2013

Replication stress (RS) is a source of DNA damage that has been linked to cancer and aging, which is suppressed by the ATR kinase. In mice, reduced ATR levels in a model of the ATR-Seckel syndrome lead to RS and accelerated aging. Similarly, ATR-Seckel embryonic fibroblasts (MEF) accumulate RS and undergo cellular senescence. We previously showed that senescence of ATR-Seckel MEF cannot be rescued by p53-deletion. Here, we show that the genetic ablation of the INK4a/Arf locus fully rescues senescence on ATR mutant MEF, but also that induced by other conditions that generate RS such as low doses of hydroxyurea or ATR inhibitors. In addition, we show that a persistent exposure to RS leads to increased levels of INK4a/Arf products, revealing that INK4a/ARF behaves as a bona fide RS checkpoint. Our data reveal an unknown role for INK4a/ARF in limiting the expansion of cells suffering from persistent replication stress, linking this well-known tumor suppressor to the maintenance of genomic integrity.

Further info: Cell Cycle
New Insights into FoxE1 Functions: Identification of Direct FoxE1 Targets in Thyroid Cells.
Fernández LP, López-Márquez A, Martínez AM, Gómez-López G, Santisteban P.
PLoS One. 2013 May 13;8(5):e62849.

FoxE1 is a thyroid-specific forkhead transcription factor essential for thyroid gland development, as well as for the maintenance of the thyroid differentiated state in adults. FoxE1 recognizes and binds to a short DNA sequence present in thyroglobulin (Tg) and thyroperoxidase (Tpo) promoters, but FoxE1 binding to regulatory regions other than Tg and Tpo promoters remains almost unexplored. Improving knowledge of the regulatory functions of FoxE1 is necessary to clarify its role in endocrine syndromes and cancer susceptibility. In order to further investigate downstream FoxE1 targets, we performed a genome-wide expression screening after knocking-down FoxE1 and obtained new insights into FoxE1 transcriptional networks in thyroid follicular cells. After validation, we confirmed Adamts9, Cdh1, Duox2 and S100a4 as upregulated genes and Casp4, Creld2, Dusp5, Etv5, Hsp5a, Nr4a2 and Tm4sf1 as downregulated genes when FoxE1 was silenced. In promoter regions of putative FoxE1-regulated genes and also in the promoters of the classical thyroid genes Nis, Pax8 and Titf1, we performed an in silico search of the FoxE1 binding motif that was in close proximity to the NF1/CTF binding sequence, as previously described for other forkhead factors. Using chromatin immunoprecipitation we detected specific in vivo FoxE1 binding to novel regulatory regions in two relevant thyroid genes, Nis and Duox2. Moreover, we demonstrated simultaneous binding of FoxE1 and NF1/CTF to the Nis upstream enhancer region, as well as a clear functional activation of the Nis promoter by both transcription factors. In search for potential downstream mediators of FoxE1 function in thyroid cells, we identified two novel direct FoxE1 target genes. To our knowledge, this is the first evidence regarding the implication of Nis and Duox2 in executing the transcriptional program triggered by FoxE1. Furthermore, this study points out the important role of FoxE1 in the regulation of a large number of genes in thyroid cells.

Further info: PLoS One
RUbioSeq: a suite of parallelized pipelines to automate exome variation and bisulfite-seq analyses.
Rubio-Camarillo M, Gómez-López G, Fernández JM, Valencia A, Pisano DG.
Bioinformatics. 2013 Apr 28

RUbioSeq has been developed to facilitate the primary and secondary analysis of resequencing projects by providing an integrated software suite of parallelized pipelines to detect exome variants (SNVs and CNVs) and to perform Bisulfite-seq analyses automatically. RUbioSeq's variant analysis results have been already validated and published. AVAILABILITY: http://rubioseq.sourceforge.net/

Further info: Bioinformatics Journal
ARID1A alterations are associated with FGFR3-wild type, poor-prognosis, urothelial bladder tumors.
Cristina Balbás-Martínez, María Rodríguez-Pinilla, Ariel Casanova, Orlando Domínguez,Pisano DG, Gómez G, Josep Lloreta, José A. Lorente, Nuria Malats, Francisco X. Real.
PLoS One 8(5): e62483. doi:10.1371/journal.pone.0062483

Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological, genetic, and epigenetic levels. Exome sequencing has identified ARID1A as a novel tumor suppressor gene coding for a chromatin remodeling protein that is mutated in UBC. Here, we assess ARID1A alterations in two series of patients with UBC. In the first tumor series, we analyze exons 2–20 in 52 primary UBC and find that all mutant tumors belong to the aggressive UBC phenotype (high grade non-muscle invasive and muscle invasive tumors) (P = 0.05). In a second series (n = 84), we assess ARID1A expression using immunohistochemistry, a surrogate for mutation analysis, and find that loss of expression increases with higher stage/grade, it is inversely associated with FGFR3 overexpression (P = 0.03) but it is not correlated with p53 overexpression (P = 0.30). We also analyzed the expression of cytokeratins in the same set of tumor and find, using unsupervised clustering, that tumors with ARID1A loss of expression are generally KRT5/6-low. In this patient series, loss of ARID1A expression is also associated with worse prognosis, likely reflecting the higher prevalence of losses found in tumors of higher stage and grade. The independent findings in these two sets of patients strongly support the notion that ARID1A inactivation is a key player in bladder carcinogenesis occurring predominantly in FGFR3 wild type tumors.

Further info: PLoS One

2012

Medullary thyroid carcinoma accounts for 2% to 5% of thyroid malignancies, of which 75% are sporadic and the remaining 25% are hereditary and related to multiple endocrine neoplasia type 2 syndrome. Despite a genotype-phenotype correlation with specific germline RET mutations, knowledge of pathways specifically associated with each mutation and with non-RET-mutated sporadic MTC remains lacking. Gene expression patterns have provided a tool for identifying molecular events related to specific tumor types and to different clinical features that could help identify novel therapeutic targets. Using transcriptional profiling of 49 frozen MTC specimens classified as RET mutation, we identified PROM1, LOXL2, GFRA1, and DKK4 as related to RET(M918T) and GAL as related to RET(634) mutation. An independent series of 19 frozen and 23 formalin-fixed, paraffin-embedded (FFPE) MTCs was used for validation by RT-qPCR. Two tissue microarrays containing 69 MTCs were available for IHC assays. According to pathway enrichment analysis and gene ontology biological processes, genes associated with the MTC(M918T) group were involved mainly in proliferative, cell adhesion, and general malignant metastatic effects and with Wnt, Notch, NFκB, JAK/Stat, and MAPK signaling pathways. Assays based on silencing of PROM1 by siRNAs performed in the MZ-CRC-1 cell line, harboring RET(M918T), caused an increase in apoptotic nuclei, suggesting that PROM1 is necessary for survival of these cells. This is the first report of PROM1 overexpression among primary tumors.

Further info: PubMed
Downregulation of specific miRNAs in hyperdiploid multiple myeloma mimics the oncogenic effect of IgH translocations occurring in the non-hyperdiploid subtype.
Rio-Machin A, Ferreira BI, Henry T, Gómez-López G, Agirre X, Alvarez S, Rodriguez-Perales S, Prosper F, Calasanz MJ, Martínez J, Fonseca R, Cigudosa JC.
Leukemia. 2012 Oct 22. doi: 10.1038/leu.2012.302.

Currently, multiple myeloma (MM) patients are broadly grouped into a non-hyperdiploid (nh-MM) group, highly enriched for IgH translocations, or into a hyperdiploid (h-MM) group, which is typically characterized by trisomies of some odd-numbered chromosomes. We compared the micro RNA (miRNA) expression profiles of these two groups and we identified 16 miRNAs that were downregulated in the h-MM group, relative to the nh-MM group. We found that target genes of the most differentially expressed miRNAs are directly involved in the pathogenesis of MM; specifically, the inhibition of hsa-miR-425, hsa-miR-152 and hsa-miR-24, which are all downregulated in h-MM, leads to the overexpression of CCND1, TACC3, MAFB, FGFR3 and MYC, which are the also the oncogenes upregulated by the most frequent IgH chromosomal translocations occurring in nh-MM. Importantly, we showed that the downregulation of these specific miRNAs and the upregulation of their targets also occur simultaneously in primary cases of h-MM. These data provide further evidence on the unifying role of cyclin D pathways deregulation as the key mechanism involved in the development of both groups of MM. Finally, they establish the importance of miRNA deregulation in the context of MM, thereby opening up the potential for future therapeutic approaches based on this molecular mechanism.

Further info: Leukemia Journal
ChiTaRS: a database of human, mouse and fruit fly chimeric transcripts and RNA-sequencing data..
Frenkel-Morgenstern M, Gorohovski A, Lacroix V, Rogers M, Ibanez K, Boullosa C, Andres Leon Eduardo, Ben-Hur A, Valencia A.
Nucleic Acids Res. 2012 Nov 9.

Chimeric RNAs that comprise two or more different transcripts have been identified in many cancers and among the Expressed Sequence Tags (ESTs) isolated from different organisms; they might represent functional proteins and produce different disease phenotypes. The ChiTaRS database of Chimeric Transcripts and RNA-Sequencing data (http://chitars.bioinfo.cnio.es/) collects more than 16 000 chimeric RNAs from humans, mice and fruit flies, 233 chimeras confirmed by RNA-seq reads and ∼2000 cancer breakpoints. The database indicates the expression and tissue specificity of these chimeras, as confirmed by RNA-seq data, and it includes mass spectrometry results for some human entries at their junctions. Moreover, the database has advanced features to analyze junction consistency and to rank chimeras based on the evidence of repeated junction sites. Finally, 'Junction Search' screens through the RNA-seq reads found at the chimeras' junction sites to identify putative junctions in novel sequences entered by users. Thus, ChiTaRS is an extensive catalog of human, mouse and fruit fly chimeras that will extend our understanding of the evolution of chimeric transcripts in eukaryotes and can be advantageous in the analysis of human cancer breakpoints.

Further info: Nucleic Acids Res. journal

Current genetic evidence in mice indicates that SIRT1 has potent tumor suppressor activity in a variety of cancer models, with no evidence yet for SIRT1 oncogenic activity in vivo. We report here that transgenic Sirt1 expression is oncogenic in murine thyroid and prostate carcinogenesis initiated by Pten-deficiency. Based on mRNA expression analyses of pre-tumoral murine thyroids, we find that SIRT1 increases c-MYC transcriptional programs. Moreover, we show higher c-MYC protein levels in murine thyroid cancers from Sirt1 transgenic mice. Similarly, SIRT1 is overexpressed in human thyroid cancers and it is positively correlated with c-MYC protein levels. Finally, we show in cultured thyroid cancer cells that SIRT1 stabilizes c-MYC protein. These results implicate SIRT1 as a new candidate target for the treatment of thyroid carcinomas.

Further info: Oncogene journal
Therapeutic Effect of γ-Secretase Inhibition in Kras(G12V)-Driven Non-Small Cell Lung Carcinoma by Derepression of DUSP1 and Inhibition of ERK.
Maraver A, Fernandez-Marcos PJ, Herranz D, Cañamero M, Muñoz-Martin M, Gómez-López G, Mulero F, Megías D, Sanchez-Carbayo M, Shen J, Sanchez-Cespedes M, Palomero T, Ferrando A, Serrano M.
Cancer Cell. 2012 Aug 14;22(2):222-34.

Here, we have investigated the role of the Notch pathway in the generation and maintenance of Kras(G12V)-driven non-small cell lung carcinomas (NSCLCs). We demonstrate by genetic means that γ-secretase and RBPJ are essential for the formation of NSCLCs. Of importance, pharmacologic treatment of mice carrying autochthonous NSCLCs with a γ-secretase inhibitor (GSI) blocks cancer growth. Treated carcinomas present reduced HES1 levels and reduced phosphorylated ERK without changes in phosphorylated MEK. Mechanistically, we show that HES1 directly binds to and represses the promoter of DUSP1, encoding a dual phosphatase that is active against phospho-ERK. Accordingly, GSI treatment upregulates DUSP1 and decreases phospho-ERK. These data provide proof of the in vivo therapeutic potential of GSIs in primary NSCLCs.

Further info: Cancer Cell
Distinct DNA methylomes of newborns and centenarians.
Heyn H, Li N, Ferreira HJ, Moran S, Pisano DG, Gomez A, Diez J, Sanchez-Mut JV, Setien F, Carmona FJ, Puca AA, Sayols S, Pujana MA, Serra-Musach J, Iglesias-Platas I, Formiga F, Fernandez AF, Fraga MF, Heath SC, Valencia A, Gut IG, Wang J, Esteller M.
Proc Natl Acad Sci U S A. 2012 Jun 11.

Human aging cannot be fully understood in terms of the constrained genetic setting. Epigenetic drift is an alternative means of explaining age-associated alterations. To address this issue, we performed whole-genome bisulfite sequencing (WGBS) of newborn and centenarian genomes. The centenarian DNA had a lower DNA methylation content and a reduced correlation in the methylation status of neighboring cytosine-phosphate-guanine (CpGs) throughout the genome in comparison with the more homogeneously methylated newborn DNA. The more hypomethylated CpGs observed in the centenarian DNA compared with the neonate covered all genomic compartments, such as promoters, exonic, intronic, and intergenic regions. For regulatory regions, the most hypomethylated sequences in the centenarian DNA were present mainly at CpG-poor promoters and in tissue-specific genes, whereas a greater level of DNA methylation was observed in CpG island promoters. We extended the study to a larger cohort of newborn and nonagenarian samples using a 450,000 CpG-site DNA methylation microarray that reinforced the observation of more hypomethylated DNA sequences in the advanced age group. WGBS and 450,000 analyses of middle-age individuals demonstrated DNA methylomes in the crossroad between the newborn and the nonagenarian/centenarian groups. Our study constitutes a unique DNA methylation analysis of the extreme points of human life at a single-nucleotide resolution level.

Further info: PNAS
UNG shapes the specificity of AID-induced somatic hypermutation
Pablo Pérez-Durán, Laura Belver, Virginia G. de Yébenes, Pilar Delgado, David G. Pisano, Almudena R. Ramiro.
J Exp Med. 2012 Jun 4.

Secondary diversification of antibodies through somatic hypermutation (SHM) and class switch recombination (CSR) is a critical component of the immune response. Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes. The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR). Central to this processing is the activity of uracil-N-glycosylase (UNG), an enzyme normally involved in error-free base excision repair. We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions. Loss- and gain-of-function experiments showed that UNG activity can promote both error-prone and high fidelity repair of U:G lesions. Unexpectedly, the balance between these alternative outcomes was influenced by the sequence context of the deaminated cytosine, with individual hotspots exhibiting higher susceptibility to UNG-triggered error-free or error-prone resolution. These results reveal UNG as a new molecular layer that shapes the specificity of AID-induced mutations and may provide new insights into the role of AID in cancer development.

Further info: The Journal of Experimental Medicine
New Mutations in Chronic Lymphocytic Leukemia Identified by Target Enrichment and Deep Sequencing.
Elena Doménech, Gonzalo Gómez-López, Daniel Gzlez-Peña, Mar López, Beatriz Herreros, Juliane Menezes, Natalia Gómez-Lozano, Angel Carro, Osvaldo Graña, David G. Pisano, Orlando Domínguez, José A. García-Marco, Miguel A. Piris, Margarita Sánchez-Beato.
PLoS ONE 7(6): e38158. doi:10.1371/journal.pone.0038158

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease without a well-defined genetic alteration responsible for the onset of the disease. Several lines of evidence coincide in identifying stimulatory and growth signals delivered by B-cell receptor (BCR), and co-receptors together with NFkB pathway, as being the driving force in B-cell survival in CLL. However, the molecular mechanism responsible for this activation has not been identified. Based on the hypothesis that BCR activation may depend on somatic mutations of the BCR and related pathways we have performed a complete mutational screening of 301 selected genes associated with BCR signaling and related pathways using massive parallel sequencing technology in 10 CLL cases. Four mutated genes in coding regions (KRAS, SMARCA2, NFKBIE and PRKD3) have been confirmed by capillary sequencing. In conclusion, this study identifies new genes mutated in CLL, all of them in cases with progressive disease, and demonstrates that next-generation sequencing technologies applied to selected genes or pathways of interest are powerful tools for identifying novel mutational changes.

Further info: PLoS One Journal
The specific contributions of cohesin-SA1 to cohesion and gene expression: Implications for cancer and development.
Cuadrado A, Remeseiro S, Gómez-López G, Pisano DG, Losada A.
Cell Cycle. 2012 Jun 15;11(12).

Besides its well-established role in sister chromatid cohesion, cohesin has recently emerged as major player in the organization of interphase chromatin. Such important function is related to its ability to entrap two DNA segments also in cis, thereby facilitating long-range DNA looping which is crucial for transcriptional regulation, organization of replication factories and V(D)J recombination. Vertebrate somatic cells have two different versions of cohesin, containing Smc1, Smc3, Rad21/Scc1 and either SA1 or SA2, but their functional specificity has been largely ignored. We recently generated a knockout mouse model for the gene encoding SA1, and found that this protein is essential to complete embryonic development. Cohesin-SA1 mediates cohesion at telomeres, which is required for their replication. Telomere defects in SA1- deficient cells provoke chromosome segregation errors resulting in aneuploidy despite robust centromere cohesion. This aneuploidy could explain why heterozygous animals have an earlier onset of tumorigenesis. In addition, the genome-wide distribution of cohesin changes dramatically in the absence of SA1, and the complex shows reduced accumulation at promoters and CTCF sites. As a consequence, gene expression is altered, leading to downregulation of biological processes related to a developmental disorder linked to cohesin function, the Cornelia de Lange Syndrome (CdLS). These results point out a prominent role of cohesin-SA1 in transcriptional regulation, with clear implications in the etiology of CdLS.

Further info:http://www.landesbioscience.com/journals/cc/article/20318/
Src kinases catalytic activity regulates proliferation, migration and invasiveness of MDA-MB-231 breast cancer cells.
Sánchez-Bailón MP, Calcabrini A, Gómez-Domínguez D, Morte B, Martín-Forero E, Gómez-López G, Molinari A, Wagner KU, Martín-Pérez J.
Cell Signal. 2012 Jun;24(6):1276-86.

SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1–S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.

Further info: http://www.sciencedirect.com/science/article/pii/S0898656812000708
Genome Wide Analysis Of Pax8 Binding Provides New Insights Into Thyroid Functions.
Sergio Ruiz-Llorente, Enrique Carrillo de Santa Pau, Ana Sastre-Perona, Cristina Montero-Conde,Gonzalo Gomez-Lopez,James A Fagin, Alfonso Valencia, David G Pisano, Pilar Santisteban.
BMC Genomics 2012, 13:147 doi:10.1186/1471-2164-13-147.

The transcription factor Pax8 is essential for the differentiation of thyroid cells. However, there are few data on genes transcriptionally regulated by Pax8 other than thyroid-related genes. To better understand the role of Pax8 in the biology of thyroid cells, we obtained transcriptional profiles of Pax8-silenced PCCl3 thyroid cells using whole genome expression arrays and integrated these signals with global cis-regulatory sequencing studies performed by ChIP-Seq analysis.Exhaustive analysis of Pax8 immunoprecipitated peaks demonstrated preferential binding to intragenic regions and CpG-enriched islands, which suggests a role of Pax8 in transcriptional regulation of orphan CpG regions. In addition, ChIP-Seq allowed us to identify Pax8 partners, including proteins involved in tertiary DNA structure (CTCF) and chromatin remodeling (Sp1), and these direct transcriptional interactions were confirmed in vivo. Moreover, both factors modulate Pax8-dependent transcriptional activation of the sodium iodide symporter (Nis) gene promoter. We ultimately combined putative and novel Pax8 binding sites with actual target gene expression regulation to define Pax8-dependent genes. Functional classification suggests that Pax8-regulated genes may be directly involved in important processes of thyroid cell function such as cell proliferation and differentiation, apoptosis, cell polarity, motion and adhesion, and a plethora of DNA/protein-related processes. Our study provides novel insights into the role of Pax8 in thyroid biology, exerted through transcriptional regulation of important genes involved in critical thyrocyte processes. In addition, we found new transcriptional partners of Pax8, which functionally cooperate with Pax8 in the regulation of thyroid gene transcription. Besides, our data demonstrate preferential location of Pax8 in non-promoter CpG regions. These data point to an orphan CpG island-mediated mechanism that represents a novel role of Pax8 in the transcriptional output of the thyrocyte.

Further info: BMC Genomics
Genetic inactivation of Cdk7 leads to cell cycle arrest and induces premature aging due to adult stem cell exhaustion.
Ganuza M, Sáiz-Ladera C, Cañamero M, Gómez G, Schneider R, Blasco MA, Pisano D, Paramio JM, Santamaría D, Barbacid M.
EMBO J. 2012 Apr 13. doi: 10.1038/emboj.2012.94.

Cyclin-dependent kinase (Cdk)7, the catalytic subunit of the Cdk-activating kinase (CAK) complex has been implicated in the control of cell cycle progression and of RNA polymerase II (RNA pol II)-mediated transcription. Genetic inactivation of the Cdk7 locus revealed that whereas Cdk7 is completely dispensable for global transcription, is essential for the cell cycle via phosphorylation of Cdk1 and Cdk2. In vivo, Cdk7 is also indispensable for cell proliferation except during the initial stages of embryonic development. Interestingly, widespread elimination of Cdk7 in adult tissues with low proliferative indexes had no phenotypic consequences. However, ablation of conditional Cdk7 alleles in tissues with elevated cellular turnover led to the efficient repopulation of these tissues with Cdk7-expressing cells most likely derived from adult stem cells that may have escaped the inactivation of their targeted Cdk7 alleles. This process, a physiological attempt to maintain tissue homeostasis, led to the attrition of adult stem cell pools and to the appearance of age-related phenotypes, including telomere shortening and early death.

Further info: EMBO Journal
A unique role of cohesin-SA1 in gene regulation and development.
Remeseiro S, Cuadrado A,Gómez-López G, Pisano DG, Losada A.
EMBO J. 2012 Mar 13. doi: 10.1038/emboj.2012.60.

Vertebrates have two cohesin complexes that consist of Smc1, Smc3, Rad21/Scc1 and either SA1 or SA2, but their functional specificity is unclear. Mouse embryos lacking SA1 show developmental delay and die before birth. Comparison of the genome-wide distribution of cohesin in wild-type and SA1-null cells reveals that SA1 is largely responsible for cohesin accumulation at promoters and at sites bound by the insulator protein CTCF. As a consequence, ablation of SA1 alters transcription of genes involved in biological processes related to Cornelia de Lange syndrome (CdLS), a genetic disorder linked to dysfunction of cohesin. We show that the presence of cohesin-SA1 at the promoter of myc and of protocadherin genes positively regulates their expression, a task that cannot be assumed by cohesin-SA2. Lack of SA1 also alters cohesin-binding pattern along some gene clusters and leads to dysregulation of genes within. We hypothesize that impaired cohesin-SA1 function in gene expression underlies the molecular aetiology of CdLS.

Further info: EMBO Journal
The Tumor Suppressor and Chromatin Remodeling Factor BRG1 Antagonizes Myc Activity and Promotes Cell Differentiation in Human Cancer.
Romero OA, Setien F, John S, Gimenez-Xavier P, Gómez-López G, Pisano D, Condom E, Villanueva A, Hager GL, Sanchez-Cespedes M.
EMBO Mol Med. 2012 Mar 7. doi: 10.1002/emmm.201200236.

BRG1, a member of the SWI/SNF complex, is mutated in cancer, but it is unclear how it promotes tumorigenesis. We report that re-expression of BRG1 in lung cancer cells up-regulates lung-specific transcripts, restoring the gene expression signature of normal lung. Using cell lines from several cancer types we found that those lacking BRG1 do not respond to retinoic acid (RA) or glucocorticoids (GC), while restoration of BRG1 restores sensitivity. Conversely, in SH-SY5Y cells, a paradigm of RA-dependent differentiation, abrogation of BRG1 prevented the response to RA. Further, our data suggest an antagonistic functional connection between BRG1 and MYC, whereby refractoriness to RA and GC by BRG1 inactivation involves deregulation of MYC activity. Mechanistically, some of these effects are mediated by BRG1 binding to MYC and MYC-target promoters. The BRG1-MYC antagonism was also evident in primary tumors. Finally, BRG1 restoration significantly dampened invasion and progression and decreased MYC in lung cancer cells orthotopically implanted in nude mice. Thus, BRG1 inactivation enables cancer cells to sustain undifferentiated gene expression programs and prevent its response to environmental stimuli.

Further info: EMBO Molecular Medicine
Pten Positively Regulates Brown Adipose Function, Energy Expenditure, and Longevity.
Ana Ortega-Molina, Alejo Efeyan, Elena Lopez-Guadamillas, Maribel Muñoz-Martin, Gonzalo Gómez-López, Marta Cañamero, Francisca Mulero, Joaquin Pastor, Sonia Martinez, Eduardo Romanos, M. Mar Gonzalez-Barroso, Eduardo Rial, Angela M. Valverde, James R. Bischoff, Manuel Serrano.
Cell Metabolism Volume 15, Issue 3, 382-394. 2012.

Aging in worms and flies is regulated by the PI3K/Akt/Foxo pathway. Here we extend this paradigm to mammals. Ptentg mice carrying additional genomic copies of Pten are protected from cancer and present a significant extension of life span that is independent of their lower cancer incidence. Interestingly, Ptentg mice have an increased energy expenditure and protection from metabolic pathologies. The brown adipose tissue (BAT) of Ptentg mice is hyperactive and presents high levels of the uncoupling protein Ucp1, which we show is a target of Foxo1. Importantly, a synthetic PI3K inhibitor also increases energy expenditure and hyperactivates the BAT in mice. These effects can be recapitulated in isolated brown adipocytes and, moreover, implants of Ptentg fibroblasts programmed with Prdm16 and Cebpβ form subcutaneous brown adipose pads more efficiently than wild-type fibroblasts. These observations uncover a role of Pten in promoting energy expenditure, thus decreasing nutrient storage and its associated damage.

Further info: http://www.cell.com/cell-metabolism/retrieve/pii/S1550413112000484
MicroRNA signatures in B-cell lymphomas.
L Di Lisio, M Sanchez-Beato, Gómez-López G, M E Rodríguez, S Montes-Moreno, M Mollejo, J Menárquez, M A Martínez, F J Alves,D G Pisano, M A Piris, N Martínez.
Blood Cancer Journal (2012) 2, e57; doi:10.1038/bcj.2012.1

Accurate lymphoma diagnosis, prognosis and therapy still require additional markers. We explore the potential relevance of microRNA (miRNA) expression in a large series that included all major B-cell non-Hodgkin lymphoma (NHL) types. The data generated were also used to identify miRNAs differentially expressed in Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) samples. A series of 147 NHL samples and 15 controls were hybridized on a human miRNA one-color platform containing probes for 470 human miRNAs. Each lymphoma type was compared against the entire set of NHLs. BL was also directly compared with DLBCL, and 43 preselected miRNAs were analyzed in a new series of routinely processed samples of 28 BLs and 43 DLBCLs using quantitative reverse transcription-polymerase chain reaction. A signature of 128 miRNAs enabled the characterization of lymphoma neoplasms, reflecting the lymphoma type, cell of origin and/or discrete oncogene alterations. Comparative analysis of BL and DLBCL yielded 19 differentially expressed miRNAs, which were confirmed in a second confirmation series of 71 paraffin-embedded samples. The set of differentially expressed miRNAs found here expands the range of potential diagnostic markers for lymphoma diagnosis, especially when differential diagnosis of BL and DLBCL is required.

Further info: http://www.nature.com/bcj/journal/v2/n2/full/bcj20121a.html

Epstein-Barr virus microRNAs repress BCL6 expression in diffuse large B-cell lymphoma. 
D Martín-Pérez, P. Vargiu, S. Montes-Moreno, Eduardo Andres León, S. M. Rodríguez-Pinilla, L. D. Lisio, N. Martínez, R. Rodríguez, M. Mollejo, J. Castellvi, David G. Pisano, M. Sánchez-Beato, and M. A. Piris. 
Leukemia 2012 Jan 26.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma, accounting for 30–40% of all newly diagnosed lymphomas. DLBCL is considered a heterogeneous disease, with some specific clinicopathological variants of DLBCLs being associated with the presence of the EBV.1 EBV is a lymphotropic virus that has been implicated in the development of several lymphoid malignancies, mainly Burkitt lymphoma (BL) and Hodgkin's lymphoma and with low prevalence in DLBCL.1 BCL6 is a key transcriptional repressor during normal B-cell differentiation that has been shown to repress NF-kB in some DLBCLs.2 In some B-cell lymphomas, BCL6 expression was inversely correlated with LMP1 expression, and some evidences suggest that LMP1 can cause downregulation of BCL6(ref. 3), but other possible mechanisms have not been studied. We have found a strong inverse correlation between BCL6 protein expression and EBV infection (P<0.001, Figures 1a and b) in a series of 149 DLBCL samples, where only one out of 34 EBV-positive cases (2.94%) expressed BCL6, although 87 out of 115 EBV-negative cases expressed BCL6 (75.65%). However, this correlation was independent of LMP1 because 54% of EBER-positive samples were LMP1-negative (Spearman, P-value: 0.18). Little is known about the mechanisms that cause the absence of BCL6 in EBV-positive DLBCL; however, the possibility that EBV-encoded miRNAs could contribute to BCL6 repression has never been explored.

Further info: http://www.nature.com/leu/journal/vaop/ncurrent/full/leu2011189a.html#aff2

The AML1-ETO fusion protein, which is present in 10-15% of cases of acute myeloid leukemia, is known to repress myeloid differentiation genes through DNA binding and recruitment of chromatin-modifying proteins and transcription factors in target genes. ChIP-chip analysis of human hematopoietic stem/progenitor cells transduced with the AML1-ETO fusion gene enabled us to identify 1168 AML1-ETO target genes, 103 of which were co-occupied by histone deacetylase 1 (HDAC1) and had lost the hyperacetylation mark at histone H4, and 264 showed a K9 trimethylation at histone H3. Enrichment of genes involved in hematopoietic differentiation and in specific signaling pathways was observed in the presence of these epigenetic modifications associated with an 'inactive' chromatin status. Furthermore, AML1-ETO target genes had a significant correlation between the chromatin marks studied and transcriptional silencing. Interestingly, AML1 binding sites were absent on a large number of selected AML1-ETO promoters and an Sp1 binding site was found in over 50% of them. Reversible silencing induced by the fusion protein in the presence of AML1 and/or Sp1 transcription factor binding site was confirmed. Therefore, this study provides a global analysis of AML1-ETO functional chromatin modifications and identifies the important role of Sp1 in the DNA binding pattern of AML1-ETO, suggesting a role for Sp1-targeted therapy in this leukemia subtype.

Further info: http://www.nature.com/leu/journal/vaop/ncurrent/abs/leu2011376a.html

2011

Cdc14 is an essential phosphatase in yeast but its role in the mammalian cell cycle remains obscure. We report here that Cdc14b-knockout cells display unscheduled induction of multiple cell cycle regulators resulting in early entry into DNA replication and mitosis from quiescence. Cdc14b dephosphorylates Ser5 at the C-terminal domain (CTD) of RNA polymerase II, a major substrate of cyclin-dependent kinases. Lack of Cdc14b results in increased CTD-Ser5 phosphorylation, epigenetic modifications that mark active chromatin, and transcriptional induction of cell cycle regulators. These data suggest a function for mammalian Cdc14 phosphatases in the control of transcription during the cell cycle.

Further info: http://www.nature.com/srep/2011/111212/srep00189/full/srep00189.html
Integration of BRCA1-mediated miRNA and mRNA profiles reveals microRNA regulation of TRAF2 and NFκB pathway.
Tanic M, Zajac M, Gómez-López G, Benítez J, Martínez-Delgado B.
Breast Cancer Res Treat. 2011 Dec 14.

Microarray-based techniques are being useful to obtain miRNA and gene expression signatures associated with different tumors. BRCA1 deregulation is a frequent event in the pathogenesis of breast as well as other cancers. In addition to DNA repair functions of BRCA1, it is involved in a wide range of cellular processes such as cell cycle, chromatin remodeling or transcription. However, the molecular events underlying BRCA1-associated tumorigenesis are still largely unknown. In order to deepen our understanding of BRCA1-associated tumorigenesis, we integrated data from mRNA and miRNA microarray experiments on HCC1937 breast cancer cell line, and the isogenic HCC1937 stably expressing BRCA1, to obtain significant miRNA-mRNA relationships associated with the presence of BRCA1 gene. By using bioinformatic integration of gene and miRNA expression data, we found significant miRNA-gene relationships underlying the array signatures. We additionally evaluated the role of these statistically significant pairs at the biological pathways level and identified MAPK and NF-κB pathways associated with these expression changes. Furthermore, we experimentally validated miRNAs induced by BRCA1 that commonly regulate TRAF2, a key regulator of NF-κB and MAPK pathways. We demonstrate that miR-146a, miR-99b and miR-205, induced in HCC1937 BRCA1-expressing cells, bind and regulate TRAF2 gene. In addition, re-expression of miR-146a, miR-99b or miR-205 in HCC1937 BRCA1-null cells was sufficient to modulate NF-κB activity. Our results demonstrate that integration of mRNA and miRNA associated with BRCA1 expression was useful to discover new miRNA-gene interactions as molecular events underlying BRCA1-mediated tumorigenesis.

Further info: http://www.springerlink.com/content/f225r81l61362173/
Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia.
Víctor Quesada, Laura Conde, Neus Villamor, Gonzalo R Ordóñez, Pedro Jares, Laia Bassaganyas, Andrew J Ramsay, Sílvia Beà, Magda Pinyol, Alejandra Martínez-Trillos, Mónica López-Guerra, Dolors Colomer, Alba Navarro, Tycho Baumann, Marta Aymerich, María Rozman, Julio Delgado, Eva Giné, Jesús M Hernández, Marcos González-Díaz, Diana A Puente, Gloria Velasco, José M P Freije, José M C Tubío, Romina Royo, Josep L Gelpí, Modesto Orozco, David G Pisano, Jorge Zamora, Miguel Vázquez, Alfonso Valencia, Heinz Himmelbauer, Mónica Bayés, Simon Heath, Marta Gut, Ivo Gut, Xavier Estivill, Armando López-Guillermo, Xose S Puente, Elías Campo and Carlos López-Otín.
Nat Genet. 2011 Dec 11

Here we perform whole-exome sequencing of samples from 105 individuals with chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults in Western countries. We found 1,246 somatic mutations potentially affecting gene function and identified 78 genes with predicted functional alterations in more than one tumor sample. Among these genes, SF3B1, encoding a subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), is somatically mutated in 9.7% of affected individuals. Further analysis in 279 individuals with CLL showed that SF3B1 mutations were associated with faster disease progression and poor overall survival. This work provides the first comprehensive catalog of somatic mutations in CLL with relevant clinical correlates and defines a large set of new genes that may drive the development of this common form of leukemia. The results reinforce the idea that targeting several well-known genetic pathways, including mRNA splicing, could be useful in the treatment of CLL and other malignancies.

Further info: http://www.nature.com/ng/journal/vaop/ncurrent/full/ng.1032.html
Long-Range Epigenetic Silencing Associates with Deregulation of Ikaros Targets in Colorectal Cancer Cells.

Javierre B, Rodriguez-Ubreva J, Al-Shahrour F, Corominas M, Graña O, Ciudad L, Agirre X, Pisano DG, Valencia A, Roman-Gomez J, Calasanz M, Prosper F, Esteller M, Gonzalez-Sarmiento R, Ballestar E.
Mol Cancer Res.Published on line 7th July 2011.

Transcription factors (TFs) are common targets of epigenetic inactivation in human cancer. Promoter hypermethylation and subsequent silencing of TFs can lead to further deregulation of their targets. In this study, we explored the potential epigenetic deregulation in cancer of Ikaros family genes, which code for essential TFs in cell differentiation and exhibit genetic defects in hematological neoplasias. Unexpectedly, our analysis revealed that Ikaros undergoes very specific promoter hypermethylation in colorectal cancer, including in all the cell lines studied and around 64% of primary colorectal adenocarcinomas, with increasing proportions in advanced Duke's stages. Ikaros hypermethylation occurred in the context of a novel long-range epigenetic silencing (LRES) region. Reintroduction of Ikaros in colorectal cancer cells, ChIP-chip analysis and validation in primary samples led us to identify a number of direct targets that are possibly related with colorectal cancer progression. Our results not only provide the first evidence that LRES can have functional specific effects in cancer, but also identify several deregulated Ikaros targets that may contribute to progression in colorectal adenocarcinoma.

Further info:http://mcr.aacrjournals.org/
PileLineGUI: a desktop environment for handling genome position files in next-generation sequencing studies.
Hugo López-Fernández, Daniel Glez-Peña, Miguel Reboiro-Jato, Gonzalo Gómez-López, David G Pisano and Florentino Fdez-Riverola.
Nucleic Acids Res. (Web Server Issue). Jul;39 Suppl 2:W562-6. Published on line 6th June, 2011.

Next-generation sequencing (NGS) technologies are making sequence data available on an unprecedented scale. In this context, new catalogs of Single Nucleotide Polymorphism and mutations generated by resequencing studies are usually stored in genome position files (e.g. Variant Call Format, SAMTools pileup, BED, GFF) comprising of large lists of genomic positions, which are difficult to handle by researchers. Here, we present PileLineGUI, a novel desktop application primarily designed for manipulating, browsing and analysing genome position files (GPF), with specific support to somatic mutation finding studies. The developed tool also integrates a new genome browser module specially designed for inspecting GPFs. PileLineGUI is free, multiplatform and designed to be intuitively used by biomedical researchers. PileLineGUI is available at: http://sing.ei.uvigo.es/pileline/pilelinegui.html.

Further info: http://nar.oxfordjournals.org/content/early/2011/06/06/nar.gkr439.full
Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia.
Xose S. Puente, Magda Pinyol, Víctor Quesada, Laura Conde, Gonzalo R. Ordóñez, Neus Villamor, Georgia Escaramis, Pedro Jares, Sílvia Beà, Marcos González-Díaz, Laia Bassaganyas, Tycho Baumann, Manel Juan, Mónica López-Guerra, Dolors Colomer, José M. C. Tubío, Cristina López, Alba Navarro, Cristian Tornador, Marta Aymerich, María Rozman, Jesús M. Hernández, Diana A. Puente, José M. P. Freije, Gloria Velasco, Ana Gutiérrez-Fernández, Dolors Costa, Anna Carrió, Sara Guijarro, Anna Enjuanes, Lluís Hernández, Jordi Yagüe, Pilar Nicolás, Carlos M. Romeo-Casabona, Heinz Himmelbauer, Ester Castillo, Juliane C. Dohm, Silvia de Sanjosé, Miguel A. Piris, Enrique de Alava, Jesús San Miguel, Romina Royo, Josep L. Gelpí, David Torrents, Modesto Orozco,David G Pisano, Alfonso Valencia, Roderic Guigó, Mónica Bayés, Simon Heath, Marta Gut, Peter Klatt, John Marshall, Keiran Raine, Lucy A. Stebbings, P. Andrew Futreal, Michael R. Stratton, Peter J. Campbell, Ivo Gut, Armando López-Guillermo, Xavier Estivill, Emili Montserrat, Carlos López-Otín and Elías Campo.
Nature. Published on line 5th June, 2011.

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

Further info: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10113.html
MicroRNA expression in diffuse large B-cell lymphoma treated with chemoimmunotherapy.
Montes-Moreno S, Martinez N, Sanchez-Espiridión B, Díaz Uriarte R, Rodriguez ME, Saez A, Montalbán C, Gómez G, Pisano DG, García JF, Conde E, Gonzalez-Barca E, Lopez A, Mollejo M, Grande C, Martinez MA, Dunphy C, Hsi ED, Rocque GB, Chang J, Go RS, Visco C, Xu-Monette Z, Young KH, Piris MA.
Blood 2011 Jul 28;118(4):1034-40. 

DLBCL prognostication requires additional biological markers. MicroRNAs (miRNAs) may constitute markers for cancer diagnosis, outcome or therapy response. Here we have analyzed the miRNA expression profile in a retrospective multicenter series of DLBCL patients (258 cases) uniformly treated with chemoimmunotherapy. Findings were correlated with OS and PFS. miRNA and gene expression profiles were studied using microarrays in an initial set of 36 cases. A selection of miRNAs associated with either DLBCL molecular subtypes (GCB/ABC) or clinical outcome were studied by multiplex RT-PCR in a test group of 240 cases with available FFPE diagnostic sample, divided into a training set (123 patients), which was used to derive miRNA-based and combined (with IPI score) Cox regression models, and an independent validation series (117 patients). A model based on miRNA expression predicts OS and PFS. This model improves the prediction based on clinical variables. Combined models with IPI score identify a high-risk group of patients with a 2-year OS and PFS probability of less than 50%. In summary, a precise miRNA signature is associated with poor clinical outcome in chemoimmunotherapy-treated DLBCL patients. This information improves IPI-based prediction and identifies a subgroup of candidate patients for alternative therapeutic regimens to those of standard therapies.

Further info: http://www.ncbi.nlm.nih.gov/pubmed/21633089
A DNA methylation fingerprint of 1,628 human samples.
Fernandez AF, Assenov Y, Martin-Subero J, Balint B, Siebert R, Taniguchi H, Yamamoto H, Hidalgo M, Tan AC, Galm O, Ferrer I, Sanchez-Cespedes M, Villanueva A, Carmona J, Sanchez-Mut JV, Berdasco M, Moreno V, Capella G, Monk D, Ballestar E, Ropero S, Martinez R, Sanchez-Carbayo M, Prosper F, Agirre X, Fraga MF, Graña O, Perez-Jurado L, Mora J, Puig S, Prat J, Badimon L, Puca AA, Meltzer SJ, Lengauer T, Bridgewater J, Bock C, Esteller M.
Genome Res. 2011 May 25.

DNA methylation is the best characterized of the different layers that make up the epigenetic setting. 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. The recently arrived single-base-resolution technologies for DNA methylation are extremely helpful tools, but are not yet applicable and affordable for studying large groups of subjects. 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 1,628 human samples where we interrogated 1,505 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 where 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 obtained DNA methylation fingerprints might be useful for translational purposes by showing that are able to identify the tumor type origin of Cancers of Unknown Primary (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.

Further info: http://genome.cshlp.org/content/early/2011/05/25/gr.119867.110.long
Epigenetic regulation of Nanog expression by Ezh2 in pluripotent stem cells.
Villasante A, Piazzolla D, Li H, Gómez-López G, Djabali M, Serrano M.
Cell Cycle 2011 May 1;10(9).

Nanog levels in pluripotent stem cells are heterogeneous and this is thought to reflect two different and interchangeable cell states, respectively poised to self-renew (Nanog-high subpopulation) or to differentiate (Nanog-low subpopulation). However, little is known about the mechanisms responsible for this pattern of Nanog expression. Here, we have examined the impact of the histone methyltransferase Ezh2 on pluripotent stem cells and on Nanog expression. Interestingly, induced pluripotent stem (iPS) cells lacking Ezh2 presented higher levels of Nanog due to a relative expansion of the Nanog-high subpopulation, and this was associated to severe defects in differentiation. Moreover, we found that the Nanog promoter in embryonic stem (ES) cells and iPS cells coexists in two alternative univalent chromatin configurations, either H3K4me3 or H3K27me3, the latter being dependent on the presence of functional Ezh2. Finally, the levels of expression of Ezh2, as well as the amount of H3K27me3 present at the Nanog promoter, were higher in the Nanog-low subpopulation of ES/iPS cells. Together, these data indicate that Ezh2 directly regulates the epigenetic status of the Nanog promoter affecting the balance of Nanog expression in pluripotent stem cells and, therefore, the equilibrium between self-renewal and differentiation.

Further info: http://www.landesbioscience.com/journals/cc/article/15658/
Combinatorial effects of microRNAs to suppress the Myc oncogenic pathway.
Bueno MJ, Gómez de Cedrón M, Gómez-López G, Perez de Castro I, Di Lisio L, Montes S, Martinez N, Guerrero M, Sanchez-Martinez R, Santos J, Pisano DG, Piris MA, Fernández-Piqueras J, Malumbres M.
Blood 2011, Apr 8.

Many mammalian transcripts contain target sites for multiple miRNAs although it is not clear to what extent miRNAs may coordinately regulate single genes. We have mapped the interactions between downregulated miRNAs and overexpressed target protein-coding genes in murine and human lymphomas. Myc, one of the hallmark oncogenes in these lymphomas, stands out as the upregulated gene with the highest number of genetic interactions with downregulated miRNAs in mouse lymphomas. The regulation of Myc by several of these miRNAs is confirmed by cellular and reporter assays. The same approach indentifies MYC and multiple Myc targets as a preferential target of downregulated miRNAs in human Burkitt's lymphoma, a pathology characterized by translocated MYC oncogenes. These results indicate that several miRNAs must be coordinately downregulated in order to enhance critical oncogenes such as Myc. Some of these Myc-targeting miRNAs are repressed by Myc, suggesting that these tumors are a consequence of the unbalanced activity of Myc versus miRNAs.

Further info: http://bloodjournal.hematologylibrary.org/content/early/2011/04/08/blood-2010-10-315432.abstract

Assessment of copy number variation using the Illumina Infinium 1M SNP-array: a comparison of methodological approaches in the Spanish Bladder Cancer/EPICURO study. 
Gaëlle Marenne, Benjamín Rodríguez-Santiago, Montserrat García Closas, Luis Pérez-Jurado, Nathaniel Rothman, Daniel Rico, Guillermo Pita, David G. Pisano, Manolis Kogevinas, Debra T. Silverman, Alfonso Valencia, Francisco X. Real, Stephen J. Chanock, Emmanuelle Génin, Núria Malats. Hum Mutat (2011) vol. 32 (2) pp. 240-8. High-throughput single nucleotide polymorphism (SNP)-array technologies allow to investigate copy number variants (CNVs) in genome-wide scans and specific calling algorithms have been developed to determine CNV location and copy number. We report the results of a reliability analysis comparing data from 96 pairs of samples processed with CNVpartition, PennCNV, and QuantiSNP for Infinium Illumina Human 1Million probe chip data. We also performed a validity assessment with multiplex ligation-dependent probe amplification (MLPA) as a reference standard. The number of CNVs per individual varied according to the calling algorithm. Higher numbers of CNVs were detected in saliva than in blood DNA samples regardless of the algorithm used. All algorithms presented low agreement with mean Kappa Index (KI) 0.62). Our results indicate that the current calling algorithms should be improved for high performance CNV analysis in genome-wide scans. 

 
Further info: http://onlinelibrary.wiley.com/doi/10.1002/humu.21398/abstract

Genomic position (GP) files currently used in next-generation sequencing (NGS) studies are always difficult to manipulate due to their huge size and the lack of appropriate tools to properly manage them. The structure of these flat files is based on representing one line per position that has been covered by at least one aligned read, imposing significant restrictions from a computational performance perspective.
PileLine implements a flexible command-line toolkit providing specific support to the management, filtering, comparison and annotation of GP files produced by NGS experiments. PileLine tools are coded in Java and run on both UNIX (Linux, Mac OS) and Windows platforms. The set of tools comprising PileLine are designed to be memory efficient by performing fast seek on-disk operations over sorted GP files.
Our novel toolbox has been extensively tested taking into consideration performance issues. It is publicly available at http://sourceforge.net/projects/pilelinetools under the GNU LGPL license. Full documentation including common use cases and guided analysis workflows is available at http://sing.ei.uvigo.es/pileline.

Further info: www.biomedcentral.com/1471-2105/12/31

Expression and functional validation of new p38α transcriptional targets in tumorigenesis.
Swat A, Dolado I, Igea A, Gómez-López G, Pisano DG, Cuadrado A, Nebreda AR.
Biochem J. Feb 24;434(3):549-58. 2011. p38α MAP kinase plays an important tumor suppressor role, which is mediated by both its negative effect on cell proliferation and its pro-apoptotic activity. Surprisingly, most tumor suppressor mechanisms coordinated by p38α have been reported to occur at the post- translational level. This contrasts with the important role of p38α in the regulation of transcription and the profound changes in gene expression that normally occur during tumorigenesis. We have analyzed whole genome expression profiles of Ras-transformed wild- type and p38α-deficient cells and have identified 202 genes that are potentially regulated by p38α in transformed cells. Expression analysis has confirmed the regulation of these genes by p38α in tumors, and functional validation has identified several of them as likely mediators of the tumor suppressor effect of p38α on Ras-induced transformation. Interestingly, about 10% of the genes that are negatively regulated by p38α in transformed cells contribute to EGF receptor signalling. Our results suggest that inhibition of EGF receptor signalling by transcriptional targets of p38α is an important function of this signalling pathway in the context of tumor suppression.

 
Further info: http://www.biochemj.org/bj/imps/abs/BJ20101410.htm#

Massive parallel DNA pyrosequencing analysis of the tumor suppressor BRG1/SMARCA4 in lung primary tumors.
Rodríguez-Nieto S, Cañada A, Pros E, Pinto AI, Torres-Lanzas J, Lopez-Rios F, Sánchez-Verde L, Pisano DG, Sánchez-Cespedes M.
Hum Mutat. 2010 Dec 7. The tumor suppressor gene, SMARCA4 (or BRG1), which encodes the ATPase component of the chromatin remodeling complex SWI/SNF, is commonly inactivated by mutations and deletions in lung cancer cell lines. However, SMARCA4 alterations appear to be rare in lung primary tumors. Ultra-deep sequencing technologies provide a promising alternative to achieve a sensitivity superior to that of current sequencing strategies. Here we used ultra-deep pyrosequencing to screen for mutations over the entire SMARCA4 coding region in 12 lung tumors without detectable BRG1 protein. While automatic-fluorescence-based sequencing detected one somatic mutation (p.K586X), the pyrosequencing revealed additional variants, thus increasing the sensitivity. One of the variants, which affected a consensus splice site, was confirmed by individual cloning of PCR products, ruling out the possibility of PCR or pyrosequencing artifacts. This mutation, confirmed to be somatic, was present at a frequency of ten percent, suggesting normal cell contamination in the tumor. Our analysis also allowed us to determine the sensitivity and to identify some limitations of the technology. In conclusion, in addition to cell lines, SMARCA4 is biallelically inactivated in a significant proportion of lung primary tumors, thereby constituting one of the most important genes contributing to the development of this type of cancer.

Further info: http://onlinelibrary.wiley.com/doi/10.1002/humu.21415/abstract;jsessionid=A8C042A523D2E07303479A7AFA4A6768.d02t01


Transcriptional profiling reveals different pseudo-hypoxic signatures in SDHB and VHL-related pheocromocytomas.
Elena López-Jiménez, Gonzalo Gómez-López, L Javier Leandro-García, Iván Muñoz, Francesca Schiavi, Cristina Montero-Conde, Aguirre A de Cubas, Ricardo Ramires, Iñigo Landa, Susanna Leskelä, Agnieszka Maliszewska, Lucía Inglada-Pérez, Leticia de la Vega, Cristina Rodríguez-Antona, Rocío Letón, Carmen Bernal, José M de Campos, Cristina Diez-Tascón, Mario F Fraga, Cesar Boullosa, David G Pisano, Giuseppe Opocher, Mercedes Robledo, Alberto Cascón.
Mol. Endocrinol. (2010) Dec;24(12):2382-91.

Further info: http://www.biomedcentral.com/1755-8794/3/44


Transcriptional characteristics of familial non-BRCA1/BRCA2 breast tumors.
Ricardo Fernández-Ramires, Gonzalo Gómez, Iván Muñoz-Repeto, Loris de Cecco, Gemma Llort, Alicia Cazorla, Ignacio Blanco, Manuela Gariboldi, Marco Alessandro Pierotti, Javier Benítez, Ana Osorio.
Int J Cancer (2010)In order to better understand the alterations present in the group of the so- called BRCAX tumors, we have used a cDNA microarray containing genes related to tumorigenesis and analyzed a series of 49 tumors consisting of 13 BRCA1, 14 BRCAX and 22 sporadic. We have confirmed that the BRCAX tumors are heterogeneous and can be divided in at least two main subgroups, so-called A and B, transcriptionally distinguishable and with different altered pathways within each of the groups. We have found that BRCAX-A and B subgroups, can be classified as Luminal A and B respectively, taking into account the intrinsic phenotypes defined for the sporadic breast tumors. We have found that, at the somatic level, the BRCAX-B tumors are identical to their sporadic Luminal B counterparts, while BRCAX-A, despite having a luminal A phenotype, show additional genomic alterations. We have found twenty-one de-regulated genes in the BRCAX-A group that we have called "The BRCAX Susceptibility Pathway" and suggested it as a candidate to search for new genes involved in the inherited susceptibility underlying the disease in this group.

Further info: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=20715112&retmode=ref&cmd=prlinks

Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.
Paula Martinez, Maria Thanasoula, Ana R Carlos, Gonzalo Gómez-López, Agueda M Tejera, Stefan Schoeftner, Orlando Dominguez, David G Pisano, Madalena Tarsounas, Maria A Blasco.
Nature Cell Biology (2010) vol. 12 (8) pp. 768-80

Rap1 is a component of the shelterin complex at mammalian telomeres, but its in vivo role in telomere biology has remained largely unknown to date. Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We generated cells and mice deleted for Rap1; mice with Rap1 deletion in stratified epithelia were viable but had shorter telomeres and developed skin hyperpigmentation in adulthood. By performing chromatin immunoprecipitation coupled with ultrahigh-throughput sequencing, we found that Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG)(2) consensus motif. Extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. More than 70% of extratelomeric Rap1-binding sites were in the vicinity of genes, and 31% of the genes deregulated in Rap1-null cells contained Rap1-binding sites, suggesting a role for Rap1 in transcriptional control. These findings place a telomere protein at the interface between telomere function and transcriptional regulation.

Further info: http://www.nature.com/ncb/journal/v12/n8/abs/ncb2081.html

waviCGH: a web application for the analysis and visualization of genomic copy number alterations.
Angel Carro, Daniel Rico, Oscar M Rueda, Ramón Díaz-Uriarte, David G Pisano.
Nucleic Acids Res (2010) vol. 38 Suppl pp. W182-7

waviCGH is a versatile web server for the analysis and comparison of genomic copy number alterations in multiple samples from any species. waviCGH processes data generated by high density SNP-arrays, array-CGH or copy-number calls generated by any technique. waviCGH includes methods for pre-processing of the data, segmentation, calling of gains and losses, and minimal common regions determination over a set of experiments. The server is a user-friendly interface to the analytical methods, with emphasis on results visualization in a genomic context. Analysis tools are introduced to the user as the different steps to follow in an experimental protocol. All the analysis steps generate high quality images and tables ready to be imported into spreadsheet programs. Additionally, for human, mouse and rat, altered regions are represented in a biological context by mapping them into chromosomes in an integrated cytogenetic browser. waviCGH is available at http://wavi.bioinfo.cnio.es.

Further info: http://nar.oxfordjournals.org/cgi/content/full/38/suppl_2/W182?view=long&pmid=20507915

The EMBRACE web service collection.
Steve Pettifer, Jon Ison, Matús Kalas, Dave Thorne, Philip McDermott, Inge Jonassen, Ali Liaquat, José M Fernández, Jose M Rodriguez, INB-Partners, David G Pisano, Christophe Blanchet, Mahmut Uludag, Peter Rice, Edita Bartaseviciute, Kristoffer Rapacki, Maarten Hekkelman, Olivier Sand, Heinz Stockinger, Andrew B Clegg, Erik Bongcam-Rudloff, Jean Salzemann, Vincent Breton, Teresa K Attwood, Graham Cameron, Gert Vriend.
Nucleic Acids Res (2010) vol. 38 Suppl pp. W683-8

The EMBRACE (European Model for Bioinformatics Research and Community Education) web service collection is the culmination of a 5-year project that set out to investigate issues involved in developing and deploying web services for use in the life sciences. The project concluded that in order for web services to achieve widespread adoption, standards must be defined for the choice of web service technology, for semantically annotating both service function and the data exchanged, and a mechanism for discovering services must be provided. Building on this, the project developed: EDAM, an ontology for describing life science web services; BioXSD, a schema for exchanging data between services; and a centralized registry (http://www.embraceregistry.net) that collects together around 1000 services developed by the consortium partners. This article presents the current status of the collection and its associated recommendations and standards definitions.

Further info: http://nar.oxfordjournals.org/cgi/content/full/38/suppl_2/W683?view=long&pmid=20462862

Mantle cell lymphoma: transcriptional regulation by microRNAs.
L Di Lisio, G Gómez-López, M Sánchez-Beato, C Gómez-Abad, M E Rodríguez, R Villuendas, B I Ferreira, Carro, D Rico, M Mollejo, M A Martínez, J Menárguez, A Díaz-Alderete, J Gil, J C Cigudosa, David G Pisano, M A Piris, N Martínez.
Leukemia (2010) vol. 24 (7) pp. 1335-42

Mantle cell lymphoma (MCL) pathogenesis is still partially unexplained. We investigate the importance of microRNA (miRNA) expression as an additional feature that influences MCL pathway deregulation and may be useful for predicting patient outcome. Twenty-three MCL samples, eight cell lines and appropriate controls were screened for their miRNAs and gene expression profiles and DNA copy-number changes. MCL patients exhibit a characteristic signature that includes 117 miRNA (false discovery rate <0.05). Combined analysis of miRNAs and the gene expression profile, paired with bioinformatics target prediction (miRBase and TargetScan), revealed a series of genes and pathways potentially targeted by a small number of miRNAs, including essential pathways for lymphoma survival such as CD40, mitogen-activated protein kinase and NF-kappaB. Functional validation in MCL cell lines demonstrated NF-kappaB subunit nuclear translocation to be regulated by the expression of miR-26a. The expression of 12 selected miRNAs was studied by quantitative PCR in an additional series of 54 MCL cases. Univariate analysis identified a single miRNA, miR-20b, whose lack of expression distinguished cases with a survival probability of 56% at 60 months. In summary, using a novel bioinformatics approach, this study identified miRNA changes that contribute to MCL pathogenesis and markers of potential utility in MCL diagnosis and clinical prognostication.

Further info: http://www.nature.com/leu/journal/v24/n7/abs/leu201091a.html

MidA is a putative methyltransferase that is required for mitochondrial complex I function.
Sergio Carilla-Latorre, M Esther Gallardo, Sarah J Annesley, Javier Calvo-Garrido, Osvaldo Graña, Sandra L Accari, Paige K Smith, Alfonso Valencia, Rafael Garesse, Paul R Fisher, Ricardo Escalante.
J Cell Sci (2010) vol. 123 (Pt 10) pp. 1674-83

Dictyostelium and human MidA are homologous proteins that belong to a family of proteins of unknown function called DUF185. Using yeast two-hybrid screening and pull-down experiments, we showed that both proteins interact with the mitochondrial complex I subunit NDUFS2. Consistent with this, Dictyostelium cells lacking MidA showed a specific defect in complex I activity, and knockdown of human MidA in HEK293T cells resulted in reduced levels of assembled complex I. These results indicate a role for MidA in complex I assembly or stability. A structural bioinformatics analysis suggested the presence of a methyltransferase domain; this was further supported by site-directed mutagenesis of specific residues from the putative catalytic site. Interestingly, this complex I deficiency in a Dictyostelium midA(-) mutant causes a complex phenotypic outcome, which includes phototaxis and thermotaxis defects. We found that these aspects of the phenotype are mediated by a chronic activation of AMPK, revealing a possible role of AMPK signaling in complex I cytopathology.

Further info: http://jcs.biologists.org/cgi/content/full/123/10/1674

Serum and tissue profiling in bladder cancer combining protein and tissue arrays.
Esteban Orenes-Piñero, Rodrigo Barderas, Daniel Rico, J Ignacio Casal, David G Pisano, Jose Navajo, Ferran Algaba, Josep Maria Piulats, Marta Sanchez-Carbayo.
J Proteome Res (2010) vol. 9 (1) pp. 164-73

Aiming at identifying biomarkers for bladder cancer, the serum proteome was explored in a pilot study through a profiling approach using protein arrays. Supervised analyses identified a panel 171 immunogenic proteins differentially expressed between patients with bladder cancer (n = 12) and controls without the disease (n = 10). The microanatomical expression patterns of novel immunogenic proteins, especially dynamin and clusterin, were found significantly associated with histopathologic variables and overall survival, as confirmed by immunohistochemistry using an independent series of bladder tumors contained in tissue microarrays (n = 289). Thus, the protein arrays approach has identified a panel of immunogenic candidates that may potentially play a role as diagnostic biomarkers, especially for muscle invasive disease. Moreover, the protein expression patterns of dynamin and clusterin in bladder tumors were shown to adjunct for histopathologic staging and clinical outcome prognosis.

Further info: http://pubs.acs.org/doi/abs/10.1021/pr900273u

Inference of functional relations in predicted protein networks with a machine learning approach.
Beatriz García-Jiménez, David Juan, Iakes Ezkurdia, Eduardo Andrés-León, Alfonso Valencia.
PLoS ONE (2010) vol. 5 (4).

BACKGROUND: Molecular biology is currently facing the challenging task of functionally characterizing the proteome. The large number of possible protein-protein interactions and complexes, the variety of environmental conditions and cellular states in which these interactions can be reorganized, and the multiple ways in which a protein can influence the function of others, requires the development of experimental and computational approaches to analyze and predict functional associations between proteins as part of their activity in the interactome. METHODOLOGY/PRINCIPAL FINDINGS: We have studied the possibility of constructing a classifier in order to combine the output of the several protein interaction prediction methods. The AODE (Averaged One-Dependence Estimators) machine learning algorithm is a suitable choice in this case and it provides better results than the individual prediction methods, and it has better performances than other tested alternative methods in this experimental set up. To illustrate the potential use of this new AODE-based Predictor of Protein InterActions (APPIA), when analyzing high-throughput experimental data, we show how it helps to filter the results of published High-Throughput proteomic studies, ranking in a significant way functionally related pairs. Availability: All the predictions of the individual methods and of the combined APPIA predictor, together with the used datasets of functional associations are available at http://ecid.bioinfo.cnio.es/. CONCLUSIONS: We propose a strategy that integrates the main current computational techniques used to predict functional associations into a unified classifier system, specifically focusing on the evaluation of poorly characterized protein pairs. We selected the AODE classifier as the appropriate tool to perform this task. AODE is particularly useful to extract valuable information from large unbalanced and heterogeneous data sets. The combination of the information provided by five prediction interaction prediction methods with some simple sequence features in APPIA is useful in establishing reliability values and helpful to prioritize functional interactions that can be further experimentally characterized.

Further info: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=20376314&retmode=ref&cmd=prlinks

Whole genome analysis of p38 SAPK-mediated gene expression upon stress.
Isabel Ferreiro, Manel Joaquin, Abul Islam, Gonzalo Gomez-Lopez, Montserrat Barragan, Luís Lombardía, Orlando Domínguez, David G Pisano, Nuria Lopez-Bigas, Angel R Nebreda, Francesc Posas. BMC Genomics (2010) vol. 11.

BACKGROUND: Cells have the ability to respond and adapt to environmental changes through activation of stress-activated protein kinases (SAPKs). Although p38 SAPK signalling is known to participate in the regulation of gene expression little is known on the molecular mechanisms used by this SAPK to regulate stress-responsive genes and the overall set of genes regulated by p38 in response to different stimuli. RESULTS: Here, we report a whole genome expression analyses on mouse embryonic fibroblasts (MEFs) treated with three different p38 SAPK activating-stimuli, namely osmostress, the cytokine TNFalpha and the protein synthesis inhibitor anisomycin. We have found that the activation kinetics of p38alpha SAPK in response to these insults is different and also leads to a complex gene pattern response specific for a given stress with a restricted set of overlapping genes. In addition, we have analysed the contribution of p38alpha the major p38 family member present in MEFs, to the overall stress-induced transcriptional response by using both a chemical inhibitor (SB203580) and p38alpha deficient (p38alpha-/-) MEFs. We show here that p38 SAPK dependency ranged between 60% and 88% depending on the treatments and that there is a very good overlap between the inhibitor treatment and the ko cells. Furthermore, we have found that the dependency of SAPK varies depending on the time the cells are subjected to osmostress. CONCLUSIONS: Our genome-wide transcriptional analyses shows a selective response to specific stimuli and a restricted common response of up to 20% of the stress up-regulated early genes that involves an important set of transcription factors, which might be critical for either cell adaptation or preparation for continuous extra-cellular changes. Interestingly, up to 85% of the up-regulated genes are under the transcriptional control of p38 SAPK. Thus, activation of p38 SAPK is critical to elicit the early gene expression program required for cell adaptation to stress.

Further info: http://www.biomedcentral.com/1471-2164/11/144



2009

Telomere shortening relaxes X chromosome inactivation and forces global transcriptome alterations.
Stefan Schoeftner, Raquel Blanco, Isabel Lopez de Silanes, Purificación Muñoz, Gonzalo Gómez-López, Juana M Flores, Maria A Blasco.
Proc Natl Acad Sci USA (2009) vol. 106 (46) pp. 19393-8.
Telomeres are heterochromatic structures at chromosome ends essential for chromosomal stability. Telomere shortening and the accumulation of dysfunctional telomeres are associated with organismal aging. Using telomerase-deficient TRF2-overexpressing mice (K5TRF2/Terc(-/-)) as a model for accelerated aging, we show that telomere shortening is paralleled by a gradual deregulation of the mammalian transcriptome leading to cumulative changes in a defined set of genes, including up-regulation of the mTOR and Akt survival pathways and down-regulation of cell cycle and DNA repair pathways. Increased DNA damage from dysfunctional telomeres leads to reduced deposition of H3K27me3 onto the inactive X chromosome (Xi), impaired association of the Xi with telomeric transcript accumulations (Tacs), and reactivation of an X chromosome-linked K5TRF2 transgene that is subjected to X-chromosome inactivation in female mice with sufficiently long telomeres. Exogenously induced DNA damage also disrupts Xi-Tacs, suggesting DNA damage at the origin of these alterations. Collectively, these findings suggest that critically short telomeres activate a persistent DNA damage response that alters gene expression programs in a nonstochastic manner toward cell cycle arrest and activation of survival pathways, as well as impacts the maintenance of epigenetic memory and nuclear organization, thereby contributing to organismal aging.

Further info: http://www.pnas.org/content/106/46/19393.long

Assessment of domain boundary predictions and the prediction of intramolecular contacts in CASP8. 
Ezkurdia I, Graña O, Izarzugaza JM, Tress ML. 
Proteins. 2009;77 Suppl 9:196-209.


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.

Further info: http://www.ncbi.nlm.nih.gov/pubmed/19714769

Functional signatures identified in B-cell non-Hodgkin lymphoma profiles.
Mohit Aggarwal, Margarita Sánchez-Beato, Gonzalo Gómez-López, Fátima Al-Shahrour, Nerea Martínez, Antonia Rodríguez, Elena Ruiz-Ballesteros, Francisca I Camacho, Alberto Pérez-Rosado, Paloma de la Cueva, María J Artiga, David G Pisano, Eva Kimby, Joaquín Dopazo, Raquel Villuendas, Miguel A Piris.
Leuk Lymphoma (2009) vol. 50 (10) pp. 1699-708

Gene-expression profiling in B-cell lymphomas has provided crucial data on specific lymphoma types, which can contribute to the identification of essential lymphoma survival genes and pathways. In this study, the gene-expression profiling data of all major B-cell lymphoma types were analyzed by unsupervised clustering. The transcriptome classification so obtained, was explored using gene set enrichment analysis generating a heatmap for B-cell lymphoma that identifies common lymphoma survival mechanisms and potential therapeutic targets, recognizing sets of coregulated genes and functional pathways expressed in different lymphoma types. Some of the most relevant signatures (stroma, cell cycle, B-cell receptor (BCR)) are shared by multiple lymphoma types or subclasses. A specific attention was paid to the analysis of BCR and coregulated pathways, defining molecular heterogeneity within multiple B-cell lymphoma types.

Further info: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=19863341&retmode=ref&cmd=prlinks

WhichGenes: a web-based tool for gathering, building, storing and exporting gene sets with application in gene set enrichment analysis. 
Daniel Glez-Peña, Gonzalo Gómez-López, David G Pisano, Florentino Fdez-Riverola.
Nucleic Acids Res (2009) vol. 37 (Web Server issue) pp. W329-34

WhichGenes is a web-based interactive gene set building tool offering a very simple interface to extract always-updated gene lists from multiple databases and unstructured biological data sources. While the user can specify new gene sets of interest by following a simple four-step wizard, the tool is able to run several queries in parallel. Every time a new set is generated, it is automatically added to the private gene-set cart and the user is notified by an e-mail containing a direct link to the new set stored in the server. WhichGenes provides functionalities to edit, delete and rename existing sets as well as the capability of generating new ones by combining previous existing sets (intersection, union and difference operators). The user can export his sets configuring the output format and selecting among multiple gene identifiers. In addition to the user-friendly environment, WhichGenes allows programmers to access its functionalities in a programmatic way through a Representational State Transfer web service. WhichGenes front-end is freely available at http://www.whichgenes.org/, WhichGenes API is accessible at http://www.whichgenes.org/api/.

Further info: http://nar.oxfordjournals.org/cgi/content/full/37/suppl_2/W329?view=long&pmid=19406925

The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer.
Agustin F Fernandez, Cecilia Rosales, Pilar Lopez-Nieva, Osvaldo Graña, Esteban Ballestar, Santiago Ropero, Jesus Espada, Sonia A Melo, Amaia Lujambio, Mario F Fraga, Irene Pino, Biola Javierre, Francisco J Carmona, Francesco Acquadro, Renske D M Steenbergen, Peter J F Snijders, Chris J Meijer, Pascal Pineau, Anne Dejean, Belen Lloveras, Gabriel Capella, Josep Quer, Maria Buti, Juan-Ignacio Esteban, Helena Allende, Francisco Rodriguez-Frias, Xavier Castellsague, Janos Minarovits, Jordi Ponce, Daniela Capello, Gianluca Gaidano, Juan Cruz Cigudosa, Gonzalo Gomez-Lopez, David G Pisano, Alfonso Valencia, Miguel Angel Piris, Francesc X Bosch, Ellen Cahir-McFarland, Elliott Kieff, Manel Esteller.
Genome Res (2009) vol. 19 (3) pp. 438-51

The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses.

Further info: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=19208682&retmode=ref&cmd=prlinks

Functional characterization of E- and P-cadherin in invasive breast cancer cells.
David Sarrió, José Palacios, Marta Hergueta-Redondo, Gonzalo Gómez-López, Amparo Cano, Gema Moreno-Bueno.
BMC Cancer (2009) vol. 9.

BACKGROUND: Alterations in the cadherin-catenin adhesion complexes are involved in tumor initiation, progression and metastasis. However, the functional implication of distinct cadherin types in breast cancer biology is still poorly understood. METHODS: To compare the functional role of E-cadherin and P-cadherin in invasive breast cancer, we stably transfected these molecules into the MDA-MB-231 cell line, and investigated their effects on motility, invasion and gene expression regulation. RESULTS: Expression of either E- and P-cadherin significantly increased cell aggregation and induced a switch from fibroblastic to epithelial morphology. Although expression of these cadherins did not completely reverse the mesenchymal phenotype of MDA-MB-231 cells, both E- and P-cadherin decreased fibroblast-like migration and invasion through extracellular matrix in a similar way. Moreover, microarray gene expression analysis of MDA-MB-231 cells after expression of E- and P-cadherins revealed that these molecules can activate signaling pathways leading to significant changes in gene expression. Although the expression patterns induced by E- and P-cadherin showed more similarities than differences, 40 genes were differentially modified by the expression of either cadherin type. CONCLUSION: E- and P-cadherin have similar functional consequences on the phenotype and invasive behavior of MDA-MB-231 cells. Moreover, we demonstrate for the first time that these cadherins can induce both common and specific gene expression programs on invasive breast cancer cells. Importantly, these identified genes are potential targets for future studies on the functional consequences of altered cadherin expression in human breast cancer.

Further info: http://www.biomedcentral.com/1471-2407/9/74

Translational disease interpretation with molecular networks.
Anaïs Baudot, Gonzalo Gómez-López, Alfonso Valencia.
Genome Biol (2009) vol. 10 (6) pp. 221

Molecular networks are being used to reconcile genotypes and phenotypes by integrating medical information. In this context, networks will be instrumental for the interpretation of disease at the personalized medicine level.

Further info: http://genomebiology.com/content/10/6/221

EcID: A database for the inference of functional interactions in E. coli.
Eduardo Andres Leon, Iakes Ezkurdia, Beatriz García, Alfonso Valencia, David Juan.
Nucleic Acids Res (2009) vol. 37 (Database issue) pp. D629-35

The EcID database (Escherichia coli Interaction Database) provides a framework for the integration of information on functional interactions extracted from the following sources: EcoCyc (metabolic pathways, protein complexes and regulatory information), KEGG (metabolic pathways), MINT and IntAct (protein interactions). It also includes information on protein complexes from the two E. coli high-throughput pull-down experiments and potential interactions extracted from the literature using the web services associated to the iHOP text-mining system. Additionally, EcID incorporates results of various prediction methods, including two protein interaction prediction methods based on genomic information (Phylogenetic Profiles and Gene Neighbourhoods) and three methods based on the analysis of co-evolution (Mirror Tree, In Silico 2 Hybrid and Context Mirror). EcID associates to each prediction a specifically developed confidence score. The two main features that make EcID different from other systems are the combination of co-evolution-based predictions with the experimental data, and the introduction of E. coli-specific information, such as gene regulation information from EcoCyc. The possibilities offered by the combination of the EcID database information are illustrated with a prediction of potential functions for a group of poorly characterized genes related to yeaG. EcID is available online at http://ecid.bioinfo.cnio.es.

Further info: http://nar.oxfordjournals.org/cgi/content/full/37/suppl_1/D629

2008


DAX1, a direct target of EWS/FLI1 oncoprotein, is a principal regulator of cell-cycle progression in Ewing's tumor cells.
E García-Aragoncillo, J Carrillo, E Lalli, N Agra, G Gómez-López, A Pestaña, J Alonso.
Oncogene (2008) vol. 27 (46) pp. 6034-43

The molecular hallmark of the Ewing's family of tumors is the presence of balanced chromosomal translocations, leading to the formation of chimerical transcription factors (that is, EWS/FLI1) that play a pivotal role in the pathogenesis of Ewing's tumors by deregulating gene expression. We have recently demonstrated that DAX1 (NR0B1), an orphan nuclear receptor that was not previously implicated in cancer, is induced by the EWS/FLI1 oncoprotein and is highly expressed in Ewing's tumors, suggesting that DAX1 is a biologically relevant target of EWS/FLI1-mediated oncogenesis. In this study we demonstrate that DAX1 is a direct transcriptional target of the EWS/FLI1 oncoprotein through its binding to a GGAA-rich region in the DAX1 promoter and show that DAX1 is a key player of EWS/FLI1-mediated oncogenesis. DAX1 silencing using an inducible model of RNA interference induces growth arrest in the A673 Ewing's cell line and severely impairs its capability to grow in semisolid medium and form tumors in immunodeficient mice. Gene expression profile analysis demonstrated that about 10% of the genes regulated by EWS/FLI1 in Ewing's cells are DAX1 targets, confirming the importance of DAX1 in Ewing's oncogenesis. Functional genomic analysis, validated by quantitative RT-PCR, showed that genes implicated in cell-cycle progression, such as CDK2, CDC6, MCM10 or SKP2 were similarly regulated by EWS/FLI1 and DAX1. These findings indicate that DAX1 is important in the pathogenesis of the Ewing's family of tumors, identify new functions for DAX1 as a cell-cycle progression regulator and open the possibility to new therapeutic approaches based on DAX1 function interference.

Further info: http://www.nature.com/onc/journal/v27/n46/full/onc2008203a.html

TCL1A expression delineates biological and clinical variability in B-cell lymphoma. 
M Aggarwal, R Villuendas, G Gomez-Lopez, S Rodriguez-Pinilla, M Sanchez-Beato, D Alvarez, N Martinez, A Rodriguez, M Castillo, F Camacho, S Montes-Moreno, J Garcia-Marco, E Kimby, David G Pisano, MA Piris.
Mod Pathol (2008). Feb; 22(2):206-15.

The assembly of a collection of gene-expression signatures of the major types of B-cell non-Hodgkin's lymphoma has identified increased T-cell leukemia/lymphoma 1A (TCL1) expression in multiple lymphoma types and cases, and has enabled the investigation of the functional and clinical importance of TCL1 expression. Specifically, Burkitt's lymphoma cases show a homogeneously strong expression of TCL1, whereas diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, nodal marginal zone lymphoma, and splenic marginal zone lymphoma display a striking variability in the intensity of TCL1 staining. This was validated in two independent series. A Gene-Set Enrichment Analysis of the genes correlated with TCL1A expression found that variation in the level of expression of TCL1A was significantly associated with some of the most important gene signatures recognizing B-cell lymphoma pathogenesis and heterogeneity, such as germinal center, B-cell receptor, NF-kappaB (and its target genes), death, MAP kinases, TNFR1, TOLL, and IL1R. Additionally, TCL1 expression was correlated with shorter time to treatment in chronic lymphocytic leukemia cases and shorter lymphoma-specific survival in mantle cell lymphoma series, thus indicating the clinical and biological significance of TCL1 expression, and suggesting TCL1A as a potential therapeutic target.

Further info: http://www.ncbi.nlm.nih.gov/pubmed/18820675?dopt=abstract

miR-181b negatively regulates activation-induced cytidine deaminase in B cells.
V de Yébenes, Laura Belver, David G Pisano, S González, A Villasante, C Croce, L He, A Ramiro.
J Exp Med (2008). Sep 29; 205(10), 2199-206.

Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome translocations, suggesting that the fine tuning of AID expression may be critical to restrict B cell lymphomagenesis. To determine whether microRNAs (miRNAs) play a role in the regulation of AID expression, we performed a functional screening of an miRNA library and identified miRNAs that regulate CSR. One such miRNA, miR-181b, impairs CSR when expressed in activated B cells, and results in the down-regulation of AID mRNA and protein levels. We found that the AID 3' untranslated region contains multiple putative binding sequences for miR-181b and that these sequences can be directly targeted by miR-181b. Overall, our results provide evidence for a new regulatory mechanism that restricts AID activity and can therefore be relevant to prevent B cell malignant transformation.

Further info: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=18762567&retmode=ref&cmd=prlinks

Mechanistic principles of chromatin remodeling guided by siRNAs and miRNAs.
Susana Gonzalez, David G Pisano, Manuel Serrano.
Cell Cycle (2008) vol. 7 (16) pp. 2601-8

Small RNAs can guide chromatin remodeling in mammalian cells, but the mechanisms involved are poorly understood. Previous reports have shown a requirement for overlapping transcription and the involvement of Argonaute (Ago) proteins. Here, we use the Regulatory Domain (RD) of the INK4/ARF locus as an experimental platform susceptible to siRNA-guided chromatin remodeling to interrogate about the mechanisms involved. We show that siRNA-guided chromatin remodeling of RD requires overlapping transcription, and targets the transcribed strand, but not the template strand, supporting an RNA:RNA recognition mechanism between the small RNA and the nascent RNA transcript. We found that heterochromatin formation can be triggered both by perfectly-matched double-stranded RNA precursors, as well as, by imperfectly-matched double-stranded RNA precursors. These observations, together with the fact that promoters are often subjected to overlapping transcription, open the possibility that miRNAs could also be able to guide heterochromatin formation at promoters. We demonstrate this possibility showing that miRNAs miR17-5p and miR20a from the oncomiR cluster miR-17-92 can induce heterochromatic features in promoters that undergo overlapping transcription and possess sequence complementarity to the miRNA seed region. These results unveil a new level of gene regulation by miRNAs.

Further info: http://www.landesbioscience.com/journals/cc/article/6541/

Interoperability with Moby 1.0--it's better than sharing your toothbrush!.
BioMoby Consortium, Mark D Wilkinson, Martin Senger, Edward Kawas, Richard Bruskiewich, Jerome Gouzy, Celine Noirot, Philippe Bardou, Ambrose Ng, Dirk Haase, Enrique de Andres Saiz, Dennis Wang, Frank Gibbons, Paul M K Gordon, Christoph W Sensen, Jose Manuel Rodriguez Carrasco, José M Fernández, Lixin Shen, Matthew Links, Michael Ng, Nina Opushneva, Pieter B T Neerincx, Jack A M Leunissen, Rebecca Ernst, Simon Twigger, Bjorn Usadel, Benjamin Good, Yan Wong, Lincoln Stein, William Crosby, Johan Karlsson, Romina Royo, Iván Párraga, Sergio Ramírez, Josep Lluis Gelpi, Oswaldo Trelles, David G Pisano, Natalia Jimenez, Arnaud Kerhornou, Roman Rosset, Leire Zamacola, Joaquin Tarraga, Jaime Huerta-Cepas, Jose María Carazo, Joaquin Dopazo, Roderic Guigo, Arcadi Navarro, Modesto Orozco, Alfonso Valencia, M Gonzalo Claros, Antonio J Pérez, Jose Aldana-Montes, M  Mar Rojano, Raul Fernandez-Santa Cruz, Ismael Navas, Gary Schiltz, Andrew Farmer, Damian Gessler, Heiko Schoof, Andreas Groscurth.
Brief Bioinformatics (2008) vol. 9 (3) pp. 220-31

The BioMoby project was initiated in 2001 from within the model organism database community. It aimed to standardize methodologies to facilitate information exchange and access to analytical resources, using a consensus driven approach. Six years later, the BioMoby development community is pleased to announce the release of the 1.0 version of the interoperability framework, registry Application Programming Interface and supporting Perl and Java code-bases. Together, these provide interoperable access to over 1400 bioinformatics resources worldwide through the BioMoby platform, and this number continues to grow. Here we highlight and discuss the features of BioMoby that make it distinct from other Semantic Web Service and interoperability initiatives, and that have been instrumental to its deployment and use by a wide community of bioinformatics service providers. The standard, client software, and supporting code libraries are all freely available at http://www.biomoby.org/.

Further info: http://bib.oxfordjournals.org/cgi/content/full/9/3/220

Bioinformatics and cancer research: building bridges for translational research.
Gonzalo Gómez-López, Alfonso Valencia.
Clinical & Translational Oncology. (2008) vol. 10 (2) pp. 85-95

Genome studies have revolutionised cancer research in recent years as high-throughput technologies can now be used to identify sets of genes potentially related with different processes in cancer. However, managing all this data and organising it into useful datasets is still a challenge in the bioinformatics field. Finding relationships between the molecular and genomic information and the clinical information available, within the medical informatics domain, is currently driving the development of translational research in biomedicine. The dispersion and complexity of the molecular information, the poor adherence to standards, together with the fast evolution of the experimental techniques, pose obvious challenges for the development of integrated molecular resources. In parallel, restricted access to medical information together with the gaps in the development of standard terminologies are typical limitations in the area of medical informatics. The development of research projects combining medical and molecular information together with the current efforts to standardise and integrate databases and terminologies are described in this review as a demonstration of the fruitful activity in this area.

Further info: http://www.ncbi.nlm.nih.gov/pubmed/18258507?dopt=abstract

Comparative genome profiling across subtypes of low-grade B-cell lymphoma identifies type-specific and common aberrations that target genes with a role in B-cell neoplasia.
Ferreira BI, García JF, Suela J, Mollejo M, Camacho FI, Carro A, Montes S, Piris MA, Cigudosa JC.
Haematologica. (2008) May; 93(5): 670-9.

BACKGROUND: Low-grade B-cell lymphomas are a very heterogeneous group of tumors, whose differential diagnosis is frequently compromised by the lack of specific cytogenetic or molecular features. Our objective was to search for genomic features that allow a better molecular identification of the different types of lymphoma studied. DESIGN AND METHODS: We selected a panel of 87 low-grade B-cell lymphoma tumor samples that were unambiguously diagnosed (clinically and cytogenetically) as: follicular, splenic marginal zone, nodal marginal zone, lymphoplasmacytic, mantle cell, extranodal marginal zone MALT-type lymphoma or B-cell chronic lymphocytic leukemia. All samples were subjected to the same high-resolution genomic DNA analysis (array-based comparative genomic hybridization): a whole genome platform that contained 44000 probes distributed across the genome. Genomic imbalances were recorded, compiled and analyzed. RESULTS: Eighty percent of analyzed cases showed genomic imbalances (deletions and gain/amplifications) but the frequency of these imbalances ranged from 100% in mantle cell lymphomas to 33% in MALT lymphomas. A total of 95 new genomic imbalances affecting all lymphoma subtypes, were defined. We evaluated the extension of the genomic instability, detecting distinct patterns of genomic instability within subtypes. Specific pathways, such as nuclear factor kB (gains of REL and BCL11A, and losses of COMMD3, BIRC1, IKK1 and NFKB2), Polycomb group proteins (gain of BMI1 and deletion of PCGF6), DNA repair checkpoint pathways (deletion of 16q24 involving CDT1), or miRNA with a role in B-cell lymphoma pathogenesis (MIRN15A, MIRN16-1), were targeted by this genomic instability. CONCLUSIONS: Although all subtypes of lymphomas showed gains and losses of DNA, the analysis of their genomic profiles indicated that there are specific aberrations in almost every subtype as well as frequent aberrations that are common to a large number of lymphoma types. These common aberrations target genes that are important in B-cell lymphomagenesis.

Further info: http://www.haematologica.org/cgi/content/full/93/5/670

2007

CARGO: a web portal to integrate customized biological information.
Ildefonso Cases, David G Pisano, E Andres Leon, A. Carro, G Gomez-Lopez, JM Rodriguez, Jaime Fernández-Vera, Alfonso Valencia, Ana Rojas.
Nucleic Acids Res (2007). Jul 35 (Web Server issue):W16-20.

There is a huge quantity of information generated in Life Sciences, and it is dispersed in many databases and repositories. Despite the broad availability of the information, there is a great demand for methods that are able to look for, gather and display distributed data in a standardized and friendly way. CARGO (Cancer And Related Genes Online) is a configurable biological web portal designed as a tool to facilitate, integrate and visualize results from Internet resources, independently of their native format or access method. Through the use of small agents, called widgets, supported by a Rich Internet Application (RIA) paradigm based on AJAX, CARGO provides pieces of minimal, relevant and descriptive biological information. The tool is designed to be used by experimental biologists with no training in bioinformatics. In the current state, the system presents a list of human cancer genes. Available at http://cargo.bioinfo.cnio.es.

Further info: http://nar.oxfordjournals.org/cgi/content/abstract/gkm280v1