RESUMEN
The heterochromatin protein 1 (HP1) family members are canonical effectors and propagators of gene repression mediated by histone H3 lysine 9 (H3K9) methylation. HP1γ exhibits an increased interaction with active transcription elongation-associated factors in embryonic stem cells (ESCs) compared to somatic cells. However, whether this association has a functional consequence remains elusive. Here we find that genic HP1γ colocalizes and enhances enrichment of transcription elongation-associated H3K36me3 rather than H3K9me3. Unexpectedly, sustained H3K36me3 deposition is dependent on HP1γ. HP1γ-deleted ESCs display reduced H3K36me3 enrichment, concomitant with decreased expression at shared genes which function to maintain cellular homeostasis. Both the H3K9me3-binding chromodomain and histone binding ability of HP1γ are dispensable for maintaining H3K36me3 levels. Instead, the chromoshadow together with the hinge domain of HP1γ that confer protein and nucleic acid-binding ability are sufficient because they retain the ability to interact with NSD1, an H3K36 methyltransferase. HP1γ-deleted ESCs have a slower self-renewal rate and an impaired ability to differentiate towards cardiac mesoderm. Our findings reveal a requirement for HP1γ in faithful establishment of transcription elongation in ESCs, which regulates pluripotency.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Células Madre Embrionarias/citología , N-Metiltransferasa de Histona-Lisina/genética , Células Madre Pluripotentes/citología , Diferenciación Celular/genética , Línea Celular , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Histonas/genética , Humanos , Metilación , Fosforilación/genética , Células Madre Pluripotentes/metabolismo , Unión Proteica/genética , Procesamiento Proteico-Postraduccional/genética , Factores de Transcripción/genéticaRESUMEN
The centromere is the chromosomal locus where the kinetochore forms and is critical for ensuring proper segregation of sister chromatids during cell division. A substantial amount of effort has been devoted to understanding the characteristic features and roles of the centromere, yet some fundamental aspects of the centromere, such as the complete list of elements that define it, remain obscure. It is well-known that human centromeres include a highly repetitive class of DNA known as alpha satellite, or alphoid, DNA. We present here the first DNA-centric examination of human protein-alpha satellite interactions, employing an approach known as HyCCAPP (hybridization capture of chromatin-associated proteins for proteomics) to identify the protein components of alphoid chromatin in a human cell line. Using HyCCAPP, cross-linked alpha satellite chromatin was isolated from cell lysate, and captured proteins were analyzed via mass spectrometry. After being compared to proteins identified in control pulldown experiments, 90 proteins were identified as enriched at alphoid DNA. This list included many known centromere-binding proteins in addition to multiple novel alpha satellite-binding proteins, such as LRIF1, a heterochromatin-associated protein. The ability of HyCCAPP to reveal both known as well as novel alphoid DNA-interacting proteins highlights the validity and utility of this approach.
Asunto(s)
Centrómero/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Hibridación Fluorescente in Situ/métodos , Anticuerpos Monoclonales/química , Centrómero/ultraestructura , Proteína B del Centrómero/genética , Proteína B del Centrómero/metabolismo , Cromatina/ultraestructura , Inmunoprecipitación de Cromatina , ADN/genética , Proteínas de Unión al ADN/genética , Expresión Génica , Humanos , Células K562 , Espectrometría de Masas/métodosRESUMEN
Helicobacter pylori is the main bacterial causative agent of gastroduodenal disorders and a risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. The draft genomes of 10 closely related H. pylori isolates from the multiracial Malaysian population will provide an insight into the genetic diversity of isolates in Southeast Asia. These isolates were cultured from gastric biopsy samples from patients with functional dyspepsia and gastric cancer. The availability of this genomic information will provide an opportunity for examining the evolution and population structure of H. pylori isolates from Southeast Asia, where the East meets the West.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/genética , Genoma Bacteriano , Helicobacter pylori/genética , Análisis de Secuencia de ADN , Biopsia , Dispepsia/microbiología , Infecciones por Helicobacter/microbiología , Helicobacter pylori/aislamiento & purificación , Humanos , Malasia , Datos de Secuencia Molecular , Neoplasias Gástricas/microbiologíaRESUMEN
Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a combination of epigenomic modifiers and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress toward iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenomic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.
Asunto(s)
Puntos de Control del Ciclo Celular , Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Análisis de la Célula Individual , Animales , Puntos de Control del Ciclo Celular/genética , Reprogramación Celular/genética , Regulación hacia Abajo/genética , Epigenómica , Epitelio/metabolismo , Femenino , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Mesodermo/citología , Ratones Endogámicos C57BL , Modelos Biológicos , Transducción de Señal , Regulación hacia Arriba/genéticaRESUMEN
The heterochromatin protein 1 (HP1) family is involved in various functions with maintenance of chromatin structure. During murine somatic cell reprogramming, we find that early depletion of HP1γ reduces the generation of induced pluripotent stem cells, while late depletion enhances the process, with a concomitant change from a centromeric to nucleoplasmic localization and elongation-associated histone H3.3 enrichment. Depletion of heterochromatin anchoring protein SENP7 increased reprogramming efficiency to a similar extent as HP1γ, indicating the importance of HP1γ release from chromatin for pluripotency acquisition. HP1γ interacted with OCT4 and DPPA4 in HP1α and HP1ß knockouts and in H3K9 methylation depleted H3K9M embryonic stem cell (ESC) lines. HP1α and HP1γ complexes in ESCs differed in association with histones, the histone chaperone CAF1 complex, and specific components of chromatin-modifying complexes such as DPY30, implying distinct functional contributions. Taken together, our results reveal the complex contribution of the HP1 proteins to pluripotency.
Asunto(s)
Reprogramación Celular/genética , Cromatina/genética , Células Madre Pluripotentes Inducidas/química , Complejos Multiproteicos/genética , Animales , Cromatina/química , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Endopeptidasas/química , Endopeptidasas/genética , Exorribonucleasas , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Ratones , Ratones Noqueados , Complejos Multiproteicos/química , Proteínas Nucleares/genética , Factor 3 de Transcripción de Unión a Octámeros/química , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteínas/química , Proteínas/genética , Proteínas Represoras , Ribonucleasas , Factores de TranscripciónRESUMEN
During the reprogramming of mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells, the activation of pluripotency genes such as NANOG occurs after the mesenchymal to epithelial transition. Here we report that both adult stem cells (neural stem cells) and differentiated cells (astrocytes) of the neural lineage can activate NANOG in the absence of cadherin expression during reprogramming. Gene expression analysis revealed that only the NANOG+E-cadherin+ populations expressed stabilization markers, had upregulated several cell cycle genes; and were transgene independent. Inhibition of DOT1L activity enhanced both the numbers of NANOG+ and NANOG+E-cadherin+ colonies in neural stem cells. Expressing SOX2 in MEFs prior to reprogramming did not alter the ratio of NANOG colonies that express E-cadherin. Taken together these results provide a unique pathway for reprogramming taken by cells of the neural lineage.
Asunto(s)
Linaje de la Célula , Reprogramación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Células Cultivadas , N-Metiltransferasa de Histona-Lisina , Células Madre Pluripotentes Inducidas/citología , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Proteína Homeótica Nanog/metabolismo , Células-Madre Neurales/citología , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
Reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) represents a profound change in cell fate. Here, we show that combining ascorbic acid (AA) and 2i (MAP kinase and GSK inhibitors) increases the efficiency of reprogramming from fibroblasts and synergistically enhances conversion of partially reprogrammed intermediates to the iPSC state. AA and 2i induce differential transcriptional responses, each leading to the activation of specific pluripotency loci. A unique cohort of pluripotency genes including Esrrb require both stimuli for activation. Temporally, AA-dependent histone demethylase effects are important early, whereas Tet enzyme effects are required throughout the conversion. 2i function could partially be replaced by depletion of components of the epidermal growth factor (EGF) and insulin growth factor pathways, indicating that they act as barriers to reprogramming. Accordingly, reduction in the levels of the EGF receptor gene contributes to the activation of Esrrb. These results provide insight into the rewiring of the pluripotency network at the late stage of reprogramming.