Identification of Methylated Genes Associated with Aggressive Clinicopathological Features in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is genetically characterized by the t(11;14)(q13;q32) translocation and a high number of secondary chromosomal alterations. The contribution of DNA methylation to MCL lymphomagenesis is not well known. We sought to identify epigenetically silenced genes in these tumours that might have clinical relevance. Methodology/Principal Findings: To identify potential methylated genes in MCL we initially investigated seven MCL cell lines treated with epigenetic drugs and gene expression microarray profiling. The methylation status of selected candidate genes was validated by a quantitative assay and subsequently analyzed in a series of primary MCL (n=38). After pharmacological reversion we identified 252 potentially methylated genes. The methylation analysis of a subset of these genes (n=25) in the MCL cell lines and normal B lymphocytes confirmed that 80% of them were methylated in the cell lines but not in normal lymphocytes. The subsequent analysis in primary MCL identified five genes (SOX9,HOXA9,AHR,NR2F2 ,and ROBO1) frequently methylated in these tumours. The gene methylation events tended to occur in the same primary neoplasms and correlated with higher proliferation, increased number of chromosomal abnormalities, and shorter survival of the patients. Conclusions: We have identified a set of genes whose methylation degree and gene expression levels correlate with aggressive clinicopathological features of MCL. Our findings also suggest that a subset of MCL might show a CpG island methylator phenotype (CIMP) that may influence the behaviour of the tumours.

Identification of Methylated Genes Associated with Aggressive Clinicopathological Features in Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is genetically characterized by the t(11;14)(q13;q32) translocation and a high number of secondary chromosomal alterations. The contribution of DNA methylation to MCL lymphomagenesis is not well known. We sought to identify epigenetically silenced genes in these tumours that might have clinical relevance. Methodology/Principal Findings: To identify potential methylated genes in MCL we initially investigated seven MCL cell lines treated with epigenetic drugs and gene expression microarray profiling. The methylation status of selected candidate genes was validated by a quantitative assay and subsequently analyzed in a series of primary MCL (n=38). After pharmacological reversion we identified 252 potentially methylated genes. The methylation analysis of a subset of these genes (n=25) in the MCL cell lines and normal B lymphocytes confirmed that 80% of them were methylated in the cell lines but not in normal lymphocytes. The subsequent analysis in primary MCL identified five genes (SOX9,HOXA9,AHR,NR2F2 ,and ROBO1) frequently methylated in these tumours. The gene methylation events tended to occur in the same primary neoplasms and correlated with higher proliferation, increased number of chromosomal abnormalities, and shorter survival of the patients. Conclusions: We have identified a set of genes whose methylation degree and gene expression levels correlate with aggressive clinicopathological features of MCL. Our findings also suggest that a subset of MCL might show a CpG island methylator phenotype (CIMP) that may influence the behaviour of the tumours.

Finding and characterizing Partially Methylated Domains in human haematopoietic cells

DNA cytosine methylation has been demonstrated to be a central epigenetic modification that has essential roles in a myriad of cellular processes. Some examples of these include gene regulation, DNA-protein interactions, cellular differentiation, X-inactivation, maintenance of genome integrity by suppressing transposable elements and viruses, embryogenesis, genomic imprinting and tumourigenesis. This list is increasingly growing thanks to recent advances in genome-wide technologies, like Whole Genome Bisulfite Sequencing (WGBS-Seq). The development of this technology in research has allowed the identification of new features of the DNA methylation landscape that was not possible using previous technologies, like Partially Methylated Domains (PMDs). PMDs have been found in several cell lines, as well as in both healthy and cancer primary samples. They have been described as regions with high variability in methylation levels across individual CpG sites and intermediate methylation levels on average with respect to the genome. Here, we performed an extensive search of PMDs in a big dataset of different haematopoietic primary cells from both myeloid and lymphoid lineages. We found and characterized significant PMDs in plasma B cells, confirming that PMDs are a phenomenon that is restricted to certain differentiated cells. Additionally, we found loci aberrantly hypomethylated in a myeloma sample which overlapped with plasma B cell PMDs. Genome-wide comparison of the myeloma and plasma B cell sample revealed that this is probably also the case for other loci.

Análisis comparativo de tres técnicas de derivación de células madre

Las células madre embrionarias (Embryonic Stem Cells; ESC) son células pluripotentes que presentan la capacidad de dividirse indefinidamente a la vez que mantienen la habilidad para diferenciarse a cualquier tipo celular. Aunque de manera rutinaria se derivan a partir de la masa celular interna de embriones en estadio de blastocisto, también pueden derivarse a partir de embriones en estadios precompactacionales y de embriones reconstruidos por procesos de transferencia nuclear. Debido a que durante el desarrollo embrionario temprano, momento en el que se derivan las ESC, tienen lugar profundos cambios de metilación en el genoma, tanto la derivación como el cultivo se consagran como técnicas que pueden alterar los patrones de metilación en genes regulados por impronta genómica. Con el objetivo de analizar la estabilidad epigenética de embriones preimplantacionales y ESC murinas, en este trabajo se ha optimizado un protocolo de anàlisis de los niveles de metilación mediante pirosecuenciación. Para ello se han seleccionado tres genes regulados por impronta genómica (H19/Igf2, Snrpn and Peg3), dos genes relacionados con el mantenimiento de pluripotencia en ESC (Oct4, Nanog y Sox2) y dos genes marcadores de diferenciación temprana (Cdx2 y Gata6). Nuestros resultados muestran que algunos grupos de embriones preimplantacionales presentan una hipo e hipermetilación en las regiones diferencialmente metiladas (Differentially Methylated Regions, DMRs) de los genes Snrpn y Peg3. Además, la línea de ESC analizada presentó anomalías en los tres genes regulados por impronta genómica. No obstante, el hecho de que esta línea fuera inestable a nivel cariotípico no permite establecer una relación entre el cultivo in vitro o la técnica de derivación y la inestabilidad epigenética demostrada. Por todo esto, parece pertinente analizar tanto la integridad epigenética como la estabilidad cromosómica de ESC antes de proceder a realizar ensayos clínicos en humanos.

A polymorphic DNA probe from chromosome 7 (7q22)

Source/Description: p6a-l is a O.9 kb HindIII/BamHl genomic fragment subclone or cosmic cNX.6a in pUC13. cNX.6a was isolated from a non-methylated enriched library from the CMGT cell line Cll (1,2).

Clopidogrel Pharmacogenetics, resistance to antiplatelet therapy in ischemic stroke by Epigenome Wide Association Study (EWAS).

Clopidogrel is a widely used antiplatelet drug used in preventing vascular events after suffering a first stoke. Genome-wide association studies (GWAS) has not been able to establish a clear association between polymorphisms and recurrence. Therefore in the present final master project an epigenetic approach is proposed. Using an array based technology, 450.000 CpG sites across all genome were assessed in 48 individuals (21 cases and 21 controls). Looking at differentially methylated levels between cases and controls, 58 CpG sites (DMGs) were found. Although, no clear locus was observed. Looking individually to each 49 genes, two appeared to be important to our study. TRAF3 and ADAMTS2 are gens highly related to platelet aggregation. In orther to confirm these result, a new DNA methylation study will be done in a larger cohort, using Sequenom technology.

The promoter activity of human Mfn2 depends on Sp1 in vascular smooth muscle cells

AIMS: Mitofusin-2 (Mfn2) expression is dysregulated in vascular proliferative disorders and its overexpression attenuates the proliferation of vascular smooth muscle cells (VSMCs) and neointimal lesion development after balloon angioplasty. We sought to gain insight into the mechanisms that control Mfn2 expression in VSMCs. METHODS AND RESULTS: We cloned and characterized 2 kb of the 5'-flanking region of the human Mfn2 gene. Its TATA-less promoter contains a CpG island. In keeping with this, 5'-rapid amplification of cDNA ends revealed six transcriptional start sites (TSSs), of which TSS2 and TSS5 were the most frequently used. The strong CpG island was found to be non-methylated under conditions characterized by large differences in Mfn2 gene expression. The proximal Mfn2 promoter contains six putative Sp1 motifs. Sp1 binds to the Mfn2 promoter and its overexpression activates the Mfn2 promoter in VSMCs. Chemical inhibition of Sp1 reduced Mfn2 expression, and Sp1 silencing reduced transcriptional activity of the Mfn2 promoter. In keeping with this view, Sp1 and Mfn2 mRNA levels were down-regulated in the aorta early after an atherogenic diet in apolipoprotein E-knockout mice or in VSMCs cultured in the presence of low serum. CONCLUSION: Sp1 is a key factor in maintaining basal Mfn2 transcription in VSMCs. Given the anti-proliferative actions of Mfn2, Sp1-induced Mfn2 transcription may represent a mechanism for prevention of VSMC proliferation and neointimal lesion and development.

The promoter activity of human Mfn2 depends on Sp1 in vascular smooth muscle cells

AIMS: Mitofusin-2 (Mfn2) expression is dysregulated in vascular proliferative disorders and its overexpression attenuates the proliferation of vascular smooth muscle cells (VSMCs) and neointimal lesion development after balloon angioplasty. We sought to gain insight into the mechanisms that control Mfn2 expression in VSMCs. METHODS AND RESULTS: We cloned and characterized 2 kb of the 5'-flanking region of the human Mfn2 gene. Its TATA-less promoter contains a CpG island. In keeping with this, 5'-rapid amplification of cDNA ends revealed six transcriptional start sites (TSSs), of which TSS2 and TSS5 were the most frequently used. The strong CpG island was found to be non-methylated under conditions characterized by large differences in Mfn2 gene expression. The proximal Mfn2 promoter contains six putative Sp1 motifs. Sp1 binds to the Mfn2 promoter and its overexpression activates the Mfn2 promoter in VSMCs. Chemical inhibition of Sp1 reduced Mfn2 expression, and Sp1 silencing reduced transcriptional activity of the Mfn2 promoter. In keeping with this view, Sp1 and Mfn2 mRNA levels were down-regulated in the aorta early after an atherogenic diet in apolipoprotein E-knockout mice or in VSMCs cultured in the presence of low serum. CONCLUSION: Sp1 is a key factor in maintaining basal Mfn2 transcription in VSMCs. Given the anti-proliferative actions of Mfn2, Sp1-induced Mfn2 transcription may represent a mechanism for prevention of VSMC proliferation and neointimal lesion and development.