O. sativa).Several studies have investigated the relationships between selective constraints in introns and their length, GC content and location within genes. To date, however, no such investigation has been done in plants. Studies of selective constraints in noncoding DNA have generally involved interspecific comparisons, under the assumption of the same selective pressures acting in each lineage. Such comparisons are limited to cases in which the noncoding sequences are not too strongly diverged so that reliable sequence alignments can be obtained. Here, we investigate selective constraints in a recent segmental duplication that includes 605 paralogous intron pairs that occurred about 7 million years ago in rice  there is a signature of strong purifying selection at splice control sites; (3) first introns are significantly longer and have a higher GC content than other introns; (4) the divergences of first and non-first introns are not significantly different from one another, a pattern that differs from Drosophila and mammals; and (5) short introns are more diverged than four-fold degenerate sites suggesting that selection reduces divergence at four-fold sites.Our principal findings are: (1) intronic divergence is negatively correlated with intron length, a pattern that has previously been described in Our observation of stronger selective constraints in long introns suggests that functional elements subject to purifying selection may be concentrated within long introns. Our results are consistent with the presence of strong purifying selection at splicing control sites. Selective constraints are not significantly stronger in first introns of rice, as they are in other species. Arabidopsis thaliana, based on a well-documented recent genome duplication event, intragenomic conserved noncoding sequences have also been investigated, and a unique set of noncoding DNA sequences enriched for function has been uncovered . Their  0.5 mya ,26, so t 0.5 mya , and aliP = 0.013), which parallels findings for other species investigated [et al. [After intron alignment and some necessary masking, a dataset of 605 intron pairs  was generated. The 605 pairs come from 272 duplicated gene pairs (which excluded genes that are part of a transposable element) from a recent duplication of rice chromosomes 11 and 12 , althouet al. [et al. [In this study, we employed several methods to minimize the frequency of incorrect alignments. These included amino acid-guided methods (see methods section) to anchor the coding regions of a paralogous gene pair (T-COFFEE), alignment using explicit models of indel evolution (MCALIGN2), and the use of two masking protocols for nonhomologous sites (for details see methods section). Our finals sample size of 605 intron pairs from 272 loci is compatible with other similar studies. For example, 200–300 loci were used by Keightley and Gaffney , 24 lociet al.  and 225  [et al. .Rs = -0.112, P = 0.006)  [A negative correlation between intron length and nucleotide divergence is present in our dataset (Spearman correlation coefficient sophila) .Rs = -0.271, P = 0.012; non-first: Rs = -0.089, P = 0.046). If introns are divided into two different sets according their length, there is a significant difference in divergence between short and long introns for first introns, whereas the difference is non-significant for non-first introns . We also divided the dataset into two subsets based on first or non-first introns. Similarly, no significant difference in divergence was found between first and non-first introns (P = 0.458)  of introns in a gene and divergence Fig. . The gloP = 0.95). However, significant differences between indel numbers and lengths per base or gene pair were observed , with more indels in first than non-first introns. This result indicates that the evolutionary pattern of indels seems to be somewhat different from nucleotide divergence in introns in rice. Whether this trend exists in other plants or animal species need further investigation.In addition to single nucleotide mutations, we also investigate the frequency distribution of indels in first and non-first intron. A total of 1,398 indels were identified in our dataset, and no significant difference in frequencies of indel lengths between first and non-first intron was observed , Haddrill et al. [In summary, selective constraints seem not to be specific to first intron in rice, so our results are similar to those previously reported in l et al.  found thl et al.  observedl et al. . The aboDrosophila [Drosophila [We next examined constraints near the 5' and 3' ends of introns, which contain splice control motifs . As expeosophila ,15. The osophila ,15.Gramineae genes is distinct from that of dicot genes. For example, in Gramineae genes GC content is relative high, and there is a gradient of GC content along the direction of transcription [Rs = -0.019, P = 0.649) or intron length  was observed  sites as putatively neutral standards to calculate constraint. Although exonic four-fold degenerate (4-fold) sites are often used as a standard against which to test for deviations from neutrality, sites in short introns evolve faster in our data set Table , so are In general, fractions of nucleotide differences at FEI sites are consistently higher than 4-fold sites and first introns. The transition events A↔G and T↔C changes are expected to be the most common substitutional changes in all categories of sites Table . The sitWe investigate selective constraints in a recent segmental duplication that includes 605 paralogous intron pairs that occurred about 7 million years ago in rice. Our observation of stronger selective constraints in long introns suggests that functional elements subject to purifying selection may be concentrated within long introns. Our results are consistent with the presence of strong purifying selection at splicing control sites. Selective constraints are not significantly stronger in first introns of rice, as they are in other species.). The segmental duplication was identified using a reciprocal BLASTP search with E-value < 10-14 within a distance of 100 kb between collinear gene pairs [Gene sequences and their annotations (release 5) were downloaded from the Rice Genome Annotation of TIGR  were estimated from 400 paralogous intron sequences, in which nucleotide and indel divergence are sufficiently low as to make the alignments practically unambiguous. In order to minimize the possibility of nonhomologous sites contributing to estimates of divergence, two simple masking protocols were implemented: 1) Regions that contained short aligned blocks surrounded by large gaps (>40 bp) were considered unlikely to be truly homologous and were masked off. A total of 608 pairs identified by this criteria were included for further analysis. 2) A moving window of 40 bp was used to check the degree of divergence in each alignment. Pairs containing more than 25 putatively nonparalogous sites in a window were excluded from further analyses. A total of 3 pairs was identified and excluded according to this criterion. Taken together, the final dataset used in this study contained 605 intron pairs.  were generated for each alignment by applying the Jukes-Cantor correction to the number of substitution per intronic site using the distmat program from EMBOSS package [Introns were either analyzed as complete sequences or as partial sequences after removal of putative splice control sequences . The exact limits of the control sequence are somewhat arbitrary . Diverge package .E) of substitutions in adjacent intronic sequences under the assumption that point mutation rates of each possible kind are equal at FEI sites, 4-fold and adjacent intronic DNA sites. The FEI sites are defined as sequences in introns, excluding first introns and introns of length > 232 bp, and the 6 bp/16 bp at the 5'/3' end of each intron. FEIs were treated as independent observations in the data sets and were used to predict six different substitution rate parameters , which were calculated as the rate of substitution expected under neutrality. For each possible substitution type, Let pi  be the pairwise divergence in the FEI segment, i.e.,In order to estimate selective constraint, a variation of the method of Kondrashow and Crow was employed, as in previous studies ,37,38. Fdi is the numbers of pairwise differences of type i, and Ni is the number of sites at which a change of type i could occur in one step . The expected number of substitutions in an adjacent interest segment is,where Mi is the corresponding number of intronic sites. This model assumes that symmetric mutation rates and equivalent base composition in the FEI sites and the other region of interest.where O):We calculated constraint by comparing E to numbers of observed substitutions . Standard errors and confidence for mean divergence were also calculated by bootstrapping the results by FEIs, 4-fold and intronic.XG and LF conceived and designed the experiments, XG and YW performed the experiments and analysed the data, XG, PDK and LF wrote the paper, and PDK advised on data analysis. All authors have read and approved the final manuscript.Chromosomal alignment of chromosome 11 and 12 of rice. The syntenic line at left corner corresponds to the recent duplication event. The Mummer program was used with word length 80 bp.Click here for fileA list of the 272 duplicated gene pairs used in this study. Locus names and their physical positions based on TIGR (Release 5) are listed.Click here for fileSequence alignments of the 605 intron pairs used in this study. Locus names and intron ordinal positions based on TIGR (Release 5) are listed.Click here for file


Background. We have previously shown a different local and systemic angiogenic profile of CXC chemokines in Idiopathic Pulmonary Fibrosis (IPF) patients compared to sarcoidosis. In particular, sarcoidosis showed an angiostatic microenvironment, as compared with the angiogenic cytokine milieu seen in IPF. Purpose of the Study. Our aim was to further investigate the aforementioned finding by measuring the expression of different chemokines in granulomatous and fibrotic diseases. We estimated the levels of vascular endothelial growth factor (VEGF) and its high-affinity receptor, Flt-1 (fms-like tyrosine kinase 1), in bronchoalveolar lavage fluid (BALF) of patients with IPF and pulmonary sarcoidosis. We have also investigated the mRNA expression of angiogenetic chemokines' receptors such as CXCR2 and CXCR3 and the biological axis of stromal derived factor-1α (SDF-1α or CXCL12α/CXCL12β) and receptor, CXCR4. Methods. We studied prospectively three groups of patients: (i) one group of 18 patients with IPF, (ii) one group of 16 patients with sarcoidosis, and (iii) 10 normal subjects. Results. A statistically significant increase has been detected in VEGF mRNA expression in IPF in comparison with pulmonary sarcoidosis (P = .03). In addition, a significant increase has been measured in CXCL12α in sarcoidosis in comparison to IPF (P = .02). Moreover, a statistically significant decrease has been found in Flt-1 protein levels in pulmonary sarcoidosis in comparison with IPF (P = .03). A significant increase in VEGF (P = .03) and CXCR4 (P = .03) mRNA levels has been also detected in sarcoidosis' patients when compared with healthy controls. Conclusions. Our data suggest that increased expression of Flt-1 and downregulation of CXCL12α in IPF may further support the hypothesis of a different angiogenetic profile between fibrotic and granulomatous diseases. However, further studies are needed in order to better investigate these enigmatic diseases. Angiogenesis has been implicated in the pathogenesis of several fibrotic lung conditions, including idiopathic pulmonary fibrosis (IPF). This process plays a significant role in wound healing and contributes to the fibroproliferation and extracellular matrix deposition . NeovascSarcoidosis continues to be a disease of research interest because of its complicated immune mechanisms and elusive etiology . So far,−CXC chemokines  [−CXC chemokine upregulation in comparison to IPF patients [α) sharing the same receptor, CXCR2, were found increased in IPF [Recently the chemokine receptors expressed specifically on Th1 cells were identified and it was reported that Th1 cells are characterized by the expression of CCR5 or CXC chemokine receptor 3 . While C/CXCL11) . Recent patients . On the d in IPF , while ad in IPF . fms-like tyrosine kinase 1) and KDR/Flk-1  [ Vascular endothelial growth factor (VEGF) is a potent growth factor for endothelial cells that regulates vascular permeability and the stimulation of angiogenesis. VEGF is crucial in lung development and maintenance during the adult life. However, VEGF also contributes in several acute and chronic lung disorders . The bioinase 1) , 15.  We hypothesized that the levels of VEGF and receptor Flt-1 would be higher in bronchoalveolar lavage fluid (BALF) from patients with IPF than in patients with pulmonary sarcoidosis. In order to further explore the angiogenetic balance, we measured the mRNA levels of chemokines' receptors, CXCR2 and CXCR3, as well as the biological axis of CXCL12 and cognate receptor, CXCR4.We studied prospectively three groups of patients: (i) 18 patients with IPF, (ii) 16 patients with sarcoidosis, and (iii) 10 normal subjects. Patients' and healthy controls' characteristics are shown in CO, (3) computed tomography (CT) appearances indicative of IPF with predominantly basal and subpleural microcystic or macrocystic honeycombing, with variably extensive ground-glass and reticular abnormalities but no consolidation, nodular abnormalities, or other parenchymal abnormalities (apart from centrilobular emphysema), and (4) no environmental exposure to a fibrogenic agent or connective tissue disease [The diagnosis of IPF was made in 8 cases by surgical biopsy  and the histologic diagnosis of Usual Interstitial Pneumonia (UIP) was obtained. In the remaining 10 cases the diagnosis was made on the basis of clinical and high-resolution computed tomography (HRCT) criteria: (1) bilateral basal or widespread crackles, (2) restrictive ventilatory defect or isolated depression of DL disease . AccordiSarcoidosis diagnosis was made according to the ATS/ERS/World Association of Sarcoidosis and Other Granulomatous Disorders joint statement . All patBALF was also obtained from ten healthy control patients without any past medical history, pulmonary symptoms, or abnormal radiographical findings. A former smoker was defined as having smoked at least one cigarette per day for 1 year: smoking histories were collected as part of a routine prospective clinical protocol.L,CO (corrected for the haemoglobin) using the single-breath method were performed by a computerised system . Predicted values were obtained from the standardised lung function testing of the European Coal and Steel Community, Luxembourg (1993) [All patients were evaluated spirometrically and by measurement of lung volumes, diffusion capacity, and arterial blood gases (at rest). Spirometry and lung volumes (helium-dilution technique) and Tg (1993) . ArteriaBALF was obtained from patients with IPF, sarcoidosis, and from normal healthy controls by methods previously described , 13. Bri6 cells/mL with RPMI1640 plus 2% FCS. The slide preparation was performed as previously reported [Total cell counts were determined using an improved Neubauer counting chamber and expressed as the total number of cells per mL of aspirated fluid. The pellet was washed three times with cold PBS-Dulbecco's and the cells were adjusted to a final concentration of 10reported . The cel−1, respectively.Human BALF protein levels of VEGF-A and Flt-1 were quantitated according to the manufacturer's protocol using ELISA kits (R&D Systems) and measurements were performed in the BALF supernatant. Human VEGF-A and Flt-1 had the lowest detectable limit of 5 and 10 pg·mLTotal RNA was extracted form each specimen (BALF pellet) using a power homogenizer and the TRIzol reagent  according to the manufacturer's instructions. cDNA was synthesized using the Strascript reverse transcriptase kit  as previously described .α and β and CXCR4 expression levels . To verify the results of the melt curve analysis, PCR products were analyzed by electrophoresis in 2% agarose gels, stained with ethidium bromide, and photographed on a UV light transilluminator. Primer sequences, annealing temperatures, and PCR products length for all the growth factors analyzed, as well as for GAPDH, are described in Peptide growth factors mRNA expression was measured using a real-time RT-PCR assay with SYBR-Green I. Primers were designed to span introns . Glyceran levels . Specifisample = CtGF − CtGAPDH.All reactions were run in triplicates, and peptide growth factor transcript levels were calculated and normalized to each specimen's house keeping gene mRNA (GAPDH) as well as the appropriate calibrators, using the ΔΔCt method for relative quantification. Specifically, after amplification, standard curves were constructed from samples used in a series of consecutive dilutions, for both the gene of interest (GF) and the internal control (GAPDH). Growth factor and GAPDH amplification efficiencies were the same, reaching 100%. IPF and sarcoidosis data were first normalized against variation in sample quality and quantity. Normalized values to GAPDH, ΔCts, were initially calculated using the following equation: ΔCtP-value <  .05).Peptide growth factors mRNA levels were first evaluated by the one-sample Kolmogorov-Smirnov goodness of fit test, in order to determine whether they follow a normal or no distribution. Based on the results, the nonparametric Spearman test was used to examine correlations. Proportions were compared using chi-square test. The Kruskal-Wallis followed by a posthoc analysis for pairwise significance and the Mann-Whitney U test were used as indicated to examine growth factors' and chemokines' expression status among IPF and pulmonary sarcoidosis groups. Statistical analysis was carried out using SPSS 13.0 Chicago IL, USA. Statistical significance was set at the 95% level  (P =  .2), suggesting a posttranscriptional decrease of mRNA expression  . Howeverpression . P =  .03, resp.). No significant difference has been detected between IPF and control subjects   .P =  .4) , suggesting a posttranscriptional downregulation of Flt-1 expression  . A statipression .No significant difference has been measured between healthy controls and sarcoidosis or IPF at flt-1 mRNA expression levels .α in sarcoidosis' patients in comparison with IPF samples (P =  .06) at mRNA levels of CXCR3 in IPF in comparison with sarcoidosis' patients . However, no significant difference has been measured between sarcoidosis and healthy controls at mRNA levels of the other chemokines .A significant increase has been detected in healthy controls in comparison with IPF at CXCL12α in sarcoidosis' patients in comparison with IPF. To the best of our knowledge this is the first study to investigate the local expression of the biological axis VEGF and its receptor Flt-1 in patients with IPF and Pulmonary Sarcoidosis without pulmonary fibrosis. Our major finding was an increase of the receptor Flt-1 at protein level in IPF in comparison with sarcoidosis. Although the increase in VEGF in IPF patients has not been confirmed at the posttranscriptional analysis, this may be due to high variation in the small group of patients analyzed. In order to further investigate the angiogenetic balance between granulomatous and fibrotic disorders, we measured CXC chemokines' receptors and the biological axis of CXCL12/CXCR4. Our major finding is the significant increase in CXCL12γ-induced CXC chemokines in the context of Th1-like immune disorders, such as sarcoidosis, was originally raised by Agostini et al. who documented an enhanced expression of IP-10 in sarcoid tissues and a positive relationship of BALF IP-10 levels and the degree of T-cell alveolitis, suggesting its pivotal role in ruling the migration of T-cells to sites of ongoing inflammation [γ-induced CXC chemokines are strongly involved in the immunomodulatory cascade of sarcoidosis implicating angiostasis with Th1 immune response [Recent immunological advances on sarcoidosis have revealed a T helper 1 (Th1) and T helper 2 (Th2) paradigm with predominance of the Th1 response in the immunopathogenesis of sarcoidosis , 21. Theammation . Our stuammation –25. In aresponse . Howeverresponse  describeresponse . Recent response . Importaresponse .α and its specific receptor, CXCR4, in pulmonary sarcoidosis, in comparison with IPF and controls, respectively, suggesting its major chemotactic role for lymphocytes, in accordance with the aforementioned recent and previous data [Furthermore, Sekiya et al.  demonstrous data , 31. However, there are several arguments that should be addressed. First of all, we have the small number of patients included in the study; moreover, there is the limitation of several variations regarding the age, disparity of number of males versus females in each group, smoking status, pulmonary function tests, and BALF cell differentiation between the different disease groups and healthy subjects. Secondly, this study is not a morphological one like other elegant reports in lung tissue –35. On tIn line with these findings, our study group demonstrated a distinct local angiogenic profile in patients with IPF compared to sarcoidosis patients. The latter evidence implicates angiogenesis in the fibrotic (Th2) pathway of ILDs and highlights novel noninvasive biomarkers to identify patients who are likely to develop progressive disease allowing anti-inflammatory and other treatments to be evaluated or eventually modified before they have failed. However, further studies are needed in order to better investigate these enigmatic diseases.


We provide detailed experimental validation of each step and, as a proof of principle, utilize the methodology to identify novel direct targets of the orphan nuclear receptor NR2F1 (COUP-TFI). The first step involved validation of microarray gene expression profiles obtained from wild-type and COUP-TFI−/− inner ear tissues. Secondly, we developed a bioinformatic tool to search for COUP-TFI DNA binding sites in genomes, using a classification-type Hidden Markov Model trained with 49 published COUP-TF response elements. We next obtained a ranked list of candidate in vivo direct COUP-TFI targets by integrating the microarray and bioinformatics analyses according to the degree of binding site evolutionary conservation and microarray statistical significance. Lastly, as proof-of-concept, 5 specific genes were validated for direct regulation. For example, the fatty acid binding protein 7 (Fabp7) gene is a direct COUP-TFI target in vivo because: i) we identified 2 conserved COUP-TFI binding sites in the Fabp7 promoter; ii) Fapb7 transcript and protein levels are significantly reduced in COUP-TFI−/− tissues and in MEFs; iii) chromatin immunoprecipitation demonstrates that COUP-TFI is recruited to the Fabp7 promoter in vitro and in vivo and iv) it is associated with active chromatin having increased H3K9 acetylation and enrichment for CBP and SRC-1 binding in the newborn brain.We describe a methodology to identify transcription factor binding sites and target genes in vivo direct nuclear receptor target genes. This bioinformatics tool can be modified to scan for response elements of transcription factors, cis-regulatory modules, or any flexible DNA pattern.We have developed and validated a methodology to identify  The nuclear receptor family encompasses a set of ligand-regulated transcription factors that bridge a variety of systemic endocrine signals with a tissue-specific gene regulation response Numerous efforts have recently been directed towards establishing comprehensive nuclear receptor gene regulatory networks. High-throughput methods that aim at defining the precise genomic sites where a nuclear receptor is physically associated  are derived from the chromatin immunoprecipitation (ChIP) technique −/− mouse has a high incidence of perinatal mortality, malformations in the glossopharyngeal ganglion, defects in axonal arborization, and loss of cortical layer IV due to the absence of thalamocortical connections in vitro and at e10.5 in vivo results in prolonged generation of early-born neurons at the expense of gliogenesis, suggesting that the precise temporal expression of both COUP-TF homologues is required by neural precursor stem cells (NPSC) to acquire gliogenic competency −/− mice display an intriguing inner ear phenotype consisting of a shorter cochlear duct, supernumerary outer hair cells and occasional inner hair cell duplications, decreased innervation, and postnatal degeneration of the basal turn of the organ of Corti −/− mice was recently discovered to have a chromosomal microdeletion of the entire COUP-TFI locus COUP-TFs  are members of the orphan subfamily of nuclear receptors in vivo direct targets to reveal genes and pathways that are important for inner ear development and functional maturation. Within the nuclear receptor superfamily, COUP-TFs have the highest degree of evolutionary conservation in vitro COUP-TF targets, most of them related to lipid/steroid metabolism. However, validated in vivo data is limited, and information on inner ear targets is nonexistent. In vitro analyses have characterized COUP-TFs to mainly be transcriptional repressors but they can also activate some target genes in silico genomic analysis with a Hidden Markov Model, and evolutionary conservation filtering.We have pursued the identification of COUP-TFI −/− P0 inner ears. For this purpose, we performed a total of 8 microarray experiments using Affymetrix MG-U74Av2 chips, including 2 biological and 2 experimental replicates per genotype sample. Due to limiting RNA yields from the newborn inner ear, each microarray chip was hybridized with an RNA pool from multiple tissue samples, a method that has the additional advantage of eliminating some of the normal biological variability across individuals In order to establish a list of candidate COUP-TFI gene targets, we analyzed the differential gene expression profiles of the wild-type and the COUP-TFICOUP-TFI has the lowest genotype p-value, validating our statistical approach (Table S1).Given the lack of consensus within the bioinformatics community regarding the different microarray normalization methods 2 = 0.07) - although we did observe a stronger correlation among the lower p-values (data not shown). The same trend was observed after plotting the interaction p-values, the genotype rank number for each probe, and the genotype p-values of those probes that did not have a significant interaction (p>0.01) in both methods (data not shown). In view of these facts and in order to obtain a hit list with the maximum confidence, we narrowed down our final gene list to 176 probe IDs (171 unique genes) that have the best genotype p-value correlation in the two normalization methods  . Within the 176 candidate COUP-TFI microarray targets, five gene ontology categories are significantly enriched: cell adhesion, cartilage development, regulation of progression through cell cycle, myeloid cell differentiation and cellular lipid metabolism. Most of the COUP-TF targets known today are associated to lipid/steroid metabolism, and this pathway, specifically the modulation of cholesterol levels, plays an important role in hair cell mechanotransduction and outer hair cell function Surprisingly, only 51 genes were present in both hit lists (−/− P0 mouse inner ears (n = 4). With this approach, we verified 7 out of the 10 gene expression changes with statistical significance (p<0.05)  have a lower expression level in the wild-type samples, a result consistent with the proposed mechanism of COUP-TF action as predominantly a transcriptional repressor In an effort to validate further the microarray gene hits and differentiate between direct and indirect COUP-TFI targets, we set out to identify if COUP-TF response elements were present within the nearby regulatory sequences of the microarray gene hits. This was problematic because the available public transcription factor search sites were inadequate for this purpose, since their position frequency matrices were derived from less than 20 known binding sites and they only described one type of response element, namely, the DR1 configuration. Since COUP-TFs can bind a diversity of DNA response elements direct COUP-TF target gene database consists of 55 genes and 61 binding sites from 7 different species. The specific binding regions were classified as half-sites, direct (DR), inverted (IR) and everted (ER) repeats of various spacings. Half-sites were discarded from the list, since they can be described with simple position weight matrices, and biochemical data suggests that COUP-TFs bind as dimers File S1). HMM emission and transition probabilities were then determined as described by Sandelin et al File S1).To obtain the specific COUP-TF HMM parameters, we first created a detailed database of all COUP-TF targets reported in the literature, which we annotated and verified as described in the j probability (probability of entering the match state), we used the Viterbi algorithm to analyze a 9000bp sequence containing all 49 training sites and ∼100bp of their surrounding native genomic environment. We were able to detect a high proportion of the training set (87% correctly labeled training site nucleotides) at a stringent j value of 0.01, and up to 92% correctly-labeled nucleotides with j> = 0.04  a sequence of the same length that reflects nucleotide distribution at the genomic level and 2) a sequence of the same size that reflects nucleotide distribution within 5000bp of established transcription start sites . Both experiments yielded low false positive detection rates even at high values of j , with a constant percentage of labeled nucleotides throughout the genome, except for human chromosomes 17 and 19, which had a significantly higher number of sites when correlated to the chromosome size. The human and mouse chromosome Y, on the other hand, resulted in significantly fewer HMM matches than expected from the number of sequenced base pairs in these chromosomes. The rat genome had a slightly lower COUP-TF match frequency, resulting in 1 site every 1124 genomic base pairs. The complete list of candidate COUP-TF binding sites  in the mouse genome is available in BED format directly from the authors. This is essentially a database of the DR0-DR13 sites in the entire mouse genome that can be uploaded into the UCSC Genome Browser Short nucleotide sequences can be processed directly using the GHMM python bindings non-coding bps, and only 1 site per 13,073 coding bps. This represents a 36.8-fold enrichment of candidate COUP-TF binding sites within the non-coding genomic regions. After correcting for the size of both coding and non-coding genomic compartments, we still observed an 11.63-fold enrichment in the non-coding section . At any rate, the high density of COUP-TF HMM matches resulted in at least one site within the promoters of all but 2 of our microarray hits  was ranked 15th within the candidate target gene list . Fabp7 is the brain-type member (BLBP) of the intracellular lipid binding protein family – although it is also expressed in other tissues such as the retina and mammary gland , and COUP-TFII is described to induce Fabp2  expression in vitroFabp7 is down-regulated in the COUP-TFI−/− inner ear as determined by our microarrays and contains 2 closely-spaced conserved COUP-TF HMM matches (DR0 and DR2) within its upstream promoter. As a preliminary step, we verified significant changes in the expression level of Fabp7 in the COUP-TFI−/− MEF cell line by real-time RT-PCR .To validate Fabp7 promoter in vivo. ChIP experiments were used and confirmed that COUP-TFI was enriched and physically associated to the Fabp7 promoter binding sites in the wild-type compared to COUP-TFI−/− P0 brain . These results lead to a gene regulation model , where COUP-TFI binds the conserved DR0/DR2 sites in the proximal upstream Fabp7 promoter, recruiting CBP, SRC-1 and possibly other coactivators that promote local H3K9 acetylation, creating an active chromatin environment and increasing the transcription rate of the Fabp7 gene.To identify potential candidate histone acetyltransferases that might be recruited to the in vitro validation experiments using wild-type and COUP-TFI−/− MEFs. We selected 4 additional candidate target genes that contain at least one conserved COUP-TF HMM match within their nearby regulatory regions, and first determined if they are also modulated in a COUP-TFI-dependent manner in these cell lines by performing real-time RT-PCR. Three genes  were significantly deregulated at the transcript level in COUP-TFI−/− MEFs when compared to wild-type cells, while one gene  did not show statistically significant changes in this context , a mechanism that is estimated to account for 30% of nuclear receptor-dependent gene regulation j value or consist of an atypical nucleotide distribution. At present, our genomic HMM scans are not intended to be used in isolation, but rather as one layer of evidence to guide the design of experiments, accelerating the otherwise arduous process of gene-wise transcription factor binding site validation. Furthermore, ChromAnalyzer HMM scans are meant to be an iterative process: each newfound, validated COUP-TFI DNA binding site will be incorporated into the HMM training list, and thus the COUP-TF HMM parameters will be fine-tuned to reflect the exact profile of the known COUP-TF binding sites.As the third layer of correlation, we used phylogenetic footprinting to filter the HMM hits, extracting the location of binding sites near microarray gene hits that are also conserved across evolution. Although phylogenetic footprinting aids in prioritizing target lists and increases specificity by ∼90% in vitro and in vivo of one of the highest-ranked candidate COUP-TFI targets identified by our analysis: the fatty-acid binding protein 7 (Fabp7) gene. Fabp7 is one of 9 intracellular fatty acid binding proteins, and it is expressed in the embryonic and adult nervous system, retina and mammary gland −/− mice have no anatomical neurologic abnormalities –possibly due to compensatory overexpression of other Fabp homologues-, they do exhibit increased anxiety and fear memory We first describe the detailed and mechanistic experimental validation Fabp1 gene is a PPARα transcriptional target, and FABP1 transports PPARα ligands and can physically interact with this receptor in the nucleus −9) for docosahexanoic acid (DHA) Although the role and expression pattern of Fabp7 is not known in all tissues, our results suggest that COUP-TFI lies upstream of Fapb7 and mediates the regulation of proliferation and differentiation of stem cells during neurogenesis and inner ear development. Several FABP proteins interact with orphan receptors to deliver their ligands in the nucleus, and their transcription rate is in turn regulated in a positive feedback loop by the activated nuclear receptor. For example, the in vitro - Crabp1, Sod1 and Casq1-, as well as one novel target in vivo –Foxo3a- were validated. These genes are known to be expressed in the cochlea and are relevant for inner ear physiology: Crabp1 is expressed during cochlear development and controls the intracellular concentration of free retinoids Casq1 participates in the calcium buffering system that is upregulated to protect hair cells from noise trauma and lies within DFNA49, an autosomal dominant non-syndromic hearing loss locus Sod1 are associated with early-onset hearing loss in mice Foxo3a regulates metabolic processes and is linked to longevity in species ranging from C. Elegans to humans As further ‘proof-of–concept’ of our methodology, 3 novel direct COUP-TFI targets The bioinformatics tool we describe can be modified to scan for response elements of any transcription factor, combinations of several types of response elements, or any kind of flexible DNA pattern. A primary requirement is the availability of a pool of annotated binding sites or SELEX dataset in order to train a Hidden Markov Model. We propose that given the vast number of expression datasets and experimental data already available, this approach can be readily applied to expand the pool of candidate target genes for other nuclear receptors as well.All studies involving the use of animals were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee according to policies, procedures, and regulations related to the protection of animals mandated by the federal government of the United States.Microarray hybridizations were performed on Affymetrix MG-U74Av2 chips through the BCM Microarray Core Facility. Two sets of hybridizations  were performed at a different point in time, each set consisting of one RNA pool per genotype hybridized in duplicate. Our microarray data is MIAME compliant and has been deposited into the Gene Expression Omnibus (GEO) database under the series accession number GSE16744.−18Microarray raw data was normalized using GCRMA File S2 for a list of primers). Real-time RT-PCR for COUP-TFII  was performed with Taqman probes (Applied Biosystems Mm00772789_m1). Statistical analysis was performed using a one-tailed Student's t-test and p = 0.05 as a cutoff.Whole P0 inner ears were placed in RNA Later (Ambion) after dissection, stored at 4C overnight and then at −20C until genotype was determined Table S2) correspond to the nucleotides in the plus strand of each site, including one or both half-sites , the spacer region (lower case), and 3 nucleotides upstream and downstream of the half-sites (lower case).In order to collect a reliable list of HMM training sequences, we created a database of all the known COUP-TF targets. The initial list included a total of 69 candidate direct targets (79 binding sites) and included the gene name, species, type of site (as described in the literature), binding site sequence (as described in the literature), position within the gene, activation/repression, competing elements or cofactors, tissues/cells, experiments performed, locus link number, OMIM link and Jackson lab ID. Entries judged to have insufficient experimental evidence for direct binding were eliminated. The nucleotide sequence of each binding site was verified by crosschecking with the UCSC database, and the specific genomic coordinates for each sequence were recorded. To verify the binding site classification , we used Consite  to analyze entire chromosomes for matches to any custom Hidden Markov Model. ChromAnalyzer was written as a Java wrapper that communicates with the python bindings of the GHMM C++ library We created the ChromAnalyzer Java library Mouse embryos (e12–e14) were removed from the uterus and placed in labeled 6-well plates with PBS. The corresponding yolk sac was placed in a separate tube for genotyping −/− MEFs were grown to 80% confluency in 15cm tissue culture plates. Chromatin crosslinking and immunoprecipitation were performed using the Active Motif ChIP-IT™ Express Kit, following the manufacturer's instructions except for the number of washes, which was increased to 6, and the final PCR amplification, which was quantified with ABI Prism 7000 Sequence Detection System using Power SYBR Green PCR Master Mix (Applied Biosystems), 5ul of immunoprecipitated chromatin or input, and the corresponding primer pair (refer to File S2 for a complete list of primer sets). For in vivo ChIPs, P0 brains were placed in 1% formaldehyde immediately after dissection. Tissues were fixed for 15min, and the reaction was then quenched in 0.125M glycine for 5 min. Nuclear extraction, lysis, immunoprecipitation and real-time PCR were performed as described above. Immunoprecipitations were performed overnight at 4C with 3µg of anti-COUP-TFI (Perseus Proteomics Inc. 2ZH8132H), anti-H3K9 (kind gift from Dr. Nikolai A. Timchenko), anti-SCR-1 (Abcam ab84) or anti-CBP (Abcam ab2832) antibodies. Negative IP reactions were performed with mouse IgG (Sigma), or no antibody. Results are plotted as relative binding (percent of input of the corresponding IP/percent of input of negative control IP). COUP-TFI−/− relative binding values were set to 1.Wild-type and COUP-TFIBrain and whole inner ears were flash frozen in liquid nitrogen and stored at −80C immediately after dissection. To obtain whole-cell protein extracts, tissues were lysed in 50mM HEPES (pH 7.5), 150mM NaCl, 0.5% DOC, 1% NP40, 0.05% SDS and protease inhibitor cocktail (Roche) for 30 min on ice and subsequently centrifuged at 14,000rpm for 10min. The supernatant was used for protein quantification with the Bradford assay method (Bio-Rad) and absorbance at 595nm was measured using PowerWave XS spectrophotometer (Witec AG). Seventy-five µg of inner ear total protein and 12µg of brain total protein were analyzed by SDS-PAGE using a 12.5% acrylamide gel. Proteins were transferred to a nitrocellulose membrane (Bio-Rad), and Western Blot was performed by with anti-Fabp7 antibody (Santa Cruz Biotechnology FL-132) in a 1∶200 dilution, and anti-actin antibody  at a 1∶1000 dilution, incubated overnight at 4C. Protein quantification was performed with ImageJ software Figure S1−/− tissue. A. COUP-TFII transcript levels in wild-type and COUP-TFI−/−P0 inner ears . B. COUP-TFII transcript levels in wild-type and COUP-TFI−/− P0 brain cortex .COUP-TFII transcript levels are upregulated in the COUP-TFI(0.30 MB TIF)Click here for additional data file.Table S1COUP-TFI microarray database. Complete gene expression and statistical data for all probes in the Affymetrix MG-U74Av2 microarray chip. The database is indexed by probe ID and includes the following fields from left to right: gene name, description, expression/statistics from the RMA-normalized set (5 columns), expression/statistics from the dChip-normalized set (5 columns), and a final column indicating which probes fulfill the criteria for the final candidate COUP-TFI target gene list . Individual columns for each normalized set includes the following fields from left to right: expression fold change (wild-type average/mutant average), genotype p-value, time-point  p-value, interaction p-value, and a column denoting the initial hit list, i.e., probe sets with genotype p<0.01 and interaction p>0.01 (denoted as “G no I”) in that particular normalization method.(2.90 MB PDF)Click here for additional data file.Table S2HMM training sites. Complete description of the 49 COUP-TF response elements used to calculate the COUP-TF HMM parameters. The included fields are, from left to right: name of the sequence, Locus Link number, gene name, brief gene description, species, competing elements or cofactors (as described in the literature), final HMM training sequence (formatted as described in the (0.04 MB XLS)Click here for additional data file.Table S3Complete candidate inner ear COUP-TFI target database. Genes are listed in decreasing rank order. MC = HMM match within a most conserved UCSC site, >0.3 = HMM match with a conservation score greater than 0.3, >0.7 = HMM match with a conservation score greater than 0.7.(0.06 MB XLS)Click here for additional data file.File S1COUP-TF HMM in XML format. XML file denoting the precise transition and emission parameters used for the COUP-TF HMM genomic search.(0.02 MB TXT)Click here for additional data file.File S2List of primers. This file includes two tables: 1) the list of primers used for real-time RT-PCR microarray validation; and 2) the list of primers used in chromatin immunoprecipitations coupled to real-time PCR.(0.02 MB XLS)Click here for additional data file.


Mobile group II introns insert site-specifically into DNA target sites by a mechanism termed retrohoming in which the excised intron RNA reverse splices into a DNA strand and is reverse transcribed by the intron-encoded protein. Retrohoming is mediated by a ribonucleoprotein particle that contains the intron-encoded protein and excised intron RNA, with target specificity determined largely by base pairing of the intron RNA to the DNA target sequence. This feature enabled the development of mobile group II introns into bacterial gene targeting vectors (“targetrons”) with programmable target specificity. Thus far, however, efficient group II intron-based gene targeting reactions have not been demonstrated in eukaryotes.Xenopus laevis oocyte microinjection assay, we show that group II intron RNPs can integrate efficiently into target DNAs in a eukaryotic nucleus, but the reaction is limited by low Mg2+ concentrations. By supplying additional Mg2+, site-specific integration occurs in up to 38% of plasmid target sites. The integration products isolated from X. laevis nuclei are sensitive to restriction enzymes specific for double-stranded DNA, indicating second-strand synthesis via host enzymes. We also show that group II intron RNPs containing either lariat or linear intron RNA can introduce a double-strand break into a plasmid target site, thereby stimulating homologous recombination with a co-transformed DNA fragment at frequencies up to 4.8% of target sites. Chromatinization of the target DNA inhibits both types of targeting reactions, presumably by impeding RNP access. However, by using similar RNP microinjection methods, we show efficient Mg2+-dependent group II intron integration into plasmid target sites in zebrafish (Danio rerio) embryos and into plasmid and chromosomal target sites in Drosophila melanogster embryos, indicating that DNA replication can mitigate effects of chromatinization.By using a plasmid-based Our results provide an experimental foundation for the development of group II intron-based gene targeting methods for higher organisms. Mobile group II introns employ a remarkable site-specific DNA integration mechanism, termed retrohoming, in which the excised intron RNA inserts directly into a DNA target site and is then reverse transcribed by the intron-encoded protein (IEP) Lactococcus lactis Ll.LtrB intron has been used as a model system both to study group II intron mobility and to develop gene targeting applications The For gene targeting in bacteria, the Ll.LtrB intron is introduced via a donor plasmid, which expresses an Ll.LtrB-ΔORF intron with flanking exons, followed by the LtrA protein from a position just downstream of the 3′ exon It would be highly desirable to develop analogous group II intron-based gene targeting methods for higher organisms. With the exception of mouse embryonic stem cells and a few other cell types, mammalian cells and those of other higher eukaryotes lack efficient homologous recombination systems for gene targeting To overcome such deficiencies, alternative gene targeting methods are being developed, employing different means to increase the frequency of homologous recombination. One of the best developed of such methods for human cells, homologous recombination with recombinant AAV, generally yields 0.1–15% of integrations at the desired target site Although still undeveloped for use in eukaryotes, mobile group II introns can potentially be employed in the same way as Zn-finger nucleases and meganucleases to introduce a targeted double-strand break that stimulates homologous recombination or yields mutations by inaccurate non-homologous end-joining Escherichia coli plasmid assays E. coli assays to show that group II introns could be used for genetic repair, either by inserting a functional copy of the gene into a defective gene, or by inserting functional exons preceded by a splice acceptor site to circumvent defective downstream exons Previously, we retargeted the Ll.LtrB group II intron to insert in the HIV-1 provirus and the human gene encoding the HIV-1 co-receptor CCR5 and showed that the retargeted introns insert into their target sites with high frequency and specificity in 2+ concentrations in eukaryotic cells. In the yeast Saccharomyces cerevisiae, mutations in mitochondrial Mg2+-transport proteins, which result in decreased intramitochondrial Mg2+ concentrations, strongly inhibited the splicing of all four mtDNA group II introns, while having much less effect on the splicing of group I introns in vitro reaction conditions for the Ll.LtrB intron use 5 mM Mg2+ for IEP-promoted RNA splicing and 10 mM Mg2+ for reverse splicing of RNPs into DNA target sites 2+ concentration is estimated to be 1–2 mM There was reason to believe that group II introns might be particularly sensitive to low MgXenopus laevis oocyte microinjection assay to show that group II intron RNPs can insert efficiently into a plasmid target site in eukaryotic nuclei, but require the injection of additional MgCl2. In such assays under optimal conditions, group II intron RNP integration frequencies were as high as 38% and targeting frequencies by double-strand break- stimulated homologous recombination were as high as 4.8%. Analogous RNP microinjection assays show relatively efficient Mg2+-dependent group II intron integration into plasmid target sites in D. melanogaster and zebrafish (Danio rerio) embryos and into chromosomal target sites in D. melanogaster embryos. Our results demonstrate the potential for using group II introns for gene targeting reactions in higher organisms.Here, we used a X. laevis oocyte nuclei, we used a plasmid assay patterned after one developed to assay group II intron mobility in E. coli  cloned upstream of a promoterless Rtet gene in an AmpR target plasmid. Insertion of the intron containing the T7 promoter into the target site activates the Rtet gene, enabling intron-integration efficiencies to be measured by the ratio of (TetR+AmpR)/AmpR colonies. For gene targeting applications, we generally use a streamlined 0.9-kb derivative of the Ll.LtrB intron from which ORF sequences encoding the IEP have been deleted. This Ll.LtrB-ΔORF intron has much higher integration efficiency and nuclease-resistance than does the full-length intron To assay group II intron targeting reactions in  E. coli  [4], [8]X. laevis oocyte nuclei is shown in ltrB target site was injected first, followed within 1 min by Ll.LtrB RNPs reconstituted with self-spliced Ll.LtrB-ΔORF lariat RNA and purified recombinant LtrA protein , which expresses phage T7 RNA polymerase. The cells were then plated on LB medium containing ampicillin with or without tetracycline to determine the mobility efficiency as described above.The protocol used for the assays in tein see . In all R colonies were obtained. In light of the previous studies indicating that group II introns are particularly sensitive to low Mg2+ concentrations , and controls showed no TetR colonies after heating the RNPs to 95°C for 3 min or using RNPs reconstituted with a mutant LtrA protein that lacks RT activity (RT−). The integration efficiency was proportional to the amount of RNPs injected (not shown) and with different batches of RNPs ranged up to 38% of plasmid target sites (with injection of 20 nl of 2.5 mg/ml RNPs). Finally, efficient integration was obtained only when both the RNP and target plasmid were injected into the nucleus and not when they were injected into the cytoplasm .To determine the optimal injected Mgid MgCl2 . The ord2 to the culture medium results in chromosome unfolding X. laevis oocyte nuclei and could facilitate group II intron integration, we found that addition of 10–500 mM MgCl2 to the X. laevis oocyte incubation medium resulted in little or no increase in the group II intron-integration efficiency (the maximum was 4×10−4 % with 65 mM MgCl2).Studies with bovine capillary endothelial cells showed that the addition of 65 mM MgCl2 injection mixture, which increased group II intron-integration frequencies in suboptimal oocytes to >3% (not shown). Higher dNTP concentration did not further increase the integration frequencies. In later experiments, 17 mM dNTPs were added routinely.In early experiments, there was considerable variation in the intron-integration efficiency between different batches of oocytes (insertion frequencies 0.002–7%). Later, we found that this problem could be mitigated by addition of 17–20 mM each of dATP, dCTP, dGTP, and dTTP (“dNTPs”) to the DNA/MgCl2+ and dNTP concentrations, the highest integration efficiencies were obtained at temperatures of 25–30°C  cleaves either single-stranded RNA or RNA in RNA/DNA heteroduplexes, had little if any effect  with a T7 promoter flanked by 705- and 718-bp homology arms corresponding to sequences on either side of the intron-insertion site in the recipient plasmid. After injecting the DNAs, we injected Ll.LtrB-ΔORF lariat RNPs, without an internal T7 promoter, into the same oocyte nuclei. Introduction of a double-strand break at the Ll.LtrB target site by the intron RNPs is expected to stimulate homologous recombination, resulting in insertion of donor DNA containing the T7 promoter upstream of the promoterless Rtet gene. After incubating the oocytes, nucleic acids were isolated and electroporated into E. coli, and the recombination (“targeting”) efficiency was measured by the ratio of (TetR+AmpR)/AmpR colonies, as in the group II intron-integration assays.We showed previously that the Ll.LtrB group II intron could be used in R colonies (not shown). In other experiments under the same conditions, targeting efficiencies were typically 0.3–1.5% with a maximum of 4.8%. By comparison, target plasmid that had been pre-cut with a restriction enzyme and tested in the same assay without RNPs gave a recombination frequency of 66% . Together, these findings show that Ll.LtrB RNPs can be used in X. laevis oocyte nuclei to introduce a targeted double-strand break that stimulates gene targeting by homologous recombination.As summarized in y of 66% . As expehe assay . Further<0.007%; . In the i.e., ligation of the 3′ end of the intron RNA to the 5′ end of the 3′ exon, leaving a DNA-strand break in vitro transcription without the self-splicing step required to generate lariat RNA, would facilitate the preparation of RNPs.We wished to test whether RNPs reconstituted with linear intron RNA could be used to introduce the recombinogenic double-strand break at the target site. The rationale was that while linear intron RNA cannot undergo both transesterification reactions required for full reverse splicing, it is expected to be fully competent to carry out the first transesterification, in vitro with an internally labeled double-stranded DNA oligonucleotide substrate containing the Ll.LtrB-insertion site, and the products were analyzed by electrophoresis in a denaturing polyacrylamide gel. As found previously First, we carried out biochemical assays to confirm that Ll.LtrB RNPs reconstituted with linear intron RNA could carry out the expected reactions. in vitro, we next compared the ability of RNPs reconstituted with linear or lariat intron RNA to promote gene targeting by group II intron-stimulated homologous recombination in X. laevis oocyte nuclei. Gene targeting was again assayed by recombination of a donor DNA containing a T7 promoter into the recipient plasmid target site to activate the downstream Rtet gene. As shown in 260 measurements, the RNPs containing linear intron RNA stimulated gene targeting by recombination at an efficiency of about one third that of RNPs reconstituted with lariat RNA . This difference in efficiency is within the range of variation for different RNP preparations, but could also reflect that the linear intron RNA transcribed in vitro has heterogeneous 3′ ends due to imprecise transcription termination by T3 RNA polymerase , and we observed little or no group II intron integration if extra MgCl2 was not injected with the target plasmid. For D. melanogaster, where the injection volumes were ∼0.3 nl, we obtained the highest integration efficiencies by injecting 250 mM MgCl2 with the target plasmid. Assuming an embryo volume of 7.3 nl 2+ resulting from this injection would be ∼10 mM. For zebrafish, where the injection volumes were ∼10 nl, the highest integration efficiencies were obtained by injecting 150 mM MgCl2, which assuming an embryo volume of 200 nl 2+ of 7.5 mM or higher if one corrects for the 77% of embryo volume made up by the less permeable yolk. Thus, in both Drosophila and zebrafish embryos, the calculated increase in intracellular Mg2+ concentrations required to give maximal Ll.LtrB intron integration are in the same range as that found for X. laevis oocytes (∼9 mM). These findings suggest that a similar requirement for additional Mg2+ for maximal group II intron RNP integration may be a common feature of eukaryotic organisms.As shown in 2 and RNPs were 27–52% compared to 35% for a single injection of distilled water. For D. melanogaster embryos, the hatch rate after a single injection of 100 mM MgCl2, a concentration that supports chromosomal gene targeting (see below), was 69%. However, a double injection of 100 mM MgCl2 and RNP buffer decreased the hatch rate to 31% (not shown). Thus, both D. melanogaster and zebrafish appear reasonably tolerant of the high Mg2+ concentrations required to support group II intron integration, but the double injection appears better tolerated in zebrafish than in the smaller D. melanogaster embryos. We did not notice any further decrease in embryo viability caused by the injection of RNPs beyond that caused by the injections of Mg2+ and RNP buffer. We note, however, that toxicity could be greater at higher RNP concentrations or with RNPs targeted to different sites and will need to be assessed carefully in each case.For zebrafish embryos, hatch rates after the double injection of 500 mM MgClD. melanogaster embryos to test whether the reaction conditions we developed in plasmid targeting assays could also be used for site-specific integration into chromosomal target sites. For this purpose, we used retargeted Ll.LtrB introns (“targetrons”) designed to insert at two different sites in the yellow (y) gene  gene . These tD. melanogaster embryos at the precellular blastoderm stage were injected with 100 mM Mg2+ plus 17 mM dNTPs, followed by Y18a lariat RNPs. After incubation for 1 h at 30°C followed by 48 h at 18°C, nucleic acids were extracted from 50 pooled embryos and analyzed by PCR. The PCRs showed bands corresponding to the expected 5′- and 3′-integration junctions (y gene was confirmed by sequencing the PCR products (not shown). A similar double-injection experiment showed precise integration of Y3776s lariat RNPs at their target site, detected by PCR and confirmed by sequencing of the 3′-integration junction in 40 pooled embryos. For both targetrons, integration into the chromosomal target sites was not detected without injection of Mg2+.In the first experiment , D. melaunctions , and pre2+ required for efficient DNA integration. Thus, we tested whether substitution of polyamines might enable us to lower the amount of Mg2+ that must be injected with the RNPs. Polyamines had been shown previously to reduce the Mg2+ requirement for group II intron self-splicing reactions 2+ and a polyamine cocktail containing 5 mM putrescine, 3 mM spermidine, 1 mM spermine, corresponding to polyamine ratios in D. melanogaster larvae y gene, detected by PCR in 30 pooled embryos and confirmed by sequencing the 3′-integration junction. Viability as measured by hatch rate after injection of this polaymine/Mg2+ mixture was similar to that for a single injection of distilled water. Thus, the use of polyamines to reduce the Mg2+ requirement for group II intron function provides a potential solution to the problem of double injection into D. melanogaster embryos, enabling the introduction of RNPs and required supplements in a single injection that gives chromosomal gene targeting.The decreased viability caused by double injection (see above) made it desirable to develop single-injection conditions. As indicated previously, however, group II intron RNPs tend to aggregate when injected with the relatively high concentrations of Mg2+. First, we demonstrated Mg2+-dependent group II intron integration promoted by RNPs into plasmid target sites in X. laevis oocyte nuclei and used the facile X. laevis system to optimize reaction conditions. Then, by using these conditions, we showed similar Mg2+-dependent group II intron integration into plasmid target sites in both Drosophila and zebrafish embryos, and in Drosophila, we extended the methods to demonstrate site-specific integration into chromosomal target sites. We also show that with the injection of additional Mg2+, group II intron RNPs injected into X. laevis oocyte nuclei can be used to introduce a targeted double-strand break that stimulates gene targeting by homologous recombination. Thus, our results demonstrate that group II introns can potentially be used for two types of gene targeting reactions, site-specific integration and double-strand break-stimulated homologous recombination, in higher organisms.Here, by using group II intron RNP microinjection assays, we show that group II intron-based gene targeting reactions can occur efficiently in eukaryotes, but are dependent upon the injection of additional Mg2+ concentration in eukaryotes differs from the situation in bacteria, the natural hosts of group II introns, where free Mg2+ concentrations are apparently sufficient to support efficient mobility of Ll.LtrB and other group II introns. Group II introns seem to be particularly sensitive to low Mg2+ concentrations. Studies of yeast mtDNA group II introns showed that mutations in Mg2+ transporters that result in decreased intramitochondrial Mg2+ concentrations strongly inhibit the splicing of all four group II introns, which belong to different structural subclasses, while having relatively little effect on the splicing of group I introns 2+ concentrations in yeast mitochondria use different protein co-factors to promote RNA splicing 2+ requirement for group II intron function is a property of the intron RNA. For eukaryotic model organisms, the limitation of group II intron function by low Mg2+ concentrations can be overcome by injecting additional Mg2+ or Mg2+ in combination with polyamines. Longer term but more general solutions would be to select variants of the Ll.LtrB intron or to identify other group II introns that can function efficiently at lower Mg2+ concentrations.The finding that Ll.LtrB intron RNP activity is limited by MgX. laevis oocyte nuclei appear to have the enzymatic machinery necessary to complete these reactions. Group II intron-stimulated homologous recombination is presumably completed by a single-strand annealing pathway, which has been shown previously to be active in X. laevis oocyte nuclei Although group II intron RNPs can by themselves initiate site-specific DNA integration and double-strand break-stimulated recombination, host enzymes are required to complete both processes see . An impoX. laevis oocyte nuclei. A key finding is that group II intron RNPs reconstituted with linear as well as lariat intron RNA can be used to introduce the targeted double-strand break. This is so because although linear intron RNA is incapable of complete reverse splicing, it is fully competent to carry out the first step, attachment of the intron RNA to the 5′ end of the 3′ exon, leaving a DNA strand break. For both linear and lariat intron RNA, DNA cleavage occurs by a transesterification reaction with the attachment of the intron RNA to the target DNA and not by hydrolysis, a mechanism used for group II intron ribozyme cleavage of RNA substrates in vitro transcription without the self-splicing step needed for the preparation of lariat RNA.The ability of group II introns to introduce a targeted double-strand break that stimulates gene targeting by homologous recombination was first demonstrated in bacteria in vivo by using hydrolysis rather than branch-point formation for cleavage at the 5′ splice site, leading to the production of linear intron RNA In addition to their practical applications, our findings for RNPs containing linear intron RNA also have implications for group II intron mobility mechanisms. Some group II introns splice X. laevis oocyte nuclei strongly inhibits both group II intron DNA integration and double-strand break-stimulated homologous recombination, presumably because it impedes access of the RNPs to the DNA target site. Although the inhibition by chromatinization appears greater for group II intron RNPs than was observed previously for the meganuclease I-SceI in analogous experiments in X. laevis oocyte nuclei 2+-dependent plasmid targeting in both Drosophila and zebrafish embryos, and in the Drosophila, we show that optimal conditions developed in plasmid targeting assays can be used for site-specific integration into two different chromosomal target sites in the y gene. In both cases, the site-specific integration into chromosomal DNA occurred at frequencies high enough to detect by PCR and confirm by sequencing in 50 or less pooled embryos. Unlike previously developed Drosophila gene targeting methods Unsurprisingly, our results show that chromatinization of the plasmid DNA target site in Both group II intron integration and double-strand break-stimulated homologous recombination are potentially powerful methods for gene targeting in higher organisms. As retroelements akin to retroviruses, group II introns have a highly evolved DNA integration mechanism, which enables targeted rather than random integration. Further, we showed previously that mobile group II introns could be used as vectors to integrate genetic markers or cargo genes cloned in domain IV at desired DNA target sites in bacteria in vitro transcription of wild-type Ll.LtrB-ΔORF intron RNA, which is then reconstituted with purified LtrA protein to form RNPs (“targetrons”) for gene targeting Rcam gene, and have the LtrA ORF cloned downstream of the 3′ exon. In pACD2, the Ll.LtrB-ΔORF intron contains an additional phage T7 promoter inserted in intron domain IV (DIV) for use in plasmid-based DNA-integration assays. pACD4C and pACD4G are derivatives of pACD3, which have C or G at the δ′ +1 position in the 3′ exon and were used for construction and in vitro transcription of targetrons Y18a and Y3776s, respectively D. melanogaster yellow (y) gene. The donor plasmid used for each targetron has a δ′ residue in the 3′ exon complementary to the retargeted δ residue in the intron RNA for optimal RNA splicing pACD2 and pACD3 are intron-donor plasmids used for ltrB gene from position −178 upstream to +91 downstream of the intron-insertion site) cloned upstream of a promoterless Rtet gene in an AmpR pBR322-based vector pBRR3-ltrB, the target plasmid for intron-integration assays, contains the Ll.LtrB homing site (ligated exon 1 and 2 of the − expresses RT-deficient LtrA (RT−) with the mutation YADD→YAAA at the RT active site pIMP-1P, used for expression of the LtrA protein, contains the LtrA ORF cloned downstream of a tac promoter and Φ10 Shine-Dalgarno sequence in the protein-expression vector pCYB2  GGATCCTTCTTCGGGGCGAAAACTCTCAAGG5′-CGGC)+PUFNcoIT7A (CCCTATAGTGAGTCGTATTACCATGGGTTATGGATGTGTTCAC5′-GAATTAAAAATGATATG) and PUFNcoIT7S (CCATGGTAATACGACTCACTATAGGGCATATCATTTTTAATTC5′-GTGAACACATCCATAAC)+pBRR+700BamA (GGATCCCTGGGCGGCGGCCAAAGCGGTCGGA5′-CGGC), respectively. The PUFNcoIT7A primer introduces a NcoI site  and a phage T7 promoter (underlined), and the outside primers introduce BamHI sites . The product obtained in a second PCR with the outside primers was cloned into the BamHI site of pUC19, and the 3,967-bp NcoI fragment of phage λ DNA was inserted into the NcoI site.pUC19-PUF, used to generate the linear donor DNA for group II intron-stimulated homologous recombination, contains homology arms of 705- and 718-bp upstream and downstream of the Ll.LtrB-insertion site, respectively, flanking a 3,967-bp phage λ DNA NcoI fragment and a T7 promoter sequence. To construct this plasmid, the left and right homology arms were amplified from pBRR3-ltrB with primers pBRR-700BamS (in vitro-synthesized intron RNA and purified LtrA protein by a modification of a method described previously AATTAACCCTCACTAAAGGGAATTGTGAGCGGGAGTCTAGA), which appends a T3 promoter sequence (underlined), and the 3′ primer LtrB+744a (5′ CTCCTCTAGAATCCGCTGTATCATCTAATATTCCTTTTG) or NheIR (CTAGCAGCACGCCATAGTGACTGGCG). The resulting precursor RNAs containing the Ll.LtrB-ΔORF intron and flanking exon sequences were self-spliced in 1.25 M NH4Cl, 50 mM MgCl2, 50 mM Tris-HCl, pH 7.5 for 3 h at 37°C, then ethanol-precipitated and dissolved in distilled water.Ll.LtrB-ΔORF intron RNPs were reconstituted from 2, 40 mM Tris-HCl, pH 7.5 and slowly cooling to 30°C prior to addition of 200 nM of purified LtrA protein 2, and 40 mM HEPES, pH 8.0. The RNP preparations typically contain 60–70% intron lariat RNA with the remainder being precursor RNA plus smaller amounts of linear intron and ligated exons, and they typically have a concentration of 2–3 mg RNA/ml based on O.D.260, with the highest obtained being 5.6 mg/ml.To reconstitute Ll.LtrB-ΔORF RNPs, the self-spliced RNA (100 nM) was re-natured by heating to 50 or 55°C in 10 ml of 450 mM NaCl, 5 mM MgClAATTAACCCTCACTAAAGGG TGCGCCCAGATAGGGTGTTAAGTCAAG), which appends a T3 promoter (underlined), and the 3′ primer LtrB+940a (GTGAAGTAGGGAGGTACCGCCTTGTTC), which corresponds to the 3′ end of the intron. The linear RNA was renatured and reconstituted with purified LtrA protein To make RNPs containing linear intron RNA, Ll.LtrB-ΔORF intron RNA was transcribed with phage T3 RNA polymerase from a PCR product generated from pACD3 using the 5′ primer T3-LIs  32P]ATP and phage T4 polynucleotide kinase (New England Biolabs). The 60-bp DNA substrates were prepared by annealing complementary synthetic oligonucleotides and purified using a centri-sep spin column . For assays with the internally labeled DNA substrate, the DNA  was incubated with RNPs (815 nM) for 30 min at 37°C in 20 µl of reaction medium containing 10 mM KCl, 10 mM MgCl2, and 50 mM Tris-HCl, pH 7.5. For assays with the 5′-labeled DNA substrates, the DNA (50 nM) was incubated with RNPs (100 nM) for 30 min at 37°C in 20 µl of the same reaction medium plus 0.2 mM each of dATP, dCTP, dGTP, and dTTP (referred to as dNTPs). After the incubations, the reactions were terminated by extraction with phenol-chloroform-isoamyl alcohol  followed by ethanol precipitation, and the products were analyzed by electrophoresis in denaturing 6% or 10% (w/v) polyacrylamide gels, which were dried and scanned with a phosphorimager.DNA endonuclease assays were carried out with a 129-bp DNA substrate, which was internally labeled by PCR in the presence of . Oocytes were kept in incubation medium at 16°C for at least 30 min before injection.Mature adult female frogs were obtained from Nasco . Stage VI oocytes were manually peeled from the follicle cell layer in isolation medium , treated with 0.05% collagenase  for 10 min, and rinsed in incubation medium  for 1 h at 55°C, and then extracted twice with phenol-CIA. Nucleic acids were precipitated with isopropanol and dissolved in 20–50 µl of distilled water. Two µl of the nucleic acid preparation was electroporated into X. laevis oocyte nuclei were injected with a mixture of closed-circular target plasmid pBRR3-ltrB (4–5 ng) and donor DNA (4–17 ng) in 10–20 nl of 0.5 M MgCl2, 10 mM Tris-HCl, pH 7.5, unless specified otherwise. The donor DNA was a 5.4-kb linear DNA generated by BamHI-digestion of pUC19-PUF (see above), followed by phenol-CIA extraction and ethanol precipitation. Ll.LtrB RNPs (0.9–5.6 mg/ml), without an internal T7 promoter, were injected into the same oocyte nuclei. Ten to twenty oocytes were injected and pooled for each experimental condition. The injected oocytes were incubated under conditions specified for individuals experiments, then nucleic acids were extracted and electroporated into E. coli HMS174(DE3), followed by plating on LB containing ampicillin with or without tetracycline, as for the group II intron-integration assays (see above).For group II intron-stimulated homologous recombination reactions, D. rerio strains were the AB line in vitro fertilization performed, as described 2, with 0.25% phenol red) and Ll.LtrB-ΔORF RNPs  were injected separately into 10 to 25 one-cell embryos in Steinberg's medium E. coli, as described above for X. laevis assays.Wild-type w1118 strain was grown in standard fly media at 22°C and embryos were collected on apple juice agar plates with yeast paste The 2 was injected into the posterior of the embryo, followed within 5 min by the injection of RNPs (1.6 mg/ml), using different needles to avoid prior mixing. For each experimental condition, 50 to 100 embryos were injected, incubated in a humidified chamber for 1 h at 30°C, taken up with a pipette, pooled, and homogenized in lysis buffer (see above) using a hand homogenizer. Plasmid DNAs were then extracted and transformed into E. coli, as described above for X. laevis assays.For plasmid-based group II intron-integration assays, target plasmid DNA (0.5 mg/ml) supplemented with 17 mM dATP, dCTP, dGTP, and dTTP (Invitrogen) and specified amounts of MgCly gene. Mg2+, dNTPs, and polyamines were injected prior to or mixed with the RNPs, and after the injections, the embryos were incubated, as specified for individual experiments in the legend of 5′-GTGAAGTAGGGAGGTACCGCCTTGTTC) and yellow+277a (5′-ACGATCTCCCCAAGGGCTTCAT) and nested primers LtrB+933a (5′-AGGGAGGTACCGCCTTGTTCACATTAC) and yellow+241a (5′-TACCATCACGCCAGCGGGGAA); Y18a, 3′-integration, LtrB+788s (5′-CGACTAATACGACTCACTATAGGGTC) and yellow-350s (5′-GCAAAGTTGGCCGATCTATGGGAAC) and nested primers LtrB+880s, (5′-GAAGAGGGTGGTGCAAACCAGTCAC) and yellow-160s, (5′-CGCCACGGTCCACAGAAGAG); Y3776s, 3′-integration junction: primers LtrB+788s (see above) and yellow+4325a (5′-ATGCCACCACCCAGATTGG) and nested primers LtrB+870s and yellow +4054a (5′-TGAGGTTTCTGTGGCAAGACAGGA). In each case, the PCR products were purified in a 1% agarose gel and sequenced to confirm integration at the expected target site.For chromosome targeting, precellular blastoderm stage embryos (<40-min collection time) were injected with targetron Y18a or Y3776s RNPs, which contain Ll.LtrB introns targeted to insert at sites in the Animal care met the principles and guidelines of the Institute for Laboratory Animal Research “Guide for Care and Use of Laboratory Animals” and the University of Texas at Austin Institutional Animal Care and Use Committee.Figure S1X. laevis oocyte nuclei. Target plasmid DNA (4.5 ng/18 nl) with 500 mM MgCl2 and 17 mM dNTPs was injected into X. laevis oocyte nuclei, followed by Ll.LtrB lariat RNPs (53 ng/18 nl). The oocytes were incubated at 25°C for the indicated times and then quick frozen on dry ice. Nucleic acids were extracted, and equal portions were either untreated or digested in 10 µl reactions with MluI , RNase A  or RNase H . MluI and RNase H digestions were done according to the manufacturers' protocols, and RNase A digestion was done in 50 mM NaCl, 10 mM Tris-HCl, pH 7.5. After digestion, the nucleic acids were extracted twice with phenol-CIA and ethanol precipitated in the presence of glycogen carrier prior to electroporation into E. coli HMS174(DE3). Integration efficiencies were determined as the ratio of (TetR+AmpR)/AmpR colonies. O/N, overnight.Nuclease-sensitivity of group II intron-integration products extracted from (0.12 MB TIF)Click here for additional data file.Figure S2TGAACAAGGCGGTACCTCCCTTGGCGACGCGTTGGGAAATGGCAATGATA by itself or annealed to a complementary DNA or RNA oligonucleotide, or the complementary RNA strand by itself. DNA and RNA oligonucleotides were obtained from Integrated DNA Technologies  and 5′-end labeled with [γ-32P]ATP  using phage T4 polynucleotide kinase (New England Biolabs) according to the manufacturer's protocol, then gel-purified in a denaturing 6% (w/v) polyacrylamide gel. In the ssDNA, DNA/RNA duplex, and dsDNA substrates, the DNA oligonucleotide was labeled, whereas in the RNA and RNA/DNA duplex substrates, the RNA oligonucleotide was labeled. Duplexes were formed by incubating the labeled oligonucleotide with an equal amount of unlabeled complementary strand, heating to 95°C and slow cooling to room temperature, then gel purifying in a 2% agarose gel containing Tris-borate-EDTA buffer . After incubation with the indicated enzymes, samples were extracted with phenol-CIA and ethanol precipitated in the presence of linear acrylamide carrier (58 µg/ml). Restriction enzyme digests were analyzed in a denaturing 8% (w/v) polyacrylamide gel, and RNase A and H digestions were analyzed in a denaturing 15% (w/v) polyacrylamide gel. Size markes were 5′-labeled 50- and 18-nt DNA oligonucleotides. The gels were dried and scanned with a phosphorimager.Digestion of DNA and RNA oligonucleotide substrates with restriction enzymes and ribonucleases A and H. (A) Single-stranded (ss) DNA, DNA/RNA heteroduplex, and double-stranded (ds) DNA substrates  were incubated with MluI or KpnI  in 100-µl of reaction medium for 1 h at 37°C according to the manufacturer's protocol. (B) RNA, RNA/DNA heteroduplex, and double-stranded DNA substrates  were incubated in 100-µl reactions with RNase A  or RNase H . RNase A digestion was done in 50 mM NaCl, 10 mM Tris-HCl, pH 7.5 for 5 min at room temperature, and RNase H digestion was done for 20 min at 37°C according to the manufacturer's protocol. In (A) and (B), substrates were DNA oligonucleotide 5′ (3.45 MB TIF)Click here for additional data file.


TBX5 gene and is characterized by congenital heart and preaxial radial ray upper limb defects. Most of the TBX5 mutations found in patients with HOS cause premature truncation of the primary TBX5 transcript. TBX5 missense mutations alter the three-dimensional structure of the protein and result in failed nuclear localization or reduced binding to target DNA. In this study we present our functional analyses of the novel and unusual c.1333delC mutation found in a patient with classical HOS.Autosomal dominant Holt-Oram syndrome (HOS) is caused by mutations in the The functional impact of this novel mutation was assessed by investigating the intracellular localization of the resulting TBX5 protein and its ability to activate the expression of its downstream target ANF.TBX5 frameshift mutation predicted to result in an elongated TBX5 protein with 74 miscoding amino acids and 62 supernumerary C-terminal amino acids. The c.1333delC mutation affects neither the nuclear localization, nor its colocalization with SALL4, but severely affects the activation of the ANF promoter.The deletion of the cytosine is the first The mutation c.1333delC does not locate within functional domains, but impairs the activation of the downstream target. This suggests that misfolding of the protein prevents its biological function. TBX5 gene mutations have been described  [TBX5 gene in patients with familial or sporadic HOS is 74% following application of stringent clinical criteria [Holt-Oram syndrome (HOS) is an autosomal-dominantly inherited disorder caused by mutations in the gene coding for the T-box transcription factor TBX5, which is located on chromosome 12q24 . To dateabase; ) . Nonsyndabase; ) , but somabase; ) . The detcriteria .Fgf10 expression in a synergistic manner together with Sall4 in the forelimbs via direct effects on the Fgf10 promoter [TBX5 abrogate this interaction and cause cardiac septal defects [TBX5 contains a T-box motif involved in DNA-binding and protein-protein interactions, and operates as a transcriptional activator ,11. Mutapromoter . This co defects . Transac defects . During  defects . Missens defects .de novo c.1333delC mutation identified in a patient with sporadic HOS. This mutation does not impair nuclear localization or co-localization with SALL4, but abrogates activation of the ANF promoter.Here we report on the results of functional analyses of a The now 4-year old male patient Fig.  presenteTBX5 gene including exon-intron boundaries was performed by direct DNA sequencing as described [After obtaining informed consent, genomic DNA was extracted from peripheral blood lymphocytes of the index patient and his unaffected parents according to standard protocols. Mutation analysis of the coding sequence of the escribed .TBX5 expression constructs were generated by PCR amplification using the commercially available human full-length cDNA clone . The sequence of the constructs was verified by automated sequencing . PCR fragments were subcloned into the pGEM®-T-Easy vector , digested with EcoRI and BamHI and subsequently cloned into the pEGFP-N3/pFLAG-N3 vectors . The ANF-luciferase vector was a kind gift from Benoit Bruneau .TBX5 construct carrying the c.1333delC mutation was generated with the QuikChange™ Site-directed Mutagenesis kit . Successful incorporation of the mutations was confirmed by automated sequencing.The COS-7 cells were grown in Minimal essential Medium (MEM), 10% Fetal Calf Serum, 100 μg/ml penicillin, 100 μg/ml streptomycin, 1× MEM nonessential amino acids  and maintained in a humidified environment with 5% carbon dioxide.4 cells per plate), washed in PBS after 24 h, fixed with methanol, washed in PBS and blocked for 30 min in 0.5% BSA in IF buffer  Tween 20). For the detection of intracellular SALL4 localization, coverslips were incubated with a monoclonal anti-FLAG® M2 antibody  for 1 h, washed twice with IF buffer and incubated with a fluorescence-labeled anti-mouse IgG (Fab specific)-TRITC antibody for 30 min. Cells were washed three times with IF buffer and mounted on slides with aquamount . Where indicated, cell nuclei were stained with DAPI blue. The TBX5 distribution pattern could be visualized due to fluorescence of the GFP fusion protein. Cells were analyzed by confocal laser scanning microscopy (CLSM).Co-transfected COS-7 cells were directly seeded on glass coverslips in 24-well plates  promoter. Each transfection was performed at least in triplicate in 24-well plates and all assays were done with two different DNA preparations of each construct. Transfections of adherent COS-7 cells were carried out at 60–80% confluency using 1 μg Lipofectamine™2000 reagent  per 500 ng transfected DNA. In each transfection an equimolar DNA concentration was used. Control vector pRL-SV40 , expressing the Renilla reniformis luciferase, was co-transfected for relativating transfection efficiency at a ratio of 1:100 to the total amount of transfected DNA. In order to analyze the transcriptional regulation capability of the TBX5 constructs, 300 ng ANF-luciferase plasmid and 300 ng TBX5 expression vector were used for co-transfections. DNAs of empty expression vector plasmids were used as negative control. Cell extracts were harvested 24 hours after transfection and dissolved in 125 μl Passive Lysis Buffer (Promega) per well. 20 μl were used to measure luciferase activity according to the Dual Luciferase Assay System Protocol (Promega) using a Lumat LB96P luminometer .The wild type and mutated TBX5  in the index patient, but not in his parents. The de novo deletion of the cytosine residue at cDNA position 1333 (codon 445) is predicted to result in a translational frameshift creating not a truncated but an elongated TBX5 protein of 580 amino acids due to a downstream shift of the termination codon to position 581 . Thus, the mutant TBX5 protein would contain 74 miscoding amino acids (positions 445 to 518) and 62 supernumerary C-terminal amino acids [see Additional file de novo mutational event, which most likely caused Holt-Oram syndrome in our patient. To provide evidence for the pathogenic effect of this special mutation, we carried out further functional investigations.We identified a novel heterozygous mutation in exon 9 of TBX5 was cloned into the GFP expression plasmid pEGFP-N3, whereas the coding sequence of SALL4 was inserted into the FLAG expression plasmid pFLAG-N3, resulting in TBX5wt-GFP, TBX5mut-GFP and SALL4-FLAG fusion proteins. TBX5 was shown to display a nuclear localization during embryogenesis, thereby having two distinct nuclear localization signals working cooperatively [SALL4 is a nuclear protein localized to heterochromatin and operating as a transcriptional repressor [The entire coding sequence of human wild type and mutated ratively ,19. The epressor . In the epressor .SALL4 and either wild type or mutated TBX5 constructs. SALL4 was detected by use of mouse anti-FLAG antibody and goat anti-mouse IgG-TRITC. An overlay of the SALL4 and TBX5 wild type  and an ANF-luciferase construct as reporter. The upstream region of the ANF  promoter contains a TBX5 binding site [Renilla reniformis luciferase activity, used as an internal control. The constructs were analyzed by immunoblot for appropriate expression (data not shown). Reporter gene activation significantly increased in cells transfected with wild type TBX5 compared to cells transfected with the empty expression vector. Transfection of the TBX5-GFP construct lead to the same result, indicating that neither the FLAG-tag nor the GFP-tag  have a detrimental effect on the protein function. The luciferase activity of the mutated TBX5-FLAG construct was strongly reduced to a value comparable to cells transfected with the empty pFLAG-N3 vector. An increased amount of transfected TBX5mut plasmid  did not induce luciferase activity, ruling out a dosage effect  [TBX5 missense mutations  reduce synergistic transcriptional activity of the downstream target ANF, but only the mutations p.G80R, p.R237Q, and p.R237W were shown to affect DNA-binding [de novo c.1333delC (p.H445fsX136) mutation on transcriptional activation we co-transfected COS-7 cells with mutant/wild type TBX5 expression constructs and an ANF-luciferase construct. Under the experimental conditions of the luciferase assay, the reporter gene activation through the mutated TBX5 protein was abrogated and non functional as compared to the wild type protein.Autosomal dominant Holt-Oram syndrome, linked to mutations in the T-box transcription factor gene abase; ) ,6,22. Muabase; ) . Diverse-binding . In addi-binding . To asseTBX5 gene consists of 8 coding exons and one 5' non-coding exon dispersed over 53 kilobases (kb) on chromosome 12q24. Proteins of the T-box family comprise two distinct structural and functional domains: the DNA-binding domain, referred as T-box, and a transcriptional activator or repressor domain [TBX5 mRNA bearing the single nucleotide deletion is not degraded by nonsense mediated decay. This mechanism was shown to occur only in case that the preterminal stop codon is located at least 50–55 nucleotides before the last exon/exon boundary [TBX5, the mRNA is predicted to result in a elongated protein consisting of 580 instead of 518 amino acids. It is conceivable that an altered protein configuration or steric hindrance may silence the activity of the NLS motifs. We therefore wanted to analyze the intracellular distribution of the mutated TBX5 protein. TBX5 is mainly expressed during embryonic morphogenesis and is limited to esophagus, heart, liver, lung, prostate and trachea in the adult organism. Blood samples from the patient were taken for DNA sequence analysis, but could not be used for investigations on the TBX5 protein as it is not detectable in lymphocytes. Consequently we did not have the possibility to analyze the presence and the intracellular localization of the endogenous elongated protein. Other possible TBX5 sources were not available. All experiments were therefore carried out by transfecting wild type and mutated fusion constructs into an appropriate cell line.The human r domain . The trar domain . For theboundary . Thus, mBoth wild type as well as mutated TBX5 proteins were found to localize in dot-like structures within the nucleus, and both co-localize with SALL4. The murine homologue Sall4 was previously demonstrated to localize to heterochromatin and to operate as a transcriptional repressor .TBX5 gene  so far to our knowledge. All result in shortening of the TBX5 protein. However, it is unclear how those lead to TBX5 haploinsufficiency, since the mutations will most likely not lead to nonsense-mediated mRNA decay, because they reside within the most 3' positioned exon. The unique mutation c.1333delC differs from the other mutations in exon 9 as it leads to an elongated protein. Since the mutation neither affects the intranuclear distribution of TBX5 nor its colocalization with SALL4, but abrogates activation of the ANF promoter, it is tempting to speculate that the supernumerary amino acids modulate the configuration of the protein by masking the transcriptional activator domain.Interestingly, four other truncating mutations have been detected in exon 9 of the The authors declare that they have no competing interests.JB conceived of the study, carried out the functional molecular genetic studies and drafted the manuscript. WH and UF accomplished the sequence analysis and wrote the clinical section of the manuscript. AC participated in the functional molecular genetic studies. MV and BMEJ collected the clinical data. JK coordinated the project and participated in the manuscript preparation.Written consent for publication was obtained from the patient's parents.The pre-publication history for this paper can be accessed here:The c.1333delC mutation results in an elongated protein. The heterozygous c.1333delC mutation (yellow) in exon 9 of TBX5 results in a translational frameshift creating an elongated TBX5 protein due to a downstream shift of the termination codon. The mutant TBX5 protein contains 74 miscoding amino acids and 62 supernumerary C-terminal amino acids (black letters).Click here for file


Inteins and introns are genetic elements that are removed from proteins and RNA after translation or transcription, respectively. Previous studies have suggested that these genetic elements are found in conserved parts of the host protein. To our knowledge this type of analysis has not been done for group II introns residing within a gene. Here we provide quantitative statistical support from an analyses of proteins that host inteins, group I introns, group II introns and spliceosomal introns across all three domains of life.To determine whether or not inteins, group I, group II, and spliceosomal introns are found preferentially in conserved regions of their respective host protein, conservation profiles were generated and intein and intron positions were mapped to the profiles. Fisher's combined probability test was used to determine the significance of the distribution of insertion sites across the conservation profile for each protein. For a subset of studied proteins, the conservation profile and insertion positions were mapped to protein structures to determine if the insertion sites correlate to regions of functional activity. All inteins and most group I introns were found to be preferentially located within conserved regions; in contrast, a bacterial intein-like protein, group II and spliceosomal introns did not show a preference for conserved sites.These findings demonstrate that inteins and group I introns are found preferentially in conserved regions of their respective host proteins. Homing endonucleases are often located within inteins and group I introns and these may facilitate mobility to conserved regions. Insertion at these conserved positions decreases the chance of elimination, and slows deletion of the elements, since removal of the elements has to be precise as not to disrupt the function of the protein. Furthermore, functional constrains on the targeted site make it more difficult for hosts to evolve immunity to the homing endonuclease. Therefore, these elements will better survive and propagate as molecular parasites in conserved sites. In contrast, spliceosomal introns and group II introns do not show significant preference for conserved sites and appear to have adopted a different strategy to evade loss. Inteins are intervening polypeptide sequences that are translated as part of a protein -4, and aIntrons are defined as non-coding regions of a gene that are excised during post-transcriptional processing. Since their discovery in 1977  three maEndonucleases  provide Previous work found inteins and group I introns in conserved parts of their host genes ,25-27, wOur earlier analyses of three host proteins, ATPase catalytic subunit, replication factor C (RFC), and cell division control protein 21 (CDC21), confirmed the notion that inteins appear at highly conserved sites within their host proteins . Since pp = 0.0099). The intein database InBase [p = 0.3909). Inteins in positions "a" and "b" are mapped to the structure of the ATPase catalytic subunit of Pyrococcus horikoshii OT3 ; and mapping of these sites on the structure of a RFC confirms that these sites are in conserved and centrally located regions of the protein. The 10 spliceosomal introns are not found to be in conserved sites of the protein (p = 0.2404).The replication factor C (RFC) is less than 300 amino acids long, but accommodates inteins in three different sites (a-c) and spliceosomal introns in 10 different sites Figure . InBase p = 0.0022)  Using Fisher's combined probability method to calculate the overall significance level for inteins (omitting the BIL) inserting into conserved sites is p < 0.0001 .In addition, we analyzed all proteins that were reported in InBase  as contap = 0.022)  Figure .cox1) gene found in the mitochondria of eukaryotes accommodates self-splicing group I and group II introns in more than forty different sites , and similar to spliceosomal introns, group II introns were found not to have a significant preference for conserved sites (p = 0.4176).The cytochrome C oxidase subunit I , while there is no correlation between site conservation and number of species with group II introns at each site (p = 0.2907). These findings suggest that for fungi, either group I introns tend to target conserved DNA sites more frequently, or group I introns survive in conserved sites for longer periods of time, while group II intron targeting is not strongly influenced by site conservation.A linear regression analysis was performed to determine if there is a correlation between the number of fungal species that harbour the introns in a particular site and the conservation of the intron insertion site. A weak negative correlation between site conservation and number of species with group I introns is found at each site .An additional eight proteins which host group I and group II introns were analyzed. These represent all protein families reported in the comparative RNA web site database  as contacox1 gene is present in mitochondrial genomes of several vascular plants [Marchantia, Chara, and Prototheca, the liverworts Pellia, the soil-living amoeba Dictyostelium, and the single-celled protist Monosiga  is found in the mitochondrion, and hosts many introns representing both group I and group II introns. Several studies provide evidence for horizontal gene transfer of this element between distantly related groups of eukaryotes, confirming an intron homing model of evolution [In most eukaryotes the volution ,55,56.cox1 gene introns were found in both conserved and variable regions of the host; however, introns that are found in numerous species are more often found in highly conserved sites. The only group I intron found in the cox1 gene of several vascular plants [As shown in figure r plants  is inserr plants . As the It is possible that inteins and group I introns target all sites but only the ones that end up in a conserved site are retained. The two exceptional proteins in this study, NADH dehydrogenase subunit 3 and Chlorophyll alpha apoprotein A2, may be an example of this. This mechanism would result in a preference for conserved sites even in the absence of a site specific homing mechanism. Such independent preference for conserved sites may have caused splicing elements (introns and inteins) and homing endonucleases to target the same sites, resulting in the fusion of these elements . Free-stThe fact that BILs do not show a conserved site preference  suggestsThe rate and tendencies to gain and lose introns vary considerably between lineages of eukaryotes ,43, withWe have provided statistical support for the notion that inteins and group I introns target conserved protein sites for survival. This may also provide evidence for the homing cycle that describes the life cycle of these two molecular parasites. Furthermore, our findings suggest that group II and spliceosomal introns persist in their host genes using a different evolutionary strategy.To calculate conservation profiles along a protein sequence, BLASTP  was usedTo construct archaeal/vacuolar-type ATPase catalytic subunit A protein sequence alignments sequences from 29 Eukaryotic species, 13 Archaeal species, and four Bacterial species were aligned. Ten Archaeal species and fifteen Eukaryotic species were aligned to construct the Replication factor C protein sequence alignments. Nine Archaeal species and 21 Eukaryotic species were aligned to construct the cell division control protein 21 protein sequence alignment. Sequences from two Eukaryotic species, 18 Bacterial species and four phage sequences were used to construct the DNA polymerase I protein sequence alignments. 21 Eukaryotic species and nine Archaeal species were use for the Cytochrome C Oxidase Subunit I protein alignment. All other proteins found in the InBase database   based on the probability of a random position within the conservation profile containing an equal or greater conservation score than that at the position of the intron/intein insertion. If the protein contained more than one of each parasitic element these probabilities were then combined for each protein using Fisher's combined probability test [p*) of intron/intein insertions being randomly distributed within each conservation profile. To show that no one kingdom overwhelmingly contributed to the significance of p* each kingdom was removed from the alignment and a new conservation profile was made and the Fisher's combined probability test was performed , and the degrees of freedom in the sample (n-1).Simple linear regression was used to correlate site conservation with intron/intein penetrance (the number of species infected with a specific element). Significance was then calculated using standard methods based upon the resulting correlation coefficient  and Saccharomyces cerevisiae (V00694) cox1 genes were used as query sequences.Click here for fileAccession numbers for sequences used in protein alignment. Lists of accession numbers for each protein used for each conservation profile.Click here for filePerl scripts used to calculate the conservation profiles. Perl scripts used to calculate conservation profiles.Click here for file