Osvaldo Graña

A DNA methylation fingerprint of 1628 human samples

Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases.

Reprogramming in vivo produces teratomas and iPS cells with totipotency features

Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.

EVA: evaluation of protein structure prediction servers

EVA (http://cubic.bioc.columbia.edu/eva/) is a web server for evaluation of the accuracy of automated protein structure prediction methods. The evaluation is updated automatically each week, to cope with the large number of existing prediction servers and the constant changes in the prediction methods. EVA currently assesses servers for secondary structure prediction, contact prediction, comparative protein structure modelling and threading/fold recognition. Every day, sequences of newly available protein structures in the Protein Data Bank (PDB) are sent to the servers and their predictions are collected. The predictions are then compared to the experimental structures once a week; the results are published on the EVA web pages. Over time, EVA has accumulated prediction results for a large number of proteins, ranging from hundreds to thousands, depending on the prediction method. This large sample assures that methods are compared reliably. As a result, EVA provides useful information to developers as well as users of prediction methods.

Assessment of predictions submitted for the CASP6 comparative modeling category

Here we present a full overview of the Critical Assessment of Protein Structure Prediction (CASP6) comparative modeling category. Prediction accuracy for the 43 comparative modeling targets was assessed through detailed numerical comparisons between predicted and experimental structures. Assessments using standard measures for model backbone quality and structural alignment accuracy highlighted a small number of groups with stand out predictions and these findings were backed up by statistical comparisons. We were able to carry out evaluations of side‐chain contacts predictions and side‐chain rotamer accuracy, for which one group turned out to have statistically better predictions. We also assessed the prediction quality of structurally divergent regions and biologically important sites. Interestingly we were able to show that predictors were not predicting these important functional regions with any greater accuracy than the rest of the structure. In addition we investigated the ability of predictors to build models that improve on the structural template and reached some tentative conclusions from comparisons with the previous CASP experiment. Proteins 2005;Suppl 7:27–45.

PLCG1 mutations in cutaneous T-cell lymphomas

Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of primary cutaneous T-cell lymphoproliferative processes, mainly composed of mycosis fungoides and Sézary syndrome, the aggressive forms of which lack an effective treatment. The molecular pathogenesis of CTCL is largely unknown, although neoplastic cells show increased signaling from T-cell receptors (TCRs). DNAs from 11 patients with CTCL, both normal and tumoral, were target-enriched and sequenced by massive parallel sequencing for a selection of 524 TCR-signaling-related genes. Identified variants were validated by capillary sequencing. Multiple mutations were found that affected several signaling pathways, such as TCRs, nuclear factor κB, or Janus kinase/signal transducer and activator of transcription, but PLCG1 was found to be mutated in 3 samples, 2 of which featured a redundant mutation (c.1034T>C, S345F) in exon 11 that affects the PLCx protein catalytic domain. This mutation was further analyzed by quantitative polymerase chain reaction genotyping in a new cohort of 42 patients with CTCL, where it was found in 19% of samples. Immunohistochemical analysis for nuclear factor of activated T cells (NFAT) showed that PLCG1-mutated cases exhibited strong NFAT nuclear immunostaining. Functional studies demonstrated that PLCG1 mutants elicited increased downstream signaling toward NFAT activation, and inhibition of this pathway resulted in reduced CTCL cell proliferation and cell viability. Thus, increased proliferative and survival mechanisms in CTCL may partially depend on the acquisition of somatic mutations in PLCG1 and other genes that are essential for normal T-cell differentiation.

CASP6 Assessment of Contact Prediction

Here we present the evaluation results of the Critical Assessment of Protein Structure Prediction (CASP6) contact prediction category. Contact prediction was assessed with standard measures well known in the field and the performance of specialist groups was evaluated alongside groups that submitted models with 3D coordinates. The evaluation was mainly focused on long range contact predictions for the set of new fold targets, although we analyzed predictions for all targets. Three groups with similar levels of accuracy and coverage performed a little better than the others. Comparisons of the predictions of the three best methods with those of CASP5/CAFASP3 suggested some improvement, although there were not enough targets in the comparisons to make this statistically significant. Proteins 2005;Suppl 7:214–224.

Assessment of intramolecular contact predictions for CASP7

Predictions of intramolecular residue–residue contacts were assessed as part of the seventh community‐wide Critical Assessment of Structure Prediction experiment (CASP7). As in past assessments, we focused on contacts that lie far apart in sequence as these are likely to be more informative in predicting protein structure. One lab did somewhat better than others according to our assessment, and there is some reason to think that this labs results represent progress over CASP6. In general, contacts inferred from 3D structural predictions are similar in accuracy to those predicted by contact prediction methods. However, contact prediction methods were more accurate for some targets. Proteins 2007.

Assessment of domain boundary predictions and the prediction of intramolecular contacts in CASP8.

This article details the assessment process and evaluation results for two categories in the 8th Critical Assessment of Protein Structure Prediction experiment (CASP8). The domain prediction category was evaluated with a range of scores including the Normalized Domain Overlap score and a domain boundary distance measure. Residue‐residue contact predictions were evaluated with standard CASP measures, prediction accuracy, and Xd. In the domain boundary prediction category, prediction methods still make reliable predictions for targets that have structural templates, but continue to struggle to make good predictions for the few ab initio targets in CASP. There was little indication of improvement in the domain prediction category. The contact prediction category demonstrated that there was renewed interest among predictors and despite the small sample size the results suggested that there had been an increase in prediction accuracy. In contrast to CASP7 contact specialists predicted contacts more accurately than the majority of tertiary structure predictors. Despite this small success, the lack of free modeling targets makes it unlikely that either category will be included in their present form in CASP9. Proteins 2009.

Scoring docking models with evolutionary information

We have developed methods for the extraction of evolutionary information from multiple sequence alignments for use in the study of the evolution of protein interaction networks and in the prediction of protein interaction. For Rounds 3, 4, and 5 of the CAPRI experiment, we used scores derived from the analysis of multiple sequence alignments to submit predictions for 7 of the 12 targets. Our docking models were generated with Hex and GRAMM, but all our predictions were selected using methods based on multiple sequence alignments and on the available experimental evidence. With this approach, we were able to predict acceptable level models for 4 of the targets, and for a fifth target, we located the residues involved in the binding surface. Here we detail our successes and highlight several of the limitations and problems that we faced while dealing with particular docking cases. Proteins 2005;60:275–280.

Inhibition of De Novo NAD+ Synthesis by Oncogenic URI Causes Liver Tumorigenesis through DNA Damage

Molecular mechanisms responsible for hepatocellular carcinoma (HCC) remain largely unknown. Using genetically engineered mouse models, we show that hepatocyte-specific expression of unconventional prefoldin RPB5 interactor (URI) leads to a multistep process of HCC development, whereas its genetic reduction in hepatocytes protects against diethylnitrosamine (DEN)-induced HCC. URI inhibits aryl hydrocarbon (AhR)- and estrogen receptor (ER)-mediated transcription of enzymes implicated in L-tryptophan/kynurenine/nicotinamide adenine dinucleotide (NAD(+)) metabolism, thereby causing DNA damage at early stages of tumorigenesis. Restoring NAD(+) pools with nicotinamide riboside (NR) prevents DNA damage and tumor formation. Consistently, URI expression in human HCC is associated with poor survival and correlates negatively with L-tryptophan catabolism pathway. Our results suggest that boosting NAD(+) can be prophylactic or therapeutic in HCC.