RESUMEN
Chromatin organization is disrupted genome-wide during DNA replication. On newly synthesized DNA, nucleosomes are assembled from new naive histones and old modified histones. It remains unknown whether the landscape of histone post-translational modifications (PTMs) is faithfully copied during DNA replication or the epigenome is perturbed. Here we develop chromatin occupancy after replication (ChOR-seq) to determine histone PTM occupancy immediately after DNA replication and across the cell cycle. We show that H3K4me3, H3K36me3, H3K79me3, and H3K27me3 positional information is reproduced with high accuracy on newly synthesized DNA through histone recycling. Quantitative ChOR-seq reveals that de novo methylation to restore H3K4me3 and H3K27me3 levels occurs across the cell cycle with mark- and locus-specific kinetics. Collectively, this demonstrates that accurate parental histone recycling preserves positional information and allows PTM transmission to daughter cells while modification of new histones gives rise to complex epigenome fluctuations across the cell cycle that could underlie cell-to-cell heterogeneity.
Asunto(s)
Replicación del ADN/genética , Histonas/genética , Ciclo Celular/genética , Línea Celular Tumoral , Cromatina/genética , Epigénesis Genética/genética , Femenino , Células HeLa , Humanos , Metilación , Nucleosomas/genética , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
Dynamic shifts in transcription factor binding are central to the regulation of biological processes by allowing rapid changes in gene transcription. However, very few genome-wide studies have examined how transcription factor occupancy is coordinated temporally in vivo in higher animals. Here, we quantified the genome-wide binding patterns of two key hepatocyte transcription factors, CEBPA and CEBPB (also known as C/EBPalpha and C/EBPbeta), at multiple time points during the highly dynamic process of liver regeneration elicited by partial hepatectomy in mouse. Combining these profiles with RNA polymerase II binding data, we find three temporal classes of transcription factor binding to be associated with distinct sets of regulated genes involved in the acute phase response, metabolic/homeostatic functions, or cell cycle progression. Moreover, we demonstrate a previously unrecognized early phase of homeostatic gene expression prior to S-phase entry. By analyzing the three classes of CEBP bound regions, we uncovered mutually exclusive sets of sequence motifs, suggesting temporal codes of CEBP recruitment by differential cobinding with other factors. These findings were validated by sequential ChIP experiments involving a panel of central transcription factors and/or by comparison to external ChIP-seq data. Our quantitative investigation not only provides in vivo evidence for the involvement of many new factors in liver regeneration but also points to similarities in the circuitries regulating self-renewal of differentiated cells. Taken together, our work emphasizes the power of global temporal analyses of transcription factor occupancy to elucidate mechanisms regulating dynamic biological processes in complex higher organisms.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Genes cdc , Homeostasis/genética , Regeneración Hepática/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Sitios de Unión , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Inmunoprecipitación de Cromatina , Análisis por Conglomerados , Proteínas de Unión al ADN/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Regulación de la Expresión Génica , Masculino , Ratones , Anotación de Secuencia Molecular , Motivos de Nucleótidos , Transcripción GenéticaRESUMEN
Activation of interferon genes constitutes an important anticancer pathway able to restrict proliferation of cancer cells. Here, we demonstrate that the H3K9me3 histone methyltransferase (HMT) suppressor of variegation 3-9 homolog 1 (SUV39H1) is required for the proliferation of acute myeloid leukemia (AML) and find that its loss leads to activation of the interferon pathway. Mechanistically, we show that this occurs via destabilization of a complex composed of SUV39H1 and the two H3K9me2 HMTs, G9A and GLP. Indeed, loss of H3K9me2 correlated with the activation of key interferon pathway genes, and interference with the activities of G9A/GLP largely phenocopied loss of SUV39H1. Last, we demonstrate that inhibition of G9A/GLP synergized with DNA demethylating agents and that SUV39H1 constitutes a potential biomarker for the response to hypomethylation treatment. Collectively, we uncovered a clinically relevant role for H3K9me2 in safeguarding cancer cells against activation of the interferon pathway.
RESUMEN
Differentiation of multipotent stem cells into mature cells is fundamental for development and homeostasis of mammalian tissues, and requires the coordinated induction of lineage-specific transcriptional programs and cell cycle withdrawal. To understand the underlying regulatory mechanisms of this fundamental process, we investigated how the tissue-specific transcription factors, CEBPA and CEBPE, coordinate cell cycle exit and lineage-specification in vivo during granulocytic differentiation. We demonstrate that CEBPA promotes lineage-specification by launching an enhancer-primed differentiation program and direct activation of CEBPE expression. Subsequently, CEBPE confers promoter-driven cell cycle exit by sequential repression of MYC target gene expression at the G1/S transition and E2F-meditated G2/M gene expression, as well as by the up-regulation of Cdk1/2/4 inhibitors. Following cell cycle exit, CEBPE unleashes the CEBPA-primed differentiation program to generate mature granulocytes. These findings highlight how tissue-specific transcription factors coordinate cell cycle exit with differentiation through the use of distinct gene regulatory elements.
Asunto(s)
Regulación de la Expresión Génica , Factores de Transcripción , Animales , Ciclo Celular , Diferenciación Celular/genética , Granulocitos/metabolismo , Mamíferos/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Transcription factors PU.1 and CEBPA are required for the proper coordination of enhancer activity during granulocytic-monocytic (GM) lineage differentiation to form myeloid cells. However, precisely how these factors control the chronology of enhancer establishment during differentiation is not known. Through integrated analyses of enhancer dynamics, transcription factor binding, and proximal gene expression during successive stages of murine GM-lineage differentiation, we unravel the distinct kinetics by which PU.1 and CEBPA coordinate GM enhancer activity. We find no evidence of a pioneering function of PU.1 during late GM-lineage differentiation. Instead, we delineate a set of enhancers that gain accessibility in a CEBPA-dependent manner, suggesting a pioneering function of CEBPA. Analyses of Cebpa null bone marrow demonstrate that CEBPA controls PU.1 levels and, unexpectedly, that the loss of CEBPA results in an early differentiation block. Taken together, our data provide insights into how PU.1 and CEBPA functionally interact to drive GM-lineage differentiation.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/deficiencia , Diferenciación Celular/genética , Elementos de Facilitación Genéticos/genética , Células Mieloides/citología , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Animales , Secuencia de Bases , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Línea Celular , Linaje de la Célula , Cromatina/metabolismo , Femenino , Regulación de la Expresión Génica , Granulocitos/citología , Granulocitos/metabolismo , Ratones , Monocitos/citología , Monocitos/metabolismo , Células Mieloides/metabolismo , Unión ProteicaRESUMEN
Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) is typically inefficient and has been explained by elite-cell and stochastic models. We recently reported that B cells exposed to a pulse of C/EBPα (Bα' cells) behave as elite cells, in that they can be rapidly and efficiently reprogrammed into iPSCs by the Yamanaka factors OSKM. Here we show that C/EBPα post-transcriptionally increases the abundance of several hundred proteins, including Lsd1, Hdac1, Brd4, Med1 and Cdk9, components of chromatin-modifying complexes present at super-enhancers. Lsd1 was found to be required for B cell gene silencing and Brd4 for the activation of the pluripotency program. C/EBPα also promotes chromatin accessibility in pluripotent cells and upregulates Klf4 by binding to two haematopoietic enhancers. Bα' cells share many properties with granulocyte/macrophage progenitors, naturally occurring elite cells that are obligate targets for leukaemic transformation, whose formation strictly requires C/EBPα.
Asunto(s)
Proteína alfa Potenciadora de Unión a CCAAT/genética , Reprogramación Celular/genética , Histona Demetilasas/genética , Células Madre Pluripotentes Inducidas/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Linfocitos B/metabolismo , Western Blotting , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Línea Celular , Células Cultivadas , Femenino , Perfilación de la Expresión Génica/métodos , Ontología de Genes , Células HEK293 , Histona Demetilasas/metabolismo , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Nucleares/metabolismo , Proteómica/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Nonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2. RESULTS: We developed a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes are upregulated in NMD-deficient cells and that NMD targets all major splicing classes. Importantly, NMD-dependent effects are not restricted to premature termination codon+ isoforms but also involve an abundance of splicing events that do not generate premature termination codons. Supporting their functional importance, the latter events are associated with high intronic conservation. CONCLUSIONS: Our data demonstrate that NMD regulates alternative splicing outcomes through an intricate web of splicing regulators and that its loss leads to the deregulation of a panoply of splicing events, providing novel insights into its role in core- and tissue-specific regulation of gene expression. Thus, our study extends the importance of NMD from an mRNA quality pathway to a regulator of several layers of gene expression.