RESUMO
CCAAT/enhancer binding protein-α (C/EBPα) induces transdifferentiation of B cells into macrophages at high efficiencies and enhances reprogramming into induced pluripotent stem (iPS) cells when co-expressed with the transcription factors Oct4 (Pou5f1), Sox2, Klf4 and Myc (hereafter called OSKM). However, how C/EBPα accomplishes these effects is unclear. Here we find that in mouse primary B cells transient C/EBPα expression followed by OSKM activation induces a 100-fold increase in iPS cell reprogramming efficiency, involving 95% of the population. During this conversion, pluripotency and epithelial-mesenchymal transition genes become markedly upregulated, and 60% of the cells express Oct4 within 2 days. C/EBPα acts as a 'path-breaker' as it transiently makes the chromatin of pluripotency genes more accessible to DNase I. C/EBPα also induces the expression of the dioxygenase Tet2 and promotes its translocation to the nucleus where it binds to regulatory regions of pluripotency genes that become demethylated after OSKM induction. In line with these findings, overexpression of Tet2 enhances OSKM-induced B-cell reprogramming. Because the enzyme is also required for efficient C/EBPα-induced immune cell conversion, our data indicate that Tet2 provides a mechanistic link between iPS cell reprogramming and B-cell transdifferentiation. The rapid iPS reprogramming approach described here should help to fully elucidate the process and has potential clinical applications.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Transdiferenciação Celular , Reprogramação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Células Cultivadas , Reprogramação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Citosina/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Dioxigenases , Transição Epitelial-Mesenquimal/genética , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Regulação para Cima/genéticaRESUMO
The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid malignancies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is CEBPα-induced transdifferentiation of pre-B cells into macrophages. Here we found that CEBPα binds to upstream regions of Tet2 and that the gene becomes activated. Tet2 knockdowns impaired the upregulation of macrophage markers as well as phagocytic capacity, suggesting that the enzyme is required for both early and late stage myeloid differentiation. A slightly weaker effect was seen in primary cells with a Tet2 ablation. Expression arrays of transdifferentiating cells with Tet2 knockdowns permitted the identification of a small subset of myeloid genes whose upregulation was blunted. Activation of these target genes was accompanied by rapid increases of promoter hydroxy-methylation. Our observations indicate that Tet2 helps CEBPα rapidly derepress myeloid genes during the conversion of pre-B cells into macrophages.
Assuntos
Proteínas de Ligação a DNA , Macrófagos , Células Mieloides , Células Precursoras de Linfócitos B , Proteínas Proto-Oncogênicas , Azacitidina/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular , Linhagem Celular , Transdiferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Mielopoese , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Histone methylation plays an important role in regulating gene expression. One such methylation occurs at Lys 79 of histone H3 (H3K79) and is catalyzed by the yeast DOT1 (disruptor of telomeric silencing) and its mammalian homolog, DOT1L. Previous studies have demonstrated that germline disruption of Dot1L in mice resulted in embryonic lethality. Here we report that cardiac-specific knockout of Dot1L results in increased mortality rate with chamber dilation, increased cardiomyocyte cell death, systolic dysfunction, and conduction abnormalities. These phenotypes mimic those exhibited in patients with dilated cardiomyopathy (DCM). Mechanistic studies reveal that DOT1L performs its function in cardiomyocytes through regulating Dystrophin (Dmd) transcription and, consequently, stability of the Dystrophin-glycoprotein complex important for cardiomyocyte viability. Importantly, expression of a miniDmd can largely rescue the DCM phenotypes, indicating that Dmd is a major target mediating DOT1L function in cardiomyocytes. Interestingly, analysis of available gene expression data sets indicates that DOT1L is down-regulated in idiopathic DCM patient samples compared with normal controls. Therefore, our study not only establishes a critical role for DOT1L-mediated H3K79 methylation in cardiomyocyte function, but also reveals the mechanism underlying the role of DOT1L in DCM. In addition, our study may open new avenues for the diagnosis and treatment of human heart disease.
Assuntos
Regulação para Baixo , Distrofina/metabolismo , Metiltransferases/metabolismo , Miocárdio/metabolismo , Animais , Cardiomegalia/metabolismo , Cardiomegalia/mortalidade , Cardiomegalia/patologia , Linhagem Celular , Histona-Lisina N-Metiltransferase , Humanos , Metiltransferases/deficiência , Metiltransferases/genética , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismoRESUMO
Recent studies indicate that the methylation state of histones can be dynamically regulated by histone methyltransferases and demethylases. The H3K9-specific demethylase Jhdm2a (also known as Jmjd1a and Kdm3a) has an important role in nuclear hormone receptor-mediated gene activation and male germ cell development. Through disruption of the Jhdm2a gene in mice, here we demonstrate that Jhdm2a is critically important in regulating the expression of metabolic genes. The loss of Jhdm2a function results in obesity and hyperlipidemia in mice. We provide evidence that the loss of Jhdm2a function disrupts beta-adrenergic-stimulated glycerol release and oxygen consumption in brown fat, and decreases fat oxidation and glycerol release in skeletal muscles. We show that Jhdm2a expression is induced by beta-adrenergic stimulation, and that Jhdm2a directly regulates peroxisome proliferator-activated receptor alpha (Ppara) and Ucp1 expression. Furthermore, we demonstrate that beta-adrenergic activation-induced binding of Jhdm2a to the PPAR responsive element (PPRE) of the Ucp1 gene not only decreases levels of H3K9me2 (dimethylation of lysine 9 of histone H3) at the PPRE, but also facilitates the recruitment of Ppargamma and Rxralpha and their co-activators Pgc1alpha (also known as Ppargc1a), CBP/p300 (Crebbp) and Src1 (Ncoa1) to the PPRE. Our studies thus demonstrate an essential role for Jhdm2a in regulating metabolic gene expression and normal weight control in mice.
Assuntos
Metabolismo Energético/fisiologia , Regulação da Expressão Gênica , Obesidade/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Tecido Adiposo Marrom/metabolismo , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Glicerol/metabolismo , Canais Iônicos/metabolismo , Histona Desmetilases com o Domínio Jumonji , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/metabolismo , Oxirredução , Fenótipo , Receptores Adrenérgicos beta/metabolismo , Proteína Desacopladora 1RESUMO
Transcription factor-induced lineage reprogramming or transdifferentiation experiments are essential for understanding the plasticity of differentiated cells. These experiments helped to define the specific role of transcription factors in conferring cell identity and played a key role in the development of the regenerative medicine field. We here investigated the acquisition of DNA methylation changes during C/EBPα-induced pre-B cell to macrophage transdifferentiation. Unexpectedly, cell lineage conversion occurred without significant changes in DNA methylation not only in key B cell- and macrophage-specific genes but also throughout the entire set of genes differentially methylated between the two parental cell types. In contrast, active and repressive histone modification marks changed according to the expression levels of these genes. We also demonstrated that C/EBPα and RNA Pol II are associated with the methylated promoters of macrophage-specific genes in reprogrammed macrophages without inducing methylation changes. Our findings not only provide insights about the extent and hierarchy of epigenetic events in pre-B cell to macrophage transdifferentiation but also show an important difference to reprogramming towards pluripotency where promoter DNA demethylation plays a pivotal role.
Assuntos
Transdiferenciação Celular/genética , Metilação de DNA , Epigênese Genética , Macrófagos/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Regiões Promotoras Genéticas , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Células Cultivadas , Histonas/metabolismo , Macrófagos/citologia , Camundongos , Células Precursoras de Linfócitos B/citologia , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
We used a retroviral integration screen to search for novel genes that regulate HSC function. One of the genes that conferred HSC dominance when overexpressed due to an adjacent retroviral insertion was Musashi 2 (Msi2), an RNA-binding protein that can act as a translational inhibitor. A gene-trap mouse model that inactivates the gene shows that Msi2 is more highly expressed in long-term (LT) and short-term (ST) HSCs, as well as in lymphoid myeloid primed progenitors (LMPPs), but much less in intermediate progenitors and mature cells. Mice lacking Msi2 are fully viable for up to a year or more, but exhibit severe defects in primitive precursors, most significantly a reduction in the number of ST-HSCs and LMPPs and a decrease in leukocyte numbers, effects that are exacerbated with age. Cell-cycle and gene-expression analyses suggest that the main hematopoietic defect in Msi2-defective mice is the decreased proliferation capacity of ST-HSCs and LMPPs. In addition, HSCs lacking Msi2 are severely impaired in competitive repopulation experiments, being overgrown by wild-type cells even when mutant cells were provided in excess. Our data indicate that Msi2 maintains the stem cell compartment mainly by regulating the proliferation of primitive progenitors downstream of LT-HSCs.
Assuntos
Células Progenitoras Linfoides/fisiologia , Células Progenitoras Mieloides/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Adesão Celular/fisiologia , Divisão Celular/fisiologia , Movimento Celular/fisiologia , Feminino , Testes Genéticos/métodos , Contagem de Leucócitos , Células Progenitoras Linfoides/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Insercional , Células Progenitoras Mieloides/citologia , Neomicina , Inibidores da Síntese de Proteínas , Retroviridae/genética , Baço/patologia , Timo/patologia , beta-Galactosidase/genéticaRESUMO
Polycomb group (PcG) proteins control organism development by regulating the expression of developmental genes. Transcriptional regulation by PcG proteins is achieved, at least partly, through the PRC2-mediated methylation on lysine 27 of histone H3 (H3K27) and PRC1-mediated ubiquitylation on lysine 119 of histone H2A (uH2A). As an integral component of PRC1, Bmi1 has been demonstrated to be critical for H2A ubiquitylation. Although recent studies have revealed the genome-wide binding patterns of some of the PRC1 and PRC2 components, as well as the H3K27me3 mark, there have been no reports describing genome-wide localization of uH2A. Using the recently developed ChIP-Seq technology, here, we report genome-wide localization of the Bmi1-dependent uH2A mark in MEF cells. Gene promoter averaging analysis indicates a peak of uH2A just inside the transcription start site (TSS) of well-annotated genes. This peak is enriched at promoters containing the H3K27me3 mark and represents the least expressed genes in WT MEF cells. In addition, peak finding reveals regions of local uH2A enrichment throughout the mouse genome, including almost 700 gene promoters. Genes with promoter peaks of uH2A exhibit lower-level expression when compared to genes that do not contain promoter peaks of uH2A. Moreover, we demonstrate that genes with uH2A peaks have increased expression upon Bmi1 knockout. Importantly, local enrichment of uH2A is not limited to regions containing the H3K27me3 mark. We describe the enrichment of H2A ubiquitylation at high-density CpG promoters and provide evidence to suggest that DNA methylation may be linked to uH2A at these regions. Thus, our work not only reveals Bmi1-dependent H2A ubiquitylation, but also suggests that uH2A targeting in differentiated cells may employ a different mechanism from that in ES cells.
Assuntos
Genoma , Histonas/análise , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Animais , Células Cultivadas , Ilhas de CpG , Metilação de DNA , Histonas/metabolismo , Camundongos , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1 , Proteínas do Grupo Polycomb , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
Earlier work demonstrated that the transcription factor C/EBPα can convert immature and mature murine B lineage cells into functional macrophages. Testing >20 human lymphoma and leukemia B cell lines, we found that most can be transdifferentiated at least partially into macrophage-like cells, provided that C/EBPα is expressed at sufficiently high levels. A tamoxifen-inducible subclone of the Seraphina Burkitt lymphoma line, expressing C/EBPαER, could be efficiently converted into phagocytic and quiescent cells with a transcriptome resembling normal macrophages. The converted cells retained their phenotype even when C/EBPα was inactivated, a hallmark of cell reprogramming. Interestingly, C/EBPα induction also impaired the cells' tumorigenicity. Likewise, C/EBPα efficiently converted a lymphoblastic leukemia B cell line into macrophage-like cells, again dramatically impairing their tumorigenicity. Our experiments show that human cancer cells can be induced by C/EBPα to transdifferentiate into seemingly normal cells at high frequencies and provide a proof of principle for a potential new therapeutic strategy for treating B cell malignancies.
Assuntos
Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Macrófagos/citologia , Animais , Antineoplásicos Hormonais/uso terapêutico , Antineoplásicos Hormonais/toxicidade , Linhagem Celular Tumoral , Linhagem da Célula , Transdiferenciação Celular/efeitos dos fármacos , Humanos , Leucemia/metabolismo , Leucemia/patologia , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/metabolismo , Linfoma de Células B/mortalidade , Macrófagos/metabolismo , Camundongos , Fagocitose , Tamoxifeno/uso terapêutico , Tamoxifeno/toxicidade , Transcriptoma , Transplante HeterólogoRESUMO
Once thought to be transcriptional noise, large non-coding RNAs (lncRNAs) have recently been demonstrated to be functional molecules. The cell-type-specific expression patterns of lncRNAs suggest that their transcription may be regulated epigenetically. Using a custom-designed microarray, here we examine the expression profile of lncRNAs in embryonic stem (ES) cells, lineage-restricted neuronal progenitor cells, and terminally differentiated fibroblasts. In addition, we also analyze the relationship between their expression and their promoter H3K4 and H3K27 methylation patterns. We find that numerous lncRNAs in these cell types undergo changes in the levels of expression and promoter H3K4me3 and H3K27me3. Interestingly, lncRNAs that are expressed at lower levels in ES cells exhibit higher levels of H3K27me3 at their promoters. Consistent with this result, knockdown of the H3K27me3 methyltransferase Ezh2 results in derepression of these lncRNAs in ES cells. Thus, our results establish a role for Ezh2-mediated H3K27 methylation in lncRNA silencing in ES cells and reveal that lncRNAs are subject to epigenetic regulation in a similar manner to that of the protein-coding genes.
Assuntos
Histonas/fisiologia , RNA não Traduzido/metabolismo , Animais , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Inativação Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/fisiologia , Histonas/genética , Histonas/metabolismo , Camundongos , Análise em Microsséries , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismoRESUMO
The Ink4a-Arf-Ink4b locus has a crucial role in both cellular senescence and tumorigenesis. JmjC domain-containing histone demethylase 1b (Jhdm1b, also known as Kdm2b and Fbxl10), the mammalian paralog of the histone demethylase Jhdm1a (also known as Kdm2a and Fbxl11), has been implicated in cell-cycle regulation and tumorigenesis. In this report, we show that Jhdm1b is a histone H3 lysine 36 (H3K36) demethylase. Knockdown of Jhdm1b in primary mouse embryonic fibroblasts inhibits cell proliferation and induces cellular senescence in a pRb- and p53 pathway-dependent manner. Notably, the effect of Jhdm1b on cell proliferation and cellular senescence is mediated through derepression of p15(Ink4b), as loss of p15(Ink4b) function rescues cell-proliferation defects in Jhdm1b-knockdown cells. Chromatin immunoprecipitation on ectopically expressed Jhdm1b demonstrates that Jhdm1b targets the p15(Ink4b) locus and regulates its expression in an enzymatic activity-dependent manner. Alteration of Jhdm1b level affects Ras-induced neoplastic transformation. Collectively, our results indicate that Jhdm1b is an H3K36 demethylase that regulates cell proliferation and senescence through p15(Ink4b).
Assuntos
Proliferação de Células , Senescência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Proteínas F-Box/metabolismo , Animais , Transformação Celular Neoplásica , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas F-Box/genética , Fibroblastos/citologia , Fibroblastos/fisiologia , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Histone methylation has important roles in regulating gene expression and forms part of the epigenetic memory system that regulates cell fate and identity. Enzymes that directly remove methyl marks from histones have recently been identified, revealing a new level of plasticity within this epigenetic modification system. Here we analyse the evolutionary relationship between Jumonji C (JmjC)-domain-containing proteins and discuss their cellular functions in relation to their potential enzymatic activities.