Search

Introduction: Modulation of the DNA methylation landscape during cell differentiation is a well-established phenomenon. The B-cell lineage represents a paradigmatic cellular model to study the dynamic epigenome during cell development and specification because major B-cell maturation stages are well defined and display differential phenotypic and gene expression features. Furthermore, different B-cell subpopulations show different proliferation abilities, microenvironmental influences and life spans, providing a window of opportunity to study the epigenome in the context of multiple processes. Methods: We performed whole-genome bisulfite sequencing (WGBS), high-density methylation microarrays and gene expression profiling of ten purified human B-cell subpopulations spanning the entire differentiation program, ranging from uncommitted progenitors to terminally-differentiated plasma cells. Results: The results of both WGBS and methylation microarrays indicate that B-cell ontogenesis involves an extensive and gradual reconfiguration of the DNA methylome. We uncovered that non-CpG methylation at CpApC trinucleotides is present in progenitor cells and disappears upon B-cell commitment independently of CpG demethylation. CpG methylation, in contrast, changed extensively during the entire B-cell maturation program, with one quarter of all measured CpGs showing dynamic methylation. B-cell enhancers suffered more extensive methylation changes than promoter regions, especially in the early differentiation steps up to the germinal center B-cell (gcBC) stage, and their demethylation seemed to be mediated by binding of lineage-specific transcription factors. Enhancers with dynamic methylation were related to genes involved in a large B-cell network that showed high gene expression variability throughout differentiation. In highly proliferative gcBCs, we observed a shift of dynamic methylation from regulatory towards non-functional elements; gcBCs start to undergo global demethylation of late-replicating heterochromatic regions and methylation of polycomb-repressed regions. This signature becomes particularly extensive in long-lived memory B cells and plasma cells, indicating that these changes start in highly proliferative cells and then accumulate in non-proliferative cells with extended lifespan. Conclusion: Our epigenomic analysis of the B-cell differentiation program extends our knowledge on how the DNA methylome is modulated during cell specification and maturation and offers a resource for researchers in the field, both at global and single gene levels. Disclosures No relevant conflicts of interest to declare.

Ever since the discovery of DNA methylation at cytosine residues, the role of this so called fifth base has been extensively studied and debated. Until recently, the majority of DNA methylation studies focused on the analysis of CpG islands associated to promoter regions. However, with the upcoming possibilities to study DNA methylation in a genome-wide context, this epigenetic mark can now be studied in an unbiased manner. As a result, recent studies have shown that not only promoters but also intragenic and intergenic regions are widely modulated during physiological processes and disease. In particular, it is becoming increasingly clear that DNA methylation in the gene body is not just a passive witness of gene transcription but it seems to be actively involved in multiple gene regulation processes. In this review we discuss the potential role of intragenic DNA methylation in alternative promoter usage, regulation of short and long non-coding RNAs, alternative RNA processing, as well as enhancer activity. Furthermore, we summarize how the intragenic DNA methylome is modified both during normal cell differentiation and neoplastic transformation.

This work was funded by the Spanish Ministry of Economy and Competitivity (MINECO) through the Instituto de Salud Carlos III (ISCIII), Red Tematica de Investigacion del Cancer (RTICC) and Instituto Nacional de Bioinformatica (INB) from ISCIII, and Consolider CSD2007-00050. C.L.-O. is an investigator of the Botın Foundation and E.C. of the ICREA-Academia program. N.L.-B., D.T., and A.G.-P. acknowledge funding from the Spanish Ministry of Science and Technology (grant number SAF2009-06954). S.E. is supported by a fellowship from La Caixa., Ferreira, P.G., Jares, P., Rico, D., Gómez-López, G., Martínez-Trillos, A., Villamor, N., Ecker, S., González-Pérez, A., Knowles, D.G., Monlong, J., Johnson, R., Quesada, V., Djebali, S., Papasaikas, P., López-Guerra, M., Colomer, D., Royo, C., Cazorla, M., Pinyol, M., Clot, G., Aymerich, M., Rozman, M., Kulis, M., Tamborero, D., Gouin, A., Blanc, J., Gut, M., Gut, I., Puente, X.S., Pisano, D.G., Martin-Subero, J.I., López-Bigas, N., López-Guillermo, A., Valencia, A., López-Otín, C., Campo, E., Guigó, R.

Introduction SOX11 is a transcription factor (TF) aberrantly expressed in the majority of mantle cell lymphomas (MCLs), which is generally associated with aggressive clinical behaviour. No mutations, genetic aberrations or direct correlations with differential DNA methylation at the promoter related to its expression have been found in MCL. Deeper insights into its regulation can be found by considering the three-dimensional (3D) chromatin structure. It is becoming clear that the genome can be partitioned into 3D building blocks, topologically associated domains (TADs) and that enhancer regions likely regulate genes within their TADs by 3D contacts, but do not affect genes outside their own TADs. By mapping the 3D chromatin structure, we previously identified a distant putative SOX11 enhancer showing enhancer activity and 3D contacts with the SOX11 gene in the SOX11-positive MCL cell line Z-138, but not in the SOX11-negative MCL cell line JVM-2. Aims We aimed to deepen our understanding of the differential 3D contacts and enhancer activity previously observed at the putative SOX11 enhancer in SOX11-positive versus SOX11-negative MCL cell lines by addressing the following questions: (i) Do TAD boundaries around the SOX11 locus change between SOX11-positive and -negative MCLs? (ii) How do the 3D contacts and chromatin states at this region behave in primary MCL cases and normal B cells? (iii) Is the putative SOX11 enhancer involved in SOX11 expression in other tissues? Methods We have extended our experimental analyses of the putative SOX11 enhancer by performing (i) HiC-sequencing and 3D fluorescence in situ hybridization (3D FISH) in MCL cell lines Z-138 and JVM-2, (ii) 4C-sequencing, chromatin inmmunoprecipiation followed by deep sequencing (ChIP-seq) of 6 histone marks, an Assay for Transposase-Accessible Chromatin with deep sequencing (ATAC-seq) and chromatin state modeling by chromHMM (using the 6 histone marks) in primary MCL cases and normal naive and memory B-cells. Furthermore, we have explored the activity of this region in other SOX11 expressing cell lines studied within the ENCODE Consortium. Results HiC-sequencing in the cell lines Z-138 (SOX11-positive) and JVM-2 (SOX11-negative) showed that the SOX11 locus and its putative enhancer are located within the same TAD in both samples. Hence, shifts in TAD boundaries do not seem to underlie the differential 3D chromatin interactions between the SOX11 locus and its putative enhancer in these two cell lines. By ChIP-seq and chromatin state modeling we observed that the promoter of SOX11 is poised, i.e., carrying histone marks H3K4me3 and H3K27me3, in normal naive and memory B-cells and the SOX11-negative MCL primary case. Furthermore, we observed weak enhancer activity at the putative SOX11 enhancer in normal naive and memory B-cells and the SOX11-negative MCL primary case, but strong enhancer activity, marked by the presence of H3K27ac, only in SOX11-positive samples. In addition, by ATAC-seq we identified two specific chromatin accessible regions that potentially represent the transcription factor binding sites responsible for activation of this enhancer region in SOX11-positive MCLs. By 4C-sequencing we observed that the SOX11 locus and its putative enhancer show high 3D contacts in two other SOX11-positive MCL cell lines (GRANTA-519 and JEKO-1) and in a SOX11-positive primary MCL case, but not in a SOX11-negative primary MCL case. Furthermore, the differential 3D contacts at these regions in Z-138 and JVM-2 were confirmed by 3D FISH, which is currently being performed in primary MCL cases. Interestingly, no 3D contacts were observed in normal naive and memory B cells, indicating that although the SOX11 promoter is poised within these normal B-cell subpopulations, primed looping at these regions does not exist and seems not to explain the 3D contacts we observed in SOX11-positive MCL cell lines and primary cases. When investigating chromatin states in cell lines studied by ENCODE with an active SOX11 promoter (H1-hESC, HSMM and NHLF) none of them show activity in the identified region, suggesting that the putative SOX11 enhancer is de novo activated only in the context of MCL lymphomagenesis. Conclusions We provide new evidence that the activation of a distant SOX11 putative enhancer and its 3D contacts to the SOX11 gene, is a de novo event in SOX11-positive MCL cell lines and primary cases that is likely specific for this malignancy. Disclosures No relevant conflicts of interest to declare.

Increasing amount of evidence indicates that the deregulation of non-coding elements is a common feature of cancer and therefore, its investigation may uncover new molecular oncogenic mechanisms. In multiple myeloma (MM), the altered expression of a small number of long non-coding RNAs (lncRNAs) has been associated with progression and decreased survival, suggesting that these elements may play a more important role in this disease than previously expected. Nevertheless, an extensive high-throughput analysis that characterizes the deregulation of lncRNAs in MM has not yet been performed. To characterize the transcriptome, including all genomic types of lncRNAs, of MM we performed a paired end strand-specific RNA sequencing (ssRNA-seq) in 38 purified plasma cell (PC) samples from MM patients, as well as PC samples from tonsils (TPCs, n=5) and bone marrow (BMPCs, n=3) of healthy donors as controls. Principal component analysis (PCA) demonstrated that normal PC samples from tonsil and bone marrow cluster separately, suggesting that in spite of being the same cell type, their coding and non coding transcriptomes are very different. Therefore, we selected BMPCs as the normal counterparts for comparison with BM of MM samples. PCA analysis also demonstrated that the well known heterogeneity of MM patients rely not only on the coding transcriptome but also on the lncRNA expression profile. Comparison of MM to BMPCs samples showed 70 previously annotated lncRNAs that were deregulated in MM patients, with 3 lncRNAs showing higher and 67 lower expression than normal BMPCs. Moreover, we identified 40.552 novel MM-specific lncRNAs that were present in at least 3 of the 38 patients, highlighting the magnitude of the deregulation of these non coding elements in MM. To determine the functional role of altered lncRNAs in the biology of MM plasma cells we focused on the study of LINC-MSL1 (Myeloma-Specific LncRNA 1). Analysis of the expression of this lncRNA at different stages of B-cell differentiation (Naïve, Germinal Center, Memory and PC) indicated that it is not expressed at any stage, except for a modest expression in BMPCs. Interestingly, its overexpression was detected in 40% of MM specimens when compared to normal BMPCs which was validated by qPCR in an independent cohort of MM patients. To determine whether the expression of this lncRNA is regulated by epigenetic mechanisms, we studied the DNA methylation state of this gene. DNA methylation analysis in MM demonstrated that the CpGs located upstream of LINC-MSL1 were differentially methylated in comparison with normal counterpart BMPC. These CpGs showed 70% DNA methylation in control samples, about 40% in MGUS, whereas the average of MM was about 20%, showing a remarkable hypomethylation. We validated these results by pyrosequencing, which showed a significantly lower DNA methylation at the promoter region in comparison with B cell populations from tonsil, normal BMPCs and cell lines that do not overexpress LINC-MSL1. We also have observed a gain of active chromatin states analyzed by ChiP-seq in the promoter region of LINC-MSL1 in MM patient samples. These data suggest that epigenetic mechanisms, namely the progressive hypomethylation and the gain of active histone modifications, are the cause of the overexpression of LINC-MSL1 in MM. To analyze the role of the overexpression of LINC-MSL1 in MM, we engineered two MM cell lines that show high levels of LINC-MSL1, MM.1S and MM.1R, to express shRNAs against this lncRNA. Knockdown of LINC-MSL1 by two different shRNAs resulted in a reduced proliferation of the cell lines over time. This effect was not associated with a cell cycle arrest but with a marked increased in the percentage of Annexin V-positive apoptotic cells, indicating that the overexpression of LINC-MSL1 is necessary for the survival of MM cells. All together, these data demonstrate that the alteration of lncRNAs is an important an unexplored feature that contributes to MM pathogenesis. The overexpression of LINC-MSL1 is essential for MM survival and is very specific of MM BMPCs, suggesting it could be a relevant therapeutic target. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding. Melnick:Janssen: Research Funding.

During the last years, the study of molecular alterations associated with multiple myeloma (MM) has been mostly focused on the analysis of the genome, transcriptome and DNA methylome. These analyses are showing that (epi)genetic heterogeneity and extensive perturbation of the transcriptional landscape are hallmarks of MM. Our previous analysis of the whole DNA methylome of MM revealed that this epigenetic mark globally shows a poor correlation with gene expression, and therefore did not allow us to better understand gene deregulation in MM. In contrast, the chromatin structure and histone modifications are emerging as essential epigenetic layers to understand the mechanisms underlying gene expression changes in cancer, but remain widely unexplored in MM. We have now performed a deep ChIP-seq profiling of CD138+ sorted cells from bone marrow samples obtained from four MM patients and three biological replicates of normal plasma cells (NPCs) using antibodies against H3K4me3, H3K4me1, H3K27ac, H3K36me3, H3K27me3 and H3K9me3. Different combinations of these marks allow us to segment the MM and NPC genome into functional chromatin states, including active, weak or poised promoters, active or weak enhancers, transcriptional elongation, polycomb-repressed regions and heterochromatic regions. The initial unsupervised exploration of the data showed that the chromatin landscape of MM is widely altered as compared to NPCs. A supervised analysis of chromatin states revealed that MM globally shows a more active chromatin structure than NPCs. From over 40,000 regions identified with differential chromatin structure between MM and NPCs, 88% were de novo activated in neoplastic plasma cells. Analyzing the chromatin of individual genes, we observed that there were roughly ten times more genes gaining activity upon neoplastic transformation than those acquiring repressed chromatin marks. Interestingly, the genes showing more activate chromatin were enriched with biosynthesis and metabolic processes, while genes with repressed chromatin were related to gene ontology terms related to B cell signaling. Among those genes gaining de novo activity in MM, we selected several candidates and we are currently performing functional in vitro assays to explore their implication in MM pathogenesis. Furthermore, as extensive chromatin activation is a hallmark of MM, we are currently analyzing additional 15 MM cases and NPCs by ChIP-seq for H3K27ac (marking active promoters and enhancers) and ATAC-seq (marking active regulatory regions) to validate our initial findings and explore chromatin heterogeneity in MM. Collectively, our initial exploration of histone modification profiles in MM has revealed that MM cells acquire a more active chromatin landscape, with thousands of regions gaining activation as compared to NPCs. Reversing this global activation by epigenetic drugs, such as BET inhibitors, may represent an attractive therapeutic option for MM. During the meeting, updated information will be presented, including data from all 19 MM patients studied as well as functional data from new candidate genes involved in MM pathogenesis. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

DNA methylation has an important impact on normal cell physiology, thus any defects in this mechanism may be related to the development of various diseases In this project we are interested in identifying epigeneticaliy modified genes, in general controlled by processes related to the DNA methylation, by means of a new strategy combining protomic and genomic analyses. First, the two Dimensional-Difference Gel Electrophoresis (2-DIGE) protein analyses of extracts obtained from HCT-116 wt and double knockout for DNMT1 and DNMT3b (DKO) cells revealed 34 proteins overexpressed in the condition of DNMTs depletion. From five genes with higher transcript lavels in DKO cells, comparing with HCT-116 wt. oniy AKR1B1, UCHLl and VIM are melhylated in HCT-116. As expected. the DNA methvlation 1s lost in DKO cells. The rneth,vl ation of VIM and UCHLl promoters in some cancer samples has already been repaired, thus further studies has been focused on AKRlBI. AKR1B1 expression due lo DNA methyiaton of promoter region seems to occur specilfically in the colon cancer cell Iines. which was confirmed in the DNA rnethylation status and expression analyses. performed on 32 different cancer cell lines (including colon, breast, lymphoma, leukemia, neuroblastoma, glioma and lung cancer cell Iines) as well as normal colon and normal lymphocytes samples. AKRIBI expression after treatments with DNA demethvlating agent (AZA) was rescued in 5 coloncancer cell lines (including genetic regulation of the candidate gene. The methylation status of the rest of the genes identified in proteomic analysis was checked by methylation specific PCR (MSP) experiment and all appeared to be unmethylated. The similar research has been done also bv means of Mecp2-null mouse model For 14 selected candidate genes the analyses of expression leveis, methylation Status and MeCP2 interaction with promoters are currently being performed.

Neuroblastoma (NB) pathogenesis has been reported to be closely associated with numerous genetic alterations. However, underlying DNA methylation patterns have not been extensively studied in this developmental malignancy. Here, we generated microarray-based DNA methylation profiles of primary neuroblastic tumors. Stringent supervised differential methylation analyses allowed us to identify epigenetic changes characteristic for NB tumors as well as for clinical and biological subtypes of NB. We observed that gene-specific loss of DNA methylation is more prevalent than promoter hypermethylation. Remarkably, such hypomethylation affected cancer-related biological functions and genes relevant to NB pathogenesis such as CCND1, SPRR3, BTC, EGF and FGF6. In particular, differential methylation in CCND1 affected mostly an evolutionary conserved functionally relevant 3′ untranslated region, suggesting that hypomethylation outside promoter regions may play a role in NB pathogenesis. Hypermethylation targeted genes involved in cell development and proliferation such as RASSF1A, POU2F2 or HOXD3, among others. The results derived from this study provide new candidate epigenetic biomarkers associated with NB as well as insights into the molecular pathogenesis of this tumor, which involves a marked gene-specific hypomethylation.

We describe synthesis and properties of eight dinucleotide mRNA 5' cap analogs containing imidodiphosphate moiety within 5',5'-tri- or tetraphosphate bridge (NH-analogs). The compounds were obtained by coupling an appropriate nucleoside 5'-imidodiphosphate with nucleotide P-imidazolide mediated by divalent metal chloride in anhydrous DMF. To evaluate the novel compounds as tools for studying cap-dependent processes, we determined their binding affinities for eukaryotic translation initiation factor 4E, susceptibilities to decapping pyrophosphatase DcpS and, for non-hydrolysable analogs, binding affinities to this enzyme. The results indicate that the O to NH substitution in selected positions of oligophosphate bridge ensures resistance to enzymatic decapping and suggest that interactions of NH-analogs with cap binding proteins fairly mimic interactions of unmodified parent compounds. Finally, we identified NH-analogs as potent inhibitors of cap-dependent translation in cell free system, and evaluated their utility as reagents for obtaining 5' capped mRNAs in vitro to be rather moderate.

Decapping is an essential step in multiple pathways of mRNA degradation. Previously, we synthesized mRNAs containing caps that were resistant to decapping, both to dissect the various pathways for mRNA degradation and to stabilize mRNA for more sustained protein expression. mRNAs containing an α-β CH2 group are resistant to in vitro cleavage by the decapping enzyme hDcp2 but poorly translated. mRNAs containing an S substitution at the β-phosphate are well translated but only partially resistant to hDcp2. We now describe seven new cap analogs substituted at the β-phosphate with BH3 or Se, or substituted at either the α-β or β-γ O with NH. The analogs differ in affinity for eIF4E and efficiency of in vitro incorporation into mRNA by T7 RNA polymerase. Luciferase mRNAs capped with these analogs differ in resistance to hDcp2 hydrolysis in vitro, translational efficiency in rabbit reticulocyte lysate and in HeLa cells, and stability in HeLa cells. Whereas mRNAs capped with m27,2′-OGppSpG were previously found to have the most favorable properties of translational efficiency and stability in mammalian cells, mRNAs capped with m7GppBH3pm7G are translated with the same efficiency but are more stable. Interestingly, some mRNAs exhibit a lag of up to 60 min before undergoing first-order decay (t1/2 ≅ 25 min). Only mRNAs that are efficiently capped, resistant to decapping in vitro, and actively translated have long lag phases.

DNA methylation is one of the most intensely studied epigenetic modifications in mammals. In normal cells, it assures the proper regulation of gene expression and stable gene silencing. DNA methylation is associated with histone modifications and the interplay of these epigenetic modifications is crucial to regulate the functioning of the genome by changing chromatin architecture. The covalent addition of a methyl group occurs generally in cytosine within CpG dinucleotides which are concentrated in large clusters called CpG islands. DNA methyltransferases are responsible for establishing and maintenance of methylation pattern. It is commonly known that inactivation of certain tumor-suppressor genes occurs as a consequence of hypermethylation within the promoter regions and a numerous studies have demonstrated a broad range of genes silenced by DNA methylation in different cancer types. On the other hand, global hypomethylation, inducing genomic instability, also contributes to cell transformation. Apart from DNA methylation alterations in promoter regions and repetitive DNA sequences, this phenomenon is associated also with regulation of expression of noncoding RNAs such as microRNAs that may play role in tumor suppression. DNA methylation seems to be promising in putative translational use in patients and hypermethylated promoters may serve as biomarkers. Moreover, unlike genetic alterations, DNA methylation is reversible what makes it extremely interesting for therapy approaches. The importance of DNA methylation alterations in tumorigenesis encourages us to decode the human epigenome. Different DNA methylome mapping techniques are indispensable to realize this project in the future.

DNA methylation is one of the most intensely studied epigenetic modifications in mammals. In normal cells, it assures the proper regulation of gene expression and stable gene silencing. DNA methylation is associated with histone modifications and the interplay of these epigenetic modifications is crucial to regulate the functioning of the genome by changing chromatin architecture. The covalent addition of a methyl group occurs generally in cytosine within CpG dinucleotides which are concentrated in large clusters called CpG islands. DNA methyltransferases are responsible for establishing and maintenance of methylation pattern. It is commonly known that inactivation of certain tumor-suppressor genes occurs as a consequence of hypermethylation within the promoter regions and a numerous studies have demonstrated a broad range of genes silenced by DNA methylation in different cancer types. On the other hand, global hypomethylation, inducing genomic instability, also contributes to cell transformation. Apart from DNA methylation alterations in promoter regions and repetitive DNA sequences, this phenomenon is associated also with regulation of expression of noncoding RNAs such as microRNAs that may play role in tumor suppression. DNA methylation seems to be promising in putative translational use in patients and hypermethylated promoters may serve as biomarkers. Moreover, unlike genetic alterations, DNA methylation is reversible what makes it extremely interesting for therapy approaches. The importance of DNA methylation alterations in tumorigenesis encourages us to decode the human epigenome. Different DNA methylome mapping techniques are indispensable to realize this project in the future.

Enhancer of rudimentary homolog (Drosophila) (ERH) is a small, highly conserved, nuclear protein with a unique three-dimensional structure, whose gene has been identified in animals, plants and protists, but not in fungi. Involvement of ERH in fundamental processes such as regulation of pyrimidine metabolism, cell cycle progression, transcription and cell growth control has been suggested. Here, employing a yeast two-hybrid system, a glutathione S-transferase pull-down assay and tandem MS, we demonstrate that Ciz1 is a bona fide interactor of human ERH. Ciz1 is a nuclear zinc finger protein interacting with p21(Cip1/Waf1), a universal inhibitor of cyclin-dependent kinases, and is a DNA replication factor. The region of Ciz1 necessary for the interaction with ERH spans residues 531-644, encompassing its first zinc finger motif. This region overlaps the p21(Cip1/Waf1)-binding site, suggesting that the interaction with ERH could block the binding of p21(Cip1/Waf1) by Ciz1 in the cell. When ERH and Ciz1 are coexpressed in HeLa cells, Ciz1 recruits ERH to DNA replication foci.

In the last few years studies have shown a significant increase in psychic disorders among children and adolescents. The four disorders have been shown as the most alarming. In the first place is depression and suicide which is an integral problem of it. In the second place is anxiety disorder, which is the most common disorder in the population. In the third place is attention deficit hyperactive disorder (ADHD) which represents the biggest problem for the teachers. The last and the most acute disorder is schizophrenia. In most patients it begins in the age between 15 and 25. Besides these disorders a huge problem in the society today is child abuse. In most cases it triggers the aforementioned disorders in later life. Due to the concerning results of these studies we decided to start a project for preventing the development of symptoms in children and adolescents. The aim of this project is to start a new and better education about psychic disorders in children and adolescents for teachers in primary and high schools. Moreover we are trying to educate the teachers about symptoms of disorders in the pupils in order to recognize them and to become more efficient in working with those pupils. As the studies have shown these disorders are not curable. The only thing that we can do is to control the symptoms by prescribing medicine or going to psychotherapy. But the most important thing in treatment is to start in the early stages of the disease. Recognizing the early symptoms in pupils and preventing them to develop into a disease is the best way to help children and adolescents.