RESUMO
Transposable elements (TEs) occupy nearly 40% of mammalian genomes and, whilst most are fragmentary and no longer capable of transposition, they can nevertheless contribute to cell function. TEs within genes transcribed by RNA polymerase II can be copied as parts of primary transcripts; however, their full contribution to mature transcript sequences remains unresolved. Here, using long and short read (LR and SR) RNA sequencing data, we show that 26% of coding and 65% of noncoding transcripts in human pluripotent stem cells (hPSCs) contain TE-derived sequences. Different TE families are incorporated into RNAs in unique patterns, with consequences to transcript structure and function. The presence of TE sequences within a transcript is correlated with TE-type specific changes in its subcellular distribution, alterations in steady-state levels and half-life, and differential association with RNA Binding Proteins (RBPs). We identify hPSC-specific incorporation of endogenous retroviruses (ERVs) and LINE:L1 into protein-coding mRNAs, which generate TE sequence-derived peptides. Finally, single cell RNA-seq reveals that hPSCs express ERV-containing transcripts, whilst differentiating subpopulations lack ERVs and express SINE and LINE-containing transcripts. Overall, our comprehensive analysis demonstrates that the incorporation of TE sequences into the RNAs of hPSCs is more widespread and has a greater impact than previously appreciated.
Assuntos
Retrovirus Endógenos/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Células-Tronco Pluripotentes/metabolismo , Transcriptoma , Linhagem Celular , Humanos , RNA não Traduzido/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Polycomb group (PcG) proteins maintain cell identity by repressing gene expression during development. Surprisingly, emerging studies have recently reported that a number of PcG proteins directly activate gene expression during cell fate determination process. However, the mechanisms by which they direct gene activation in pluripotency remain poorly understood. Here, we show that Phc1, a subunit of canonical polycomb repressive complex 1 (cPRC1), can exert its function in pluripotency maintenance via a PRC1-independent activation of Nanog. Ablation of Phc1 reduces the expression of Nanog and overexpression of Nanog partially rescues impaired pluripotency caused by Phc1 depletion. We find that Phc1 interacts with Nanog and activates Nanog transcription by stabilizing the genome-wide chromatin interactions of the Nanog locus. This adds to the already known canonical function of PRC1 in pluripotency maintenance via a PRC1-dependent repression of differentiation genes. Overall, our study reveals a function of Phc1 to activate Nanog transcription through regulating chromatin architecture and proposes a paradigm for PcG proteins to maintain pluripotency.
Assuntos
Cromatina/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteína Homeobox Nanog/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/fisiologia , Animais , Células Cultivadas , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Genoma Humano , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Genéticos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/fisiologia , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/deficiênciaRESUMO
Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that ß-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by ß-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.
RESUMO
The majority of mammalian genomes are devoted to transposable elements (TEs). Whilst TEs are increasingly recognized for their important biological functions, they are a potential danger to genomic stability and are carefully regulated by the epigenetic system. However, the full complexity of this regulatory system is not understood. Here, using mouse embryonic stem cells, we show that TEs are suppressed by heterochromatic marks like H3K9me3, and are also labelled by all major types of chromatin modification in complex patterns, including bivalent activatory and repressive marks. We identified 29 epigenetic modifiers that significantly deregulated at least one type of TE. The loss of Setdb1, Ncor2, Rnf2, Kat5, Prmt5, Uhrf1, and Rrp8 caused widespread changes in TE expression and chromatin accessibility. These effects were context-specific, with different chromatin modifiers regulating the expression and chromatin accessibility of specific subsets of TEs. Our work reveals the complex patterns of epigenetic regulation of TEs.
Assuntos
Cromatina/metabolismo , Elementos de DNA Transponíveis/genética , Epigênese Genética , Histonas/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Metilação de DNA/genética , Técnicas de Silenciamento de Genes , Código das Histonas , Histonas/genética , Camundongos , Células-Tronco Embrionárias MurinasRESUMO
Familial Parkinson's disease (PD) can be caused by deleterious mutations in PINK1 (encoding PINK1) in an autosomal recessive manner. Functional studies suggest that PINK1 works as a regulator of mitochondrial homeostasis. However, how loss of PINK1 induces dopaminergic neuron degeneration is still unclear. Here, we have generated a patient-derived induced pluripotent stem cell (iPSC) line with mutant PINK1 (p. I368N). This cell line will facilitate PD disease modeling in vitro and can be used for generating isogenic cell lines through gene correction.
Assuntos
Diferenciação Celular , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Mutação , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteínas Quinases/genética , Células Cultivadas , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
In the version of this Article originally published, in Fig. 2c, the '+' sign and 'OSKM' were superimposed in the label '+OSKM'. In Fig. 4e, in the labels, all instances of 'Ant' should have been 'Anti-'. And, in Fig. 7a, the label '0.0' was misplaced; it should have been on the colour scale bar. These figures have now been corrected in the online versions.
RESUMO
Somatic cell reprogramming by exogenous factors requires cooperation with transcriptional co-activators and co-repressors to effectively remodel the epigenetic environment. How this interplay is regulated remains poorly understood. Here, we demonstrate that NCoR/SMRT co-repressors bind to pluripotency loci to create a barrier to reprogramming with the four Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC), and consequently, suppressing NCoR/SMRT significantly enhances reprogramming efficiency and kinetics. The core epigenetic subunit of the NCoR/SMRT complex, histone deacetylase 3 (HDAC3), contributes to the effects of NCoR/SMRT by inducing histone deacetylation at pluripotency loci. Among the Yamanaka factors, recruitment of NCoR/SMRT-HDAC3 to genomic loci is mostly facilitated by c-MYC. Hence, we describe how c-MYC is beneficial for the early phase of reprogramming but deleterious later. Overall, we uncover a role for NCoR/SMRT co-repressors in reprogramming and propose a dual function for c-MYC in this process.
Assuntos
Reprogramação Celular , Epigênese Genética , Células-Tronco Embrionárias Murinas/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Acetilação , Animais , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Correpressor 1 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
The binding of losartan potassium, an angiotensin II receptor antagonist, to bovine serum albumin wasstudied by equilibrium dialysis method (ED) in presence or absence of palmitic acid. The study wascarried out using ranitidine and diazepam as site-1 and site-2 specific probe, respectively. Differentanalysis of binding of losartan to bovine serum albumin suggested two sets of association constants:high affinity association constant (k1 = 11.2 x 105 M-1) with low capacity (n1 = 2) and low affinityassociation (k2 = 2. 63 x 105 M-1) constant with high capacity (n2 = 10) at pH 7.4 and 27°C. Duringconcurrent administration of palmitic acid and losartan potassium in presence or absence of ranitidineor diazepam, it was that found that palmitic acid causes the release of losartan potassium from itsbinding site on BSA resulting reduced binding of losartan potassium to BSA. The increment in freefraction of losartan potassium was from 13.1% to 47.2 % upon the addition of increased concentrationof only palmitic acid at a concentration of 0 x 10-5 M to 16 x 10-5 M. In presence of ranitidine ordiazepam as site specific probes, palmitic acid further increases the free fraction of losartan potassiumwere from 22.8% to 53.4% and 35.3 to 65.5%, respectively. This data provided the evidence ofinteraction of higher concentration of palmitic acid at the binding sites on BSA changing thepharmacokinetics properties of losartan potassium(AU)