RESUMO
Glioblastoma multiforme is a brain cancer that still shows poor prognosis for patients despite the active research for new treatments. In this work, the goal is to model and simulate the evolution of tumour associated angiogenesis and the therapeutic response to glioblastoma multiforme. Multiple phenomena are modelled in order to fit different biological pathways, such as the cellular cycle, apoptosis, hypoxia or angiogenesis. This leads to a nonlinear system with 4 equations and 4 unknowns: the density of tumour cells, the [Formula: see text] concentration, the density of endothelial cells and the vascular endothelial growth factor concentration. This system is solved numerically on a mesh fitting the geometry of the brain and the tumour of a patient based on a 2D slice of MRI. We show that our numerical scheme is positive, and we give the energy estimates on the discrete solution to ensure its existence. The numerical scheme uses nonlinear control volume finite elements in space and is implicit in time. Numerical simulations have been done using the different standard treatments: surgery, chemotherapy and radiotherapy, in order to conform to the behaviour of a tumour in response to treatments according to empirical clinical knowledge. We find that our theoretical model exhibits realistic behaviours.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/terapia , Células Endoteliais/patologia , Glioblastoma/tratamento farmacológico , Glioblastoma/terapia , Humanos , Imageamento por Ressonância Magnética , Neovascularização Patológica , Fator A de Crescimento do Endotélio VascularRESUMO
Progesterone receptor (PR) expression is used as a biomarker of oestrogen receptor-α (ERα) function and breast cancer prognosis. Here we show that PR is not merely an ERα-induced gene target, but is also an ERα-associated protein that modulates its behaviour. In the presence of agonist ligands, PR associates with ERα to direct ERα chromatin binding events within breast cancer cells, resulting in a unique gene expression programme that is associated with good clinical outcome. Progesterone inhibited oestrogen-mediated growth of ERα(+) cell line xenografts and primary ERα(+) breast tumour explants, and had increased anti-proliferative effects when coupled with an ERα antagonist. Copy number loss of PGR, the gene coding for PR, is a common feature in ERα(+) breast cancers, explaining lower PR levels in a subset of cases. Our findings indicate that PR functions as a molecular rheostat to control ERα chromatin binding and transcriptional activity, which has important implications for prognosis and therapeutic interventions.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica , Receptores de Progesterona/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/genética , Cromatina/metabolismo , Variações do Número de Cópias de DNA/genética , Progressão da Doença , Receptor alfa de Estrogênio/antagonistas & inibidores , Estrogênios/metabolismo , Estrogênios/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Camundongos , Progesterona/metabolismo , Progesterona/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptores de Progesterona/genética , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. METHODS: Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. RESULTS: These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. CONCLUSIONS: These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Animais , Apoptose/fisiologia , Neoplasias da Mama/patologia , Carcinoma Basocelular/patologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Prognóstico , Proteogenômica , Células Tumorais CultivadasRESUMO
Epigenetic mechanisms are believed to play key roles in the establishment of cell-specific transcription programs. Accordingly, the modified bases 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been observed in DNA of genomic regulatory regions such as enhancers, and oxidation of 5mC into 5hmC by Ten-eleven translocation (TET) proteins correlates with enhancer activation. However, the functional relationship between cytosine modifications and the chromatin architecture of enhancers remains elusive. To gain insights into their function, 5mC and 5hmC levels were perturbed by inhibiting DNA methyltransferases and TETs during differentiation of mouse embryonal carcinoma cells into neural progenitors, and chromatin characteristics of enhancers bound by the pioneer transcription factors FOXA1, MEIS1, and PBX1 were interrogated. In a large fraction of the tested enhancers, inhibition of DNA methylation was associated with a significant increase in monomethylation of H3K4, a characteristic mark of enhancer priming. In addition, at some specific enhancers, 5mC oxidation by TETs facilitated chromatin opening, a process that may stabilize MEIS1 binding to these genomic regions.
Assuntos
5-Metilcitosina/metabolismo , Cromatina/metabolismo , Células-Tronco de Carcinoma Embrionário/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , 5-Metilcitosina/análogos & derivados , Animais , Diferenciação Celular , Cromatina/ultraestrutura , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Células-Tronco de Carcinoma Embrionário/citologia , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Histonas/genética , Histonas/metabolismo , Camundongos , Proteína Meis1/genética , Proteína Meis1/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fator de Transcrição 1 de Leucemia de Células Pré-B/genética , Fator de Transcrição 1 de Leucemia de Células Pré-B/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Control of gene transcription relies on concomitant regulation by multiple transcriptional regulators (TRs). However, how recruitment of a myriad of TRs is orchestrated at cis-regulatory modules (CRMs) to account for coregulation of specific biological pathways is only partially understood. Here, we have used mouse liver CRMs involved in regulatory activities of the hepatic TR, NR1H4 (FXR; farnesoid X receptor), as our model system to tackle this question. Using integrative cistromic, epigenomic, transcriptomic, and interactomic analyses, we reveal a logical organization where trans-regulatory modules (TRMs), which consist of subsets of preferentially and coordinately corecruited TRs, assemble into hierarchical combinations at hepatic CRMs. Different combinations of TRMs add to a core TRM, broadly found across the whole landscape of CRMs, to discriminate promoters from enhancers. These combinations also specify distinct sets of CRM differentially organized along the genome and involved in regulation of either housekeeping/cellular maintenance genes or liver-specific functions. In addition to these TRMs which we define as obligatory, we show that facultative TRMs, such as one comprising core circadian TRs, are further recruited to selective subsets of CRMs to modulate their activities. TRMs transcend TR classification into ubiquitous versus liver-identity factors, as well as TR grouping into functional families. Hence, hierarchical superimpositions of obligatory and facultative TRMs bring about independent transcriptional regulatory inputs defining different sets of CRMs with logical connection to regulation of specific gene sets and biological pathways. Altogether, our study reveals novel principles of concerted transcriptional regulation by multiple TRs at CRMs.
Assuntos
Genoma , Fígado/metabolismo , Elementos Reguladores de Transcrição , Transcrição Gênica , Algoritmos , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genômica/métodos , Camundongos , Camundongos Knockout , PPAR alfa/deficiência , PPAR alfa/genética , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
Cell identity relies on the cross-talk between genetics and epigenetics and their impact on gene expression. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) is the first step of an active DNA demethylation process occurring mainly at enhancers and gene bodies and, as such, participates in processes governing cell identity in normal and pathological conditions. Although genetic alterations are well documented in multiple myeloma (MM), epigenetic alterations associated with this disease have not yet been thoroughly analyzed. To gain insight into the biology of MM, genome-wide 5hmC profiles were obtained and showed that regions enriched in this modified base overlap with MM enhancers and super enhancers and are close to highly expressed genes. Through the definition of a MM-specific 5hmC signature, we identified FAM72D as a poor prognostic gene located on 1q21, a region amplified in high risk myeloma. We further uncovered that FAM72D functions as part of the FOXM1 transcription factor network controlling cell proliferation and survival and we evidenced an increased sensitivity of cells expressing high levels of FOXM1 and FAM72 to epigenetic drugs targeting histone deacetylases and DNA methyltransferases.
Assuntos
Mieloma Múltiplo , Proteínas/genética , Proliferação de Células/genética , Metilação de DNA , Epigênese Genética , Epigenômica , Humanos , Mieloma Múltiplo/genéticaRESUMO
Runt-related transcription factor 1 (RUNX1) is a well-known master regulator of hematopoietic lineages but its mechanisms of action are still not fully understood. Here, we found that RUNX1 localizes on active chromatin together with Far Upstream Binding Protein 1 (FUBP1) in human B-cell precursor lymphoblasts, and that both factors interact in the same transcriptional regulatory complex. RUNX1 and FUBP1 chromatin localization identified c-KIT as a common target gene. We characterized two regulatory regions, at +700 bp and +30 kb within the first intron of c-KIT, bound by both RUNX1 and FUBP1, and that present active histone marks. Based on these regions, we proposed a novel FUBP1 FUSE-like DNA-binding sequence on the +30 kb enhancer. We demonstrated that FUBP1 and RUNX1 cooperate for the regulation of the expression of the oncogene c-KIT. Notably, upregulation of c-KIT expression by FUBP1 and RUNX1 promotes cell proliferation and renders cells more resistant to the c-KIT inhibitor imatinib mesylate, a common therapeutic drug. These results reveal a new mechanism of action of RUNX1 that implicates FUBP1, as a facilitator, to trigger transcriptional regulation of c-KIT and to regulate cell proliferation. Deregulation of this regulatory mechanism may explain some oncogenic function of RUNX1 and FUBP1.
Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Regulação Leucêmica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas de Ligação a RNA/genética , Animais , Antineoplásicos/farmacologia , Sequência de Bases , Sítios de Ligação , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/química , Cromatina/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Mesilato de Imatinib/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia , Cultura Primária de Células , Ligação Proteica , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lysine Specific Demethylase 1 (LSD1) removes mono- and dimethyl groups from lysine 4 of histone H3 (H3K4) or H3K9, resulting in repressive or activating (respectively) transcriptional histone marks. The mechanisms that control the balance between these two antagonist activities are not understood. We here show that LSD1 and the orphan nuclear receptor estrogen-related receptor α (ERRα) display commonly activated genes. Transcriptional activation by LSD1 and ERRα involves H3K9 demethylation at the transcriptional start site (TSS). Strikingly, ERRα is sufficient to induce LSD1 to demethylate H3K9 in vitro. The relevance of this mechanism is highlighted by functional data. LSD1 and ERRα coregulate several target genes involved in cell migration, including the MMP1 matrix metallo-protease, also activated through H3K9 demethylation at the TSS. Depletion of LSD1 or ERRα reduces the cellular capacity to invade the extracellular matrix, a phenomenon that is rescued by MMP1 reexpression. Altogether our results identify a regulatory network involving a direct switch in the biochemical activities of a histone demethylase, leading to increased cell invasion.
Assuntos
Histona Desmetilases/metabolismo , Histonas/metabolismo , Receptores de Estrogênio/metabolismo , Movimento Celular , Regulação da Expressão Gênica , Células HEK293 , Histona Desmetilases/genética , Humanos , Lisina/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Metilação , Regiões Promotoras Genéticas , Receptores de Estrogênio/genética , Sítio de Iniciação de Transcrição , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Due to the spectacular number of studies focusing on epigenetics in the last few decades, and particularly for the last few years, the availability of a chronology of epigenetics appears essential. Indeed, our review places epigenetic events and the identification of the main epigenetic writers, readers and erasers on a historic scale. This review helps to understand the increasing knowledge in molecular and cellular biology, the development of new biochemical techniques and advances in epigenetics and, more importantly, the roles played by epigenetics in many physiological and pathological situations.
Assuntos
Epigênese Genética , Epigenômica/história , Animais , Metilação de DNA , História do Século XX , História do Século XXI , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Transcription factors (TFs) bind specifically to discrete regions of mammalian genomes called cis-regulatory elements. Among those are enhancers, which play key roles in regulation of gene expression during development and differentiation. Despite the recognized central regulatory role exerted by chromatin in control of TF functions, much remains to be learned regarding the chromatin structure of enhancers and how it is established. Here, we have analyzed on a genomic-scale enhancers that recruit FOXA1, a pioneer transcription factor that triggers transcriptional competency of these cis-regulatory sites. Importantly, we found that FOXA1 binds to genomic regions showing local DNA hypomethylation and that its cell-type-specific recruitment to chromatin is linked to differential DNA methylation levels of its binding sites. Using neural differentiation as a model, we showed that induction of FOXA1 expression and its subsequent recruitment to enhancers is associated with DNA demethylation. Concomitantly, histone H3 lysine 4 methylation is induced at these enhancers. These epigenetic changes may both stabilize FOXA1 binding and allow for subsequent recruitment of transcriptional regulatory effectors. Interestingly, when cloned into reporter constructs, FOXA1-dependent enhancers were able to recapitulate their cell type specificity. However, their activities were inhibited by DNA methylation. Hence, these enhancers are intrinsic cell-type-specific regulatory regions of which activities have to be potentiated by FOXA1 through induction of an epigenetic switch that includes notably DNA demethylation.
Assuntos
Elementos Facilitadores Genéticos , Epigenômica , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Modelos Genéticos , Neurônios/citologia , Neurônios/metabolismoRESUMO
Enhancers are developmentally controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. In this study, we show by genome-wide mapping that the newly discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine (5hmC) is dynamically associated with transcription factor binding to distal regulatory sites during neural differentiation of mouse P19 cells and during adipocyte differentiation of mouse 3T3-L1 cells. Functional annotation reveals that regions gaining 5hmC are associated with genes expressed either in neural tissues when P19 cells undergo neural differentiation or in adipose tissue when 3T3-L1 cells undergo adipocyte differentiation. Furthermore, distal regions gaining 5hmC together with H3K4me2 and H3K27ac in P19 cells behave as differentiation-dependent transcriptional enhancers. Identified regions are enriched in motifs for transcription factors regulating specific cell fates such as Meis1 in P19 cells and PPARγ in 3T3-L1 cells. Accordingly, a fraction of hydroxymethylated Meis1 sites were associated with a dynamic engagement of the 5-methylcytosine hydroxylase Tet1. In addition, kinetic studies of cytosine hydroxymethylation of selected enhancers indicated that DNA hydroxymethylation is an early event of enhancer activation. Hence, acquisition of 5hmC in cell-specific distal regulatory regions may represent a major event of enhancer progression toward an active state and participate in selective activation of tissue-specific genes.
Assuntos
Diferenciação Celular/genética , Metilação de DNA , Elementos Facilitadores Genéticos , Células 3T3-L1 , 5-Metilcitosina/análogos & derivados , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/metabolismo , Citosina/análogos & derivados , Citosina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Camundongos , Proteína Meis1 , Proteínas de Neoplasias/metabolismo , Neurogênese/genética , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: B Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is the most common pediatric cancer. Identifying key players involved in proliferation of BCP-ALL cells is crucial to propose new therapeutic targets. Runt Related Transcription Factor 1 (RUNX1) and Core-Binding Factor Runt Domain Alpha Subunit 2 Translocated To 3 (CBFA2T3, ETO2, MTG16) are master regulators of hematopoiesis and are implicated in leukemia. METHODS: We worked with BCP-ALL mononuclear bone marrow patients' cells and BCP-ALL cell lines, and performed Chromatin Immunoprecipitations followed by Sequencing (ChIP-Seq), co-immunoprecipitations (co-IP), proximity ligation assays (PLA), luciferase reporter assays and mouse xenograft models. RESULTS: We demonstrated that CBFA2T3 transcript levels correlate with RUNX1 expression in the pediatric t(12;21) ETV6-RUNX1 BCP-ALL. By ChIP-Seq in BCP-ALL patients' cells and cell lines, we found that RUNX1 is recruited on its promoter and on an enhancer of CBFA2T3 located - 2 kb upstream CBFA2T3 promoter and that, subsequently, the transcription factor RUNX1 drives both RUNX1 and CBFA2T3 expression. We demonstrated that, mechanistically, RUNX1 and CBFA2T3 can be part of the same complex allowing CBFA2T3 to strongly potentiate the activity of the transcription factor RUNX1. Finally, we characterized a CBFA2T3-mimicking peptide that inhibits the interaction between RUNX1 and CBFA2T3, abrogating the activity of this transcription complex and reducing BCP-ALL lymphoblast proliferation. CONCLUSIONS: Altogether, our findings reveal a novel and important activation loop between the transcription regulator CBFA2T3 and the transcription factor RUNX1 that promotes BCP-ALL proliferation, supporting the development of an innovative therapeutic approach based on the NHR2 subdomain of CBFA2T3 protein.
Assuntos
Antineoplásicos/farmacologia , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Peptídeos/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Antineoplásicos/química , Linhagem Celular Tumoral , Criança , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos/química , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Proteínas Repressoras/química , Proteínas Repressoras/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
MicroRNAs (miRNAs or miRs) play crucial roles in biological and pathological processes. Some miRNAs also appear as promising biomarkers and therapeutic tools. However, the epitranscriptomic regulation of miRNAs is not yet fully elucidated in all of their fields of application. We report that adenosine methylation of miR-200b-3p inhibits its repressive function toward its mRNA targets such as XIAP by blocking the formation of the miRNA/3' UTRmRNA duplex. Our data indicate that the adenosine methylation of miR-200b-3p is associated with the survival of glioblastoma patients. Collectively, our data support the idea that the adenosine methylation of miR-200b-3p can be used as a prodrug having a selective cytotoxicity against cancer cells (while being harmless to peripheral blood mononuclear cells [PBMCs], astrocytes, neurons, and hepatocytes).
RESUMO
BACKGROUND: Multiple myeloma (MM) is a heterogeneous plasma cell malignancy that remains challenging to cure. Global hypomethylation correlates with an aggressive phenotype of the disease, while hypermethylation is observed at particular regions of myeloma such as B cell-specific enhancers. The recently discovered active epigenetic mark 5-hydroxymethylCytosine (5hmC) may also play a role in tumor biology; however, little is known about its level and distribution in myeloma. In this study, we investigated the global level and the genomic localization of 5hmC in myeloma cells from 40 newly diagnosed patients, including paired relapses, and of control individuals. RESULTS: Compared to normal plasma cells, we found global 5hmC levels to be lower in myeloma (P < 0.001). Higher levels of 5hmC were found in lower grades of the International Staging System prognostic index (P < 0.05) and tend to associate with a longer overall survival (P < 0.1). From the hydroxymethylome data, we observed that the remaining 5hmC is organized in large domains overlapping with active chromatin marks and chromatin opening. We discovered that 5hmC strongly persists at key oncogenic genes such as CCND1, CCND2 and MMSET and characterized domains that are specifically hydroxymethylated in myeloma subgroups. Novel 5hmC-enriched domains were found at putative enhancers of CCND2 and MYC in newly diagnosed patients. CONCLUSIONS: 5hmC level is associated with clinical aspects of MM. Mapping 5hmC at a genome-wide level provides insights into the disease biology directly from genomic DNA, which makes it a potent mark to study epigenetics on large patient cohorts.
Assuntos
5-Metilcitosina/análogos & derivados , Genoma/genética , Mieloma Múltiplo/genética , Sequências Reguladoras de Ácido Nucleico/genética , 5-Metilcitosina/sangue , 5-Metilcitosina/química , 5-Metilcitosina/metabolismo , Cromatina/genética , Ciclina D1/metabolismo , Ciclina D2/metabolismo , Metilação de DNA , Epigênese Genética , Epigenômica , Feminino , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Fenótipo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Índice de Gravidade de DoençaRESUMO
This protocol is designed to obtain base-resolution information on the level of 5-hydroxymethylcytosine (5hmC) in CpGs without the need for bisulfite modification. It relies on (i) the capture of hydroxymethylated sequences by a procedure known as 'selective chemical labeling' (see Szulwach et al., 2012 ) and (ii) the digestion of the captured DNA by exonucleases. After Illumina sequencing of the digested DNA fragments, an ad hoc bioinformatic pipeline extracts the information for further downstream analysis.
RESUMO
The epigenetic mark 5-hydroxymethylcytosine (5hmC) is a cytosine modification that is abundant in the central nervous system of mammals and which results from 5-methylcytosine oxidation by TET enzymes. Such a mark is suggested to play key roles in the regulation of chromatin structure and gene expression. However, its precise functions still remain poorly understood and information about its distribution in non-mammalian species is still lacking. Here, the distribution of 5hmC was investigated in the brain of adult zebrafish, African claw frog, and mouse in a comparative manner. We show that zebrafish neurons are endowed with high levels of 5hmC, whereas quiescent or proliferative neural progenitors show low to undetectable levels of the modified cytosine. In the brain of larval and juvenile Xenopus, 5hmC is also detected in neurons, while ventricular proliferative cells do not display this epigenetic mark. Similarly, 5hmC is enriched in neurons compared to neural progenitors of the ventricular zone in the mouse developing cortex. Interestingly, 5hmC colocalized with the methylated DNA binding protein MeCP2 and with the active chromatin histone modification H3K4me2 in mouse neurons. Taken together, our results show an evolutionarily conserved cerebral distribution of 5hmC between fish and tetrapods and reinforce the idea that 5hmC fulfills major functions in the control of chromatin activity in vertebrate neurons. J. Comp. Neurol. 525:478-497, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
5-Metilcitosina/análogos & derivados , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Neurônios/metabolismo , 5-Metilcitosina/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/citologia , Dermoscopia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Microscopia Confocal , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Mucosa Olfatória/citologia , Mucosa Olfatória/crescimento & desenvolvimento , Mucosa Olfatória/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Xenopus , Peixe-ZebraAssuntos
Transformação Celular Neoplásica/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Homeodomínio/genética , Humanos , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Células Tumorais CultivadasRESUMO
FOXA1 is a pioneer factor that binds to enhancer regions that are enriched in H3K4 mono- and dimethylation (H3K4me1 and H3K4me2). We performed a FOXA1 rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME) screen in ERα-positive MCF-7 breast cancer cells and found histone-lysine N-methyltransferase (MLL3) as the top FOXA1-interacting protein. MLL3 is typically thought to induce H3K4me3 at promoter regions, but recent findings suggest it may contribute to H3K4me1 deposition. We performed MLL3 chromatin immunoprecipitation sequencing (ChIP-seq) in breast cancer cells, and MLL3 was shown to occupy regions marked by FOXA1 occupancy and H3K4me1 and H3K4me2. MLL3 binding was dependent on FOXA1, indicating that FOXA1 recruits MLL3 to chromatin. MLL3 silencing decreased H3K4me1 at enhancer elements but had no appreciable impact on H3K4me3 at enhancer elements. We propose a mechanism whereby the pioneer factor FOXA1 recruits the chromatin modifier MLL3 to facilitate the deposition of H3K4me1 histone marks, subsequently demarcating active enhancer elements.