RESUMO
Sperm histones represent an essential part of the paternally transmitted epigenome, but uncertainty exists about the role of those remaining in non-coding and repetitive DNA. We therefore analyzed the genome-wide distribution of the heterochromatic marker H4K20me3 in human sperm and somatic (K562) cells. To specify the function of sperm histones, we compared all H4K20me3-containing and -free loci in the sperm genome. Sperm and somatic cells possessed a very similar H4K20me3 distribution: H4K20me3 peaks occurred mostly in distal intergenic regions and repetitive gene clusters (in particular genes encoding odorant-binding factors and zinc-finger antiviral proteins). In both cell types, H4K20me3 peaks were enriched in LINEs, ERVs, satellite DNA and low complexity repeats. In contrast, H4K20me3-free nucleosomes occurred more frequently in genic regions (in particular promoters, exons, 5'-UTR and 3'-UTR) and were enriched in genes encoding developmental factors (in particular transcription activators and repressors). H4K20me3-free nucleosomes were also detected in substantial quantities in distal intergenic regions and were enriched in SINEs. Thus, evidence suggests that paternally transmitted histones may have a dual purpose: maintenance and regulation of heterochromatin and guidance towards transcription of euchromatin.
Assuntos
Histonas/genética , Sequências Repetitivas de Ácido Nucleico/genética , Espermatozoides/fisiologia , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Linhagem Celular Tumoral , DNA/genética , Éxons/genética , Genoma/genética , Heterocromatina/genética , Humanos , Células K562 , Masculino , Nucleossomos/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
Klinefelter syndrome (KS), with an incidence between 1/600 and 1/1,000, is the main genetic cause of male infertility. Due to the lack of an accurate study model, the detailed pathogenic mechanisms by which this X chromosome aneuploidy leads to KS features remain unknown. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from a patient with KS: 47XXY-iPSCs. In order to compare the potentials of both 47XXY-iPSCs and 46XY-iPSCs to differentiate into the germ cell lineage, we developed a directed differentiation protocol by testing different combinations of factors including bone morphogenetic protein 4 (BMP4), glial-derived neurotrophic factor (GDNF), retinoic acid (RA) and stem cell factor (SCF) for 42 days. Importantly, we found a reduced ability of 47XXY-iPSCs to differentiate into germ cells when compared to 46XY-iPSCs. In particular, upon germ cell differentiation of 47XXY-iPSCs, we found a reduced proportion of cells positive for BOLL, a protein required for germ cell development and spermatogenesis, as well as a reduced proportion of cells positive for MAGEA4, a spermatogonia marker. This reduced ability to generate germ cells was not associated with a decrease of proliferation of 47XXY-iPSC-derived cells but rather with an increase of cell death upon germ cell differentiation as revealed by an increase of LDH release and of capase-3 expression in 47XXY-iPSC-derived cells. Our study supports the idea that 47XXY-iPSCs provides an excellent in vitro model to unravel the pathophysiology and to design potential treatments for KS patients.
RESUMO
One of the primary aims of the Functional Annotation of ANimal Genomes (FAANG) initiative is to characterize tissue-specific regulation within animal genomes. To this end, we used chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map four histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) in eight prioritized tissues collected as part of the FAANG equine biobank from two thoroughbred mares. Data were generated according to optimized experimental parameters developed during quality control testing. To ensure that we obtained sufficient ChIP and successful peak-calling, data and peak-calls were assessed using six quality metrics, replicate comparisons, and site-specific evaluations. Tissue specificity was explored by identifying binding motifs within unique active regions, and motifs were further characterized by gene ontology (GO) and protein-protein interaction analyses. The histone marks identified in this study represent some of the first resources for tissue-specific regulation within the equine genome. As such, these publicly available annotation data can be used to advance equine studies investigating health, performance, reproduction, and other traits of economic interest in the horse.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Elementos Reguladores de Transcrição , Análise de Sequência de DNA/métodos , Animais , Genoma , Código das Histonas , Cavalos , Anotação de Sequência Molecular , Especificidade de ÓrgãosRESUMO
Accumulating evidence support a causal link between Zika virus (ZIKV) infection during gestation and congenital microcephaly. However, the mechanism of ZIKV-associated microcephaly remains unclear. We combined analyses of ZIKV-infected human fetuses, cultured human neural stem cells and mouse embryos to understand how ZIKV induces microcephaly. We show that ZIKV triggers endoplasmic reticulum stress and unfolded protein response in the cerebral cortex of infected postmortem human fetuses as well as in cultured human neural stem cells. After intracerebral and intraplacental inoculation of ZIKV in mouse embryos, we show that it triggers endoplasmic reticulum stress in embryonic brains in vivo. This perturbs a physiological unfolded protein response within cortical progenitors that controls neurogenesis. Thus, ZIKV-infected progenitors generate fewer projection neurons that eventually settle in the cerebral cortex, whereupon sustained endoplasmic reticulum stress leads to apoptosis. Furthermore, we demonstrate that administration of pharmacological inhibitors of unfolded protein response counteracts these pathophysiological mechanisms and prevents microcephaly in ZIKV-infected mouse embryos. Such defects are specific to ZIKV, as they are not observed upon intraplacental injection of other related flaviviruses in mice.
Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Microcefalia/etiologia , Microcefalia/metabolismo , Desdobramento de Proteína , Infecção por Zika virus/complicações , Zika virus/patogenicidade , Fator 3 Ativador da Transcrição/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Embrião de Mamíferos , Feto , Regulação Viral da Expressão Gênica , Humanos , Interferon-alfa/genética , Interferon-alfa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microcefalia/prevenção & controle , Microcefalia/virologia , Proteínas do Tecido Nervoso/metabolismo , Neuroblastoma/patologia , Infecção por Zika virus/patologiaAssuntos
Córtex Cerebral/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Neurogênese/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Animais , Córtex Cerebral/citologia , Retículo Endoplasmático/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/genética , Neurônios/metabolismo , Neurônios/fisiologia , Estresse Fisiológico/fisiologiaRESUMO
The unfolded protein response (UPR) is a homeostatic signaling pathway triggered by protein misfolding in the endoplasmic reticulum (ER). Beyond its protective role, it plays important functions during normal development in response to elevated demand for protein folding. Several UPR effectors show dynamic temporal and spatial expression patterns that correlate with milestones of the central nervous system (CNS) development. Here, we discuss recent studies suggesting that a dynamic regulation of UPR supports generation, maturation, and maintenance of differentiated neurons in the CNS. We further highlight studies supporting a developmental vulnerability of CNS to UPR dysregulation, which underlies neurodevelopmental disorders. We believe that a better understanding of UPR functions may provide novel opportunities for therapeutic strategies to fight ER/UPR-associated human neurological disorders.
Assuntos
Homeostase/fisiologia , Sistema Nervoso/metabolismo , Dobramento de Proteína , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , Animais , Retículo Endoplasmático/metabolismo , HumanosRESUMO
Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, notably in coding sequences, and identify consensus sites for hydroxymethylation. We found that RNA hydroxymethylation can favor mRNA translation. Tet and hydroxymethylated RNA are found to be most abundant in the Drosophila brain, and Tet-deficient fruitflies suffer impaired brain development, accompanied by decreased RNA hydroxymethylation. This study highlights the distribution, localization, and function of cytosine hydroxymethylation and identifies central roles for this modification in Drosophila.
Assuntos
Encéfalo/anormalidades , Citosina/análogos & derivados , Drosophila melanogaster/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , 5-Metilcitosina/análogos & derivados , Animais , Encéfalo/metabolismo , Linhagem Celular , Citosina/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metilação , RNA Mensageiro/genética , TranscriptomaRESUMO
The cerebral cortex contains layers of neurons sequentially generated by distinct lineage-related progenitors. At the onset of corticogenesis, the first-born progenitors are apical progenitors (APs), whose asymmetric division gives birth directly to neurons. Later, they switch to indirect neurogenesis by generating intermediate progenitors (IPs), which give rise to projection neurons of all cortical layers. While a direct lineage relationship between APs and IPs has been established, the molecular mechanism that controls their transition remains elusive. Here we show that interfering with codon translation speed triggers ER stress and the unfolded protein response (UPR), further impairing the generation of IPs and leading to microcephaly. Moreover, we demonstrate that a progressive downregulation of UPR in cortical progenitors acts as a physiological signal to amplify IPs and promotes indirect neurogenesis. Thus, our findings reveal a contribution of UPR to cell fate acquisition during mammalian brain development.
Assuntos
Córtex Cerebral/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Histona Acetiltransferases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Resposta a Proteínas não Dobradas , Animais , Linhagem da Célula , Separação Celular , Córtex Cerebral/metabolismo , Códon , Drosophila melanogaster , Células-Tronco Embrionárias/citologia , Deleção de Genes , Genótipo , Histona Acetiltransferases/genética , Humanos , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Fosforilação , Biossíntese de Proteínas , Desnaturação Proteica , Dobramento de Proteína , Transdução de Sinais , Células-Tronco/citologia , Regulação para CimaRESUMO
The TET enzymes convert methylcytosine to the newly discovered base hydroxymethylcytosine. While recent reports suggest that TETs may play a role in response to oxidative stress, this role remains uncertain, and results lack in vivo models. Here we show a global decrease of hydroxymethylcytosine in cells treated with buthionine sulfoximine, and in mice depleted for the major antioxidant enzymes GPx1 and 2. Furthermore, genome-wide profiling revealed differentially hydroxymethylated regions in coding genes, and intriguingly in microRNA genes, both involved in response to oxidative stress. These results thus suggest a profound effect of in vivo oxidative stress on the global hydroxymethylome.
Assuntos
5-Metilcitosina/metabolismo , Proteínas de Ligação a DNA/genética , Genoma , MicroRNAs/genética , Neurônios/metabolismo , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/análogos & derivados , Animais , Antimetabólitos/farmacologia , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Glutationa/antagonistas & inibidores , Glutationa/biossíntese , Glutationa Peroxidase/deficiência , Glutationa Peroxidase/genética , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Estresse Oxidativo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Glutationa Peroxidase GPX1RESUMO
Polycomb proteins play an essential role in maintaining the repression of developmental genes in self-renewing embryonic stem cells. The exact mechanism allowing the derepression of polycomb target genes during cell differentiation remains unclear. Our project aimed to identify Cbx8 binding sites in differentiating mouse embryonic stem cells. Therefore, we used a genome-wide chromatin immunoprecipitation of endogenous Cbx8 coupled to direct massive parallel sequencing (ChIP-Seq). Our analysis identified 171 high confidence peaks. By crossing our data with previously published microarray analysis, we show that several differentiation genes transiently recruit Cbx8 during their early activation. Depletion of Cbx8 partially impairs the transcriptional activation of these genes. Both interaction analysis, as well as chromatin immunoprecipitation experiments support the idea that activating Cbx8 acts in the context of an intact PRC1 complex. Prolonged gene activation results in eviction of PRC1 despite persisting H3K27me3 and H2A ubiquitination. The composition of PRC1 is highly modular and changes when embryonic stem cells commit to differentiation. We further demonstrate that the exchange of Cbx7 for Cbx8 is required for the effective activation of differentiation genes. Taken together, our results establish a function for a Cbx8-containing complex in facilitating the transition from a Polycomb-repressed chromatin state to an active state. As this affects several key regulatory differentiation genes this mechanism is likely to contribute to the robust execution of differentiation programs.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Proteínas do Grupo Polycomb/genética , Ativação Transcricional , Animais , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Células-Tronco Embrionárias/citologia , Deleção de Genes , Histonas/genética , Histonas/metabolismo , Camundongos , Proteínas de Transporte da Membrana Mitocondrial , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Ligação Proteica , Proteômica , Ubiquitinas/genética , Ubiquitinas/metabolismo , Regulação para CimaRESUMO
The importance of epigenetic mechanisms is most clearly illustrated during early development when a totipotent cell goes through multiple cell fate transitions to form the many different cell types and tissues that constitute the embryo and the adult. The exchange of a canonical H2A histone for the 'repressive' macroH2A variant is one of the most striking epigenetic chromatin alterations that can occur at the level of the nucleosome. Here, we discuss recent data on macroH2A in zebrafish and mouse embryos, in embryonic and adult stem cells and also in nuclear reprogramming. We highlight the role of macroH2A in the establishment and maintenance of differentiated states and we discuss its still poorly recognized function in transcriptional activation.
Assuntos
Histonas/genética , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Reprogramação Celular , Cromatina/metabolismo , Desenvolvimento Embrionário , Epigênese Genética , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Camundongos , Mutação , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Células-Tronco/citologia , Peixe-ZebraRESUMO
One of the most striking epigenetic alterations that occurs at the level of the nucleosome is the complete exchange of the canonical H2A histones for the macroH2A variant. Here, we provide insight into the poorly recognized function of macroH2A in transcriptional activation and demonstrate its relevance in embryonic and adult stem cells. Knockdown of macroH2A1 in mouse embryonic stem (mES) cells limited their capacity to differentiate but not their self-renewal. The loss of macroH2A1 interfered with the proper activation of differentiation genes, most of which are direct target genes of macroH2A. Additionally, macroH2A1-deficient mES cells displayed incomplete inactivation of pluripotency genes and formed defective embryoid bodies. In vivo, macroH2A1-deficient teratomas contained a massive expansion of malignant, undifferentiated carcinoma tissue. In the heterogeneous culture of primary human keratinocytes, macroH2A1 levels negatively correlated with the self-renewal capacity of the pluripotent compartment. Together these results establish macroH2A1 as a critical chromatin component that regulates the delicate balance between self-renewal and differentiation of embryonic and adult stem cells.
Assuntos
Células-Tronco Adultas/citologia , Diferenciação Celular/fisiologia , Proliferação de Células , Células-Tronco Embrionárias/citologia , Histonas/fisiologia , Células-Tronco Adultas/fisiologia , Animais , Cromatina/fisiologia , Corpos Embrioides/metabolismo , Corpos Embrioides/patologia , Células-Tronco Embrionárias/fisiologia , Humanos , Queratinócitos/citologia , Queratinócitos/fisiologia , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Teratoma/metabolismo , Teratoma/patologiaRESUMO
Elongator is an evolutionary highly conserved complex. At least two of its cellular functions rely on the intrinsic lysine acetyl-transferase activity of the elongator complex. Its two known substrates--histone H3 and α-tubulin--reflect the different roles of elongator in the cytosol and the nucleus. A picture seems to emerge in which nuclear elongator could regulate the transcriptional elongation of a subset of stress-inducible genes through acetylation of histone H3 in the promoter-distal gene body. In the cytosol, elongator-mediated acetylation of α-tubulin contributes to intracellular trafficking and cell migration. Defects in both functions of elongator have been implicated in neurodegenerative disorders.
Assuntos
Movimento Celular/fisiologia , Histona Acetiltransferases/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Transcrição Gênica , Acetilação , Animais , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Tubulina (Proteína)/metabolismoAssuntos
Proteínas de Transporte/fisiologia , Córtex Cerebral/citologia , Disautonomia Familiar/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Acetilação , Animais , Proteínas de Transporte/genética , Movimento Celular , Disautonomia Familiar/fisiopatologia , Histona Acetiltransferases/fisiologia , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Complexos Multiproteicos , Neurogênese/genética , Processamento de Proteína Pós-Traducional , Transporte Proteico , RNA Mensageiro/biossíntese , Transdução de Sinais , Fatores de Elongação da Transcrição , Tubulina (Proteína)/metabolismoRESUMO
Lysine acetylation is a post-translational modification that critically regulates gene transcription by targeting histones as well as a variety of transcription factors in the nucleus. More recent reports have also demonstrated that numerous proteins located outside the nucleus are also acetylated and that this modification has profound consequences on their functions. This review describes the latest findings on the substrates acetylated outside the nucleus and on the acetylases and deacetylates that catalyse these modifications. Protein acetylation is emerging as a major mechanism by which key proteins are regulated in many physiological processes such as migration, metabolism and aging as well as in pathological circumstances such as cancer and neurodegenerative disorders.
Assuntos
Lisina/metabolismo , Proteínas/metabolismo , Acetilação , Animais , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
The generation of cortical projection neurons relies on the coordination of radial migration with branching. Here, we report that the multisubunit histone acetyltransferase Elongator complex, which contributes to transcript elongation, also regulates the maturation of projection neurons. Indeed, silencing of its scaffold (Elp1) or catalytic subunit (Elp3) cell-autonomously delays the migration and impairs the branching of projection neurons. Strikingly, neurons defective in Elongator show reduced levels of acetylated alpha-tubulin. Reduction of alpha-tubulin acetylation via expression of a nonacetylatable alpha-tubulin mutant leads to comparable defects in cortical neurons and suggests that alpha-tubulin is a target of Elp3. This is further supported by the demonstration that Elp3 promotes acetylation and counteracts HDAC6-mediated deacetylation of this substrate in vitro. Our results uncover alpha-tubulin as a target of the Elongator complex and suggest that a tight regulation of its acetylation underlies the maturation of cortical projection neurons.
Assuntos
Movimento Celular , Córtex Cerebral/citologia , Histona Acetiltransferases/metabolismo , Neurônios/citologia , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Linhagem Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Camundongos , Complexos Multienzimáticos/metabolismo , NeurogêneseRESUMO
Elongator, a multi-subunit complex assembled by the IkappaB kinase-associated protein (IKAP)/hELP1 scaffold protein is involved in transcriptional elongation in the nucleus as well as in tRNA modifications in the cytoplasm. However, the biological processes regulated by Elongator in human cells only start to be elucidated. Here we demonstrate that IKAP/hELP1 depleted colon cancer-derived cells show enhanced basal expression of some but not all pro-apoptotic p53-dependent genes such as BAX. Moreover, Elongator deficiency causes increased basal and daunomycin-induced expression of the pro-survival serum- and glucocorticoid-induced protein kinase (SGK) gene through a p53-dependent pathway. Thus, our data collectively demonstrate that Elongator deficiency triggers the activation of p53-dependent genes harbouring opposite functions with respect to apoptosis.
Assuntos
Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica , Complexos Multiproteicos/deficiência , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/farmacologia , Apoptose/fisiologia , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Dano ao DNA , Daunorrubicina/farmacologia , Fibroblastos/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Elongação da Transcrição , Proteína Supressora de Tumor p53/genéticaRESUMO
Mutations in IKBKAP, encoding a subunit of Elongator, cause familial dysautonomia (FD), a severe neurodevelopmental disease with complex clinical characteristics. Elongator was previously linked not only with transcriptional elongation and histone acetylation but also with other cellular processes. Here, we used RNA interference (RNAi) and fibroblasts from FD patients to identify Elongator target genes and study the role of Elongator in transcription. Strikingly, whereas Elongator is recruited to both target and nontarget genes, only target genes display histone H3 hypoacetylation and progressively lower RNAPII density through the coding region in FD cells. Interestingly, several target genes encode proteins implicated in cell motility. Indeed, characterization of IKAP/hELP1 RNAi cells, FD fibroblasts, and neuronal cell-derived cells uncovered defects in this cellular function upon Elongator depletion. These results indicate that defects in Elongator function affect transcriptional elongation of several genes and that the ensuing cell motility deficiencies may underlie the neuropathology of FD patients.