and blunted glucose-stimulated insulin secretion. Thus, this study characterizes a novel protein/protein interaction domain disrupted in a KLF gene variant that associates to MODY7, contributing to our understanding of gene regulation events in complex metabolic diseases.The function of Krüppel-like factor 11 (KLF11) in the regulation of metabolic pathways is conserved from flies to human. Alterations in KLF11 function result in maturity onset diabetes of the young 7 (MODY7) and neonatal diabetes; however, the mechanisms underlying the role of this protein in metabolic disorders remain unclear. Here, we investigated how the A347S genetic variant, present in MODY7 patients, modulates KLF11 transcriptional activity. A347S affects a previously identified transcriptional regulatory domain 3 (TRD3) for which co-regulators remain unknown. Structure-oriented sequence analyses described here predicted that the KLF11 TRD3 represents an evolutionarily conserved protein domain. Combined yeast two-hybrid and protein array experiments demonstrated that the TRD3 binds WD40, WWI, WWII, and SH3 domain-containing proteins. Using one of these proteins as a model, guanine nucleotide-binding protein β2 (Gβ Drosophila gene cabut, belongs to the KLF family of transcription factors. Members of this family regulate GC promoters in organisms ranging from flies to humans ,o humans . For insosophila , 4. Disrosophila , 6. Moreosophila –11. Thuse.g. c.-331 INS variants)  , alteratariants) . The KLFariants) . However2) to the nucleus where it binds KLF11 to regulate metabolic gene targets. Combined, these studies reveal the existence of a novel KLF-mediated pathway for coupling extracellular signals to the regulation of gene expression related to metabolism and diabetes. These findings extend our understanding of transcriptional regulatory events that are disrupted in human metabolic diseases.The KLF11 A347S variant found in MODY7 diabetic patients  maps to Cell lines were obtained from the American Type Culture Collection . Cells were cultured as described previously , 17. CHO2 as well as specified deletions into pcDNA3.1/His (Invitrogen), pCMV-Tag 2B , pGEX , and EYFP vectors as described previously (6 XpressTM) KLF11 and KLF11 A347S variant as well as empty vector (Ad5CMV) were generated as recombinant adenovirus in collaboration with the Gene Transfer Vector Core at the University of Iowa.Standard molecular biology techniques were used to clone full-length KLF11, KLF11 A347S, and Gβeviously , 19. Quiin vitro translation, GST pulldown assays, immunoprecipitation, and Western blot were all done as described previously ; HP1α ; Sin3a (Santa Cruz Biotechnology); or CBP .GST and GST fusion protein purification, eviously . AntibodDrosophila) and KLF1–17 (Homo sapiens) were analyzed using a sliding window of 15 amino acids. Windows were scored for overall hydrophobicity using the Kyte-Doolittle scale  and 5′-GTGAACTTGCGGGGTTTTTCAGTATCTACGA-3′ (reverse) and sequenced at the Mayo Molecular Biology Core Facility. To eliminate false positives, isolated library prey plasmids were transformed into Y187 yeast and crossed with AH109 yeast carrying either the empty plasmid or the bait plasmid; activation of the reporter gene was assessed by growth in Synthetic Defined medium/−Trp/−Leu/−His plus 3-amino-1,2,4-triazole.As bait, the coding region of the KLF11 TRD3 (amino acids 273–351) was cloned into the pGBKT7 plasmid of the Matchmaker system (Clontech). The integrity and expression of the fusion construct were confirmed by sequencing. No autoactivation of the reporters was associated with this bait construct as determined by cotransformation of the bait with prey library vector in host AH109 cells. Sequential library scale transformations were performed from a normal bone marrow cDNA library. A total of 2.5 million clones were screened and selected on high stringency plates (Synthetic Defined medium/−Ade/−His/−Leu/−Trp) coated with 5-bromo-4-chloro-3-indolyl-β-To screen for binding between the KLF11 TRD3 and SH3 or WW domain-containing proteins, solid phase SH3 and WW domain arrays were obtained from Panomics  and processed according to the manufacturer's instructions utilizing 10–20 μg/ml purified His-KLF11 TRD3 (amino acids 273–351) for hybridization.2 and KLF11 expression constructs. The fluorescence was imaged using an LSM510 microscope . For the BiFC analysis of individual cells by confocal microscopy, HeLa and Capan2 cells were transfected with KLF11-EYFP1 and Gβ2-EYP2 expression vectors as described previously . RNA was prepared as described previously (t test (<0.005) and corrected using the Bayes approach  and human Panc1 pancreatic adenocarcinoma cells ) were plated at a density of 10eviously . Global eviously . Genes tapproach , 26. Datapproach  and Parteviously , 28. Then signal . RNA was1/2 (Santa Cruz Biotechnology). Binding activity was derived using the NimbleGen human promoter hybridization system . Peaks were detected by searching for >4 probes where signals were above the specified cutoff values (90–15%) using a 500-bp sliding window along 5 kb upstream of the transcriptional start site in human promoters. Each peak was assigned a score that is the log2 ratio of the fourth highest probe in each peak. If multiple peaks are present, the peak nearest the transcription start site is reported. Ratio data were then randomized 20 times to evaluate the false discovery rate. Only peaks with false discovery rate scores <0.2 were deemed high confidence binding sites and reported.ChIP was performed as described previously , 19, 28 m), and samples were collected at 0 and 120 min. Measurement of secreted insulin was completed with the ultrasensitive Rat Insulin ELISA kit . The INS wild type promoter-reporter construct and luciferase assays were as described previously  for 48 h prior to insulin measurements. Cells were incubated in standard growth medium prior to initiation of the time course experiment. Medium was replaced with Krebs buffer (no glucose) with samples obtained at 0, 15, 30, 45, and 60 min. After 60 min without glucose, cells were stimulated by the addition of glucose (10 m(OMIM MODY7) (A). Analysis of the KLF11 TRD3 sequence within the region affected in MODY7 (A347S) as well as the corresponding region of its fly ortholog cabut identified several proline-rich motifs . PRMs are present in many transcription factors, chromatin regulators, and epigenetic proteins and facilitate protein/protein interactions ; this model revealed that the TRD3 is located immediately upstream of the KLF DNA binding domain in the region affected by MODY7 A347S. Evolutionary analysis of the TRD3 region further demonstrated the high pressure for preservation of the domain between fly and human across the cabut/KLF family (B). Thus, combined, these results support the prediction that the TRD3 domain functions as a proline-rich module that likely mediates protein/protein interactions, a hypothesis that we subsequently tested experimentally.Previous studies in human populations have demonstrated that defined changes in the sequence of the KLF11 protein  associate to early onset type II diabetes mellitus M MODY7) A. Analysfs PRMs; B. PRMs aractions . Interes modules –32. Thes modules A; this mF family B. Thus, 1, Gβ2, WD40 repeat domain 6, and echinoderm microtubule-associated protein-like 2 in addition to SH3- , WWI- , and WWII-containing proteins  , and some have been shown to interact with proline-rich binding proteins, such as Nedd4-like protein WWP1 for KLF2  and candidate-based (protein array) approaches to identify potential TRD3-interacting proteins. For protein array experiments, we used a high TRD3/bound array protein ratio (5:1) so that proteins from the array would capture different amounts of TRD3 according to their binding affinities as detected by different intensities. Arrays also included both positive and negative controls. Thus, the identities in the autoradiographs gave an indication of relative binding strengths. These experiments showed that the KLF11 TRD3 binds to several WD40-containing proteins, including Gβl of 33) . When anfor KLF2 . Thus, f2 as a model for a TRD3-interacting protein because this interaction was identified as our most represented candidate via several clones from the yeast two-hybrid system. This is functionally relevant because in this system protein/protein interaction occurs in the eukaryotic nucleus (yeast), the compartment in which KLF11 performs its function. Additionally, the interactions of a few KLF proteins with SH3 and WW domains have been characterized previously . We demonstrated that Gβ2 indeed binds the KLF11 TRD3 in vitro (A). Furthermore, based upon the sequences recovered from the positive yeast two-hybrid clones, we utilized deletions of Gβ2 to determine the region of this protein required for interaction with KLF11. These investigations revealed that binding with KLF11 is maintained by Gβ2 deletions containing a minimum of amino acids 209–340 as observed by positive GST-KLF11/[35S]Gβ2 interaction for Gβ2Δ1–105, Δ1–120, and Δ1–208 (B). However, interaction was not detected with the N-terminal regions encompassing Gβ2(1–214), Gβ2(106–214), and Gβ2Δ1–221 (B), which presumably disrupt the protein in WD40 repeat 5. Therefore, Gβ2 with intact WD40 repeats 5–7 is necessary for interaction with KLF11.For subsequent functional studies, we chose to study Gβeviously –37, but eviously , remain eviously –41. In feviously , 42. Preeviously , 44. Thein vitro A. Furthed Δ1–208 B. Howeveβ2Δ1–221 B, which 2 can transduce signals from the membrane to the nucleus. For this purpose, HeLa cells under basal conditions were co-transfected with His-tagged full-length KLF11 and either empty FLAG control vector or FLAG-tagged Gβ2. Upon immunoprecipitation of FLAG-tagged Gβ2, KLF11 was detected (C). This result was further confirmed by immunoprecipitation of FLAG-tagged Gβ1 as this isoform of Gβ was also recovered by our yeast two-hybrid assay, which corroborated binding to KLF11 (C). To examine the influence of G protein-coupled receptor (GPCR) activation at the plasma membrane by a physiological stimulus, we first utilized isoproterenol, an agonist of β-adrenergic GPCR signaling, which regulates many functions in HeLa cells (2 upon isoproterenol treatment (C). To visualize this interaction in living cells, we used BiFC by fusing the EYFP N terminus (EYFP(1)) to KLF11 and the EYFP C-terminal portion (EYFP(2)) to Gβ2. These constructs were co-transfected in both HeLa and Capan2 cells and subsequently stimulated with isoproterenol or secretin . Co-expression of a negative control leucine zipper protein with either KLF11 or Gβ2 fused to their respective EYFP halves did not reconstitute fluorescence, confirming that the fluorescence obtained is specific and devoid of background (data not shown). Representative panels of untreated HeLa or untreated Capan2 cells are also shown (D). To complement these studies, we also utilized CHO cells stably transfected with the GPCR CCK receptor A (CHO-CCKAR) . However, upon CCK treatment of the CHO-CCKAR cells, Gβ2 translocates to the nucleus where it localizes with KLF11, suggesting that under these conditions they function in close proximity (E). Therefore, several physiological stimuli of distinct GPCRs , which trigger dissociation of β-γ heterodimers from the trimeric G protein αs-β-γ complex at the plasma membrane to allow downstream signaling, resulted in interaction between Gβ and KLF11 in the nucleus of three different cell types. Combined, these results not only confirm our yeast two-hybrid experiments but more importantly indicate that activation of GPCRs induces the translocation of the Gβ2 subunit, which by forming a complex with KLF11 likely regulates responses (e.g. gene expression).We next investigated this interaction in cultured cells and whether the KLF11-Gβdetected C. This rto KLF11 C. To exaLa cells . Interesreatment C. To vissecretin , respectsecretin . Interacco-stain D. Co-expso shown D. To comO-CCKAR)  and tranunctions . In agreed cells E. Howeveroximity E. Theref2 encompasses the A347S KLF11 variant previously identified in a family of individuals affected by MODY diabetes (2 (A). Notably, this effect is isolated to binding between KLF11 and Gβ2 as the variant did not affect the interaction of KLF11 with its other known cofactors, Sin3a, HP1α, and CBP/p300 (B). These results reveal that the KLF11 A347S variant is useful to define whether protein/protein interactions influence the function that KLF11 has in the regulation of metabolic gene expression pathways. Thus, we performed a genome-wide query using a whole genome Affymetrix expression profile (Rat Gene 2.0 ST) in INS-1 rat pancreatic β cells transduced with empty vector control, wild type KLF11, or the KLF11 A347S variant to define genes and in particular metabolic gene networks that are regulated by KLF11 but altered via the A347S variant that interrupts the function of the KLF11 TRD3 (A). Clustering of all significantly altered probes (p < 0.05) revealed distinct clusters of genes regulated by KLF11. Detailed statistical and bioinformatics analysis identified 2275 unique genes that significantly  associate with KLF11 expression, whereas 1205 genes significantly associate with KLF11 A347S expression. Therefore, congruent with its known biological function, these data demonstrate that KLF11 is a regulator of metabolic gene networks and that the A347S variant alters this profile.Because the region of interaction between KLF11 and Gβdiabetes , we exambetes . Fig. 5B displays the overall expression pattern of 32 KLF11-regulated genes with function related to insulin secretion. KLF11 significantly (p < 0.05) regulates this insulin-associated gene network, whereas their expression is altered by the KLF11 A347S variant. In addition to this gene network, a subset of other glucose- and insulin-related biological processes significantly deregulated in the presence of the A347S mutant are also shown . To validate the regulatory effects of KLF11 and the KLF11 A347S variant observed in the genome-wide array, we performed quantitative PCR on INS-1 β cells for which results are shown in Fig. 5C. This validation indicates the overall reliability of the array in identifying patterns of gene regulation by KLF11 or its variant. The effects of KLF11 and its A347S variant were also examined in another pancreatic epithelial cell line, Panc1 , which also revealed disruption of metabolic pathways associated to the insulin response. Moreover, we also validated the regulatory effect of Gβ2 on a subset of identified KLF11-regulated targets in these cells. For this purpose, we performed expression analysis using a construct that targets Gβ2 protein to the cell nucleus . These experiments demonstrated that transcripts that are significantly regulated by KLF11 are congruently activated or repressed by Gβ2 . Combined, these data support co-occupancy and regulation of gene targets by this newly identified KLF11-Gβ2 transcriptional complex.Clustering analysis revealed that in β cells expression of the KLF11 A347S variant has an impaired transcriptional regulatory function. To statistically confirm the disruptive effect of the A347S variant on β cell gene expression, gene ontology analysis of variance analysis of the data set was performed. This statistical method demonstrates that KLF11 regulates clusters of genes known to modulate the insulin response pathway, including glucose import, glycolysis, fatty acid metabolism, insulin secretion, calcium and potassium channels, MAPK activity, and PI3K activity among others (p < 0.005) the A347S variant abrogates this effect . Subsequently, we measured levels of insulin secreted from INS-1 cells after infection with adenovirus carrying empty vector, KLF11 WT, or KLF11 A347S by ELISA. Cells were cultured for 48 h postinfection and depleted of glucose by replacing medium with Krebs buffer. Next, we measured insulin secretion after 0, 15, 30, 45, and 60 min of glucose withdrawal. Congruent with the results on INS promoter activity shown in Fig. 6A, KLF11 WT produced higher insulin levels at all time points upon withdrawal of glucose . However, when compared with the effect of KLF11 WT, the A347S MODY7 variant showed an impaired response with insulin levels in the same range as the empty vector control . As expected from a glucose-inducible activator of the insulin gene, KLF11 indeed increased the total levels of this hormone to a degree that could not be further stimulated by glucose (120.8 ± 15.3% versus no glucose) when compared with empty vector control . In contrast, cells transduced with the A347S variant showed low levels of insulin secretion even in the presence of glucose . Thus, combined, these data demonstrate that although enhanced levels of KLF11 WT appear to recapitulate the glucose response by increasing basal insulin levels and promoter activity the MODY7 A347S variant is impaired in these functions.Previous functional studies have indicated that the three KLF11 MODY7 variants have impaired transcriptional activity compared with wild type . The quaAlthough the strength of modern genetics is to discover the association of genes and their variants to disease with a high level of resolution, significantly less is known regarding how the protein products work to give rise to diseases. This has been the case for those genes within the KLF family of transcriptional regulators, such as KLF11 and KLF14, which have been strongly associated to diabetes, obesity, and insulin resistance/metabolic syndrome , 48–50. in vitro binding assay (protein array), we showed that the TRD3 domain can bind to several types of Pro-rich binding modules, such as WWI, WWII, and SH3 domain-containing proteins. Interestingly, some of these proteins, such as ITCH and Nedd4-like proteins, have been shown previously to bind to KLF transcription factors (The functional importance of the region corresponding to the KLF11 TRD3 was suggested in 1999 through a careful mapping of the N-terminal domain of this protein , althoug factors –35 at thi-coupled somatostatin receptor in rat pituitary GH3 cells induces its translocation to the nucleus where it works as a corepressor through direct binding to the AF-2 domain of GR (2-adrenergic receptor by isoproterenol or the secretin receptor by secretin induces the formation of the KLF11-Gβ2 complex in the cell nucleus. Together, these results suggest that members of very distinct families of transcription factors can utilize the Gβ2 subunit to mediate their function in gene expression.The TRD3-binding proteins identified here are a wide variety of eukaryotic proteins that have a range of functions, including as adaptor/regulatory modules in signal transduction and in pre-mRNA processing, cytoskeleton assembly, gene transcriptional activation, and cell cycle control. Of further interest, Gβ is a WD40 repeat protein, a large protein family for which members are increasingly being recognized as key regulators of chromatin dynamics, transcription, and epigenetics , 52. Amoin of GR . SimilarIt is important to discuss how, when combined, the molecular and cellular data reported in this study contribute to a better understanding of the mechanisms of insulin secretion and its potential alterations in MODY7. The current model for KLF11 function in β cells indicates that glucose enhances the intracellular levels of this transcription factor, which in turns binds to the −331 site within the insulin gene and activates the synthesis of this hormone , 6. Thus2 in the nucleus has remained elusive for many decades. Fortunately, the working model that emerges by the congruent association of all data described in this study as well as those available for the GR is that extracellular signals induce the binding of WD40 proteins to these transcription factors to regulate metabolic gene networks. Consequently, disruption of this mechanism should lead to alteration in the regulation of these networks, an effect that we observed experimentally and that is congruent with a role of MODY7 A347S in human diabetes.The observations described here are also of significant biological importance because the role of GβIn summary, the current study extends the available knowledge on KLF proteins and identifies a novel evolutionarily conserved protein/protein interaction domain that is involved in the transcriptional regulation of metabolic gene networks. The biochemical contribution of this work lies in the extensive characterization of this domain, identification of Gβ as a binding partner, and the role of this domain in the regulation of metabolic gene networks. Lastly, the fact that mutations in this pathway that are known to cause diabetes impact the regulation of metabolic gene networks should be taken into consideration for understanding potential molecular mechanisms that contribute to disease phenotypes.