RESUMO
An efficient, generalizable method for genome-wide mapping of single-cell histone modifications or chromatin-binding proteins is lacking. Here, we develop CoBATCH, combinatorial barcoding and targeted chromatin release, for single-cell profiling of genomic distribution of chromatin-binding proteins in cell culture and tissue. Protein A in fusion to Tn5 transposase is enriched through specific antibodies to genomic regions, and Tn5 generates indexed chromatin fragments ready for library preparation and sequencing. Importantly, this strategy enables not only low-input epigenomic profiling in intact tissues but also measures scalable up to tens of thousands of single cells per experiment under both native and cross-linked conditions. CoBATCH produces â¼12,000 reads/cell with extremely low background. Mapping of endothelial cell lineages from ten embryonic mouse organs through CoBATCH allows for efficient deciphering of epigenetic heterogeneity of cell populations and cis-regulatory mechanisms. Thus, obviating specialized devices, CoBATCH is broadly applicable and easily deployable for single-cell profiling of protein-DNA interactions.
Assuntos
Cromatina/genética , Epigenoma , Epigenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Acetilação , Animais , Linhagem Celular , Cromatina/metabolismo , Histonas/metabolismo , Metilação , Camundongos , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
Deciphering mechanisms in cell-fate decisions requires single-cell holistic reconstructions of multidimensional epigenomic states in transcriptional regulation. Here we develop CoTECH, a combinatorial barcoding method allowing high-throughput single-cell joint detection of chromatin occupancy and transcriptome. We used CoTECH to examine bivalent histone marks (H3K4me3 and H3K27me3) with transcription from naive to primed mouse embryonic stem cells. We also derived concurrent bivalent marks in pseudosingle cells using transcriptome as an anchor for resolving pseudotemporal bivalency trajectories and disentangling a context-specific interplay between H3K4me3/H3K27me3 and transcription level. Next, we revealed the regulatory basis of endothelial-to-hematopoietic transition in two waves of hematopoietic cells and distinctive enhancer-gene-linking schemes guiding hemogenic endothelial cell emergence, indicating a unique epigenetic control of transcriptional regulation for hematopoietic stem cell priming. CoTECH provides an efficient framework for single-cell coassay of chromatin occupancy and transcription, thus enabling higher-dimensional epigenomic reconstructions.
Assuntos
Cromatina/metabolismo , Epigenômica , Análise de Célula Única/métodos , Transcriptoma , Animais , DNA/genética , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Células NIH 3T3 , RNA/genéticaRESUMO
RATIONALE: Replication-independent histone turnover has been linked to cis-regulatory chromatin domains in cultured cell lines, but its molecular underpinnings and functional relevance in adult mammalian tissues remain yet to be defined. OBJECTIVE: We investigated regulatory functions of replication-independent histone turnover in chromatin states of postmitotic cardiomyocytes from adult mouse heart. METHODS AND RESULTS: We used H2B-GFP (histone 2B-green fluorescent protein) fusion protein pulse-and-chase approaches to measure histone turnover rate in mouse cardiomyocytes. Surprisingly, we found that the short histone half-life (≈2 weeks) contrasted dramatically with the long lifetime of cardiomyocytes, and rapid histone turnover regions corresponded to cis-regulatory domains of heart genes. Interestingly, recruitment of chromatin modifiers, including Polycomb EED (embryonic ectoderm development), was positively correlated with histone turnover rate at enhancers. Mechanistically, through directly interacting with and engaging the BAF (BRG1 [Brahma-related gene-1]-associated factor) complex for nucleosome exchange for stereotyped histone modifications from the free histone pool, EED augmented histone turnover to restrain enhancer overactivation. CONCLUSIONS: We propose a model in which replication-independent histone turnover reinforces robustness of local chromatin states for adult tissue homeostasis.
Assuntos
Montagem e Desmontagem da Cromatina , Epigênese Genética , Código das Histonas , Histonas/metabolismo , Homeostase , Miócitos Cardíacos/metabolismo , Animais , Células Cultivadas , DNA Helicases/metabolismo , Replicação do DNA , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Fatores de Transcrição/metabolismoRESUMO
RATIONALE: Polycomb repressive complex 2 is a major epigenetic repressor that deposits methylation on histone H3 on lysine 27 (H3K27me) and controls differentiation and function of many cells, including cardiac myocytes. EZH1 and EZH2 are 2 alternative catalytic subunits with partial functional redundancy. The relative roles of EZH1 and EZH2 in heart development and regeneration are unknown. OBJECTIVE: We compared the roles of EZH1 versus EZH2 in heart development and neonatal heart regeneration. METHODS AND RESULTS: Heart development was normal in Ezh1-/- (Ezh1 knockout) and Ezh2f/f::cTNT-Cre (Ezh2 knockout) embryos. Ablation of both genes in Ezh1-/-::Ezh2f/f::cTNT-Cre embryos caused lethal heart malformations, including hypertrabeculation, compact myocardial hypoplasia, and ventricular septal defect. Epigenome and transcriptome profiling showed that derepressed genes were upregulated in a manner consistent with total EZH dose. In neonatal heart regeneration, Ezh1 was required, but Ezh2 was dispensable. This finding was further supported by rescue experiments: cardiac myocyte-restricted re-expression of EZH1 but not EZH2 restored neonatal heart regeneration in Ezh1 knockout. In myocardial infarction performed outside of the neonatal regenerative window, EZH1 but not EZH2 likewise improved heart function and stimulated cardiac myocyte proliferation. Mechanistically, EZH1 occupied and activated genes related to cardiac growth. CONCLUSIONS: Our work unravels divergent mechanisms of EZH1 in heart development and regeneration, which will empower efforts to overcome epigenetic barriers to heart regeneration.
Assuntos
Desenvolvimento Embrionário/fisiologia , Coração/embriologia , Coração/fisiologia , Complexo Repressor Polycomb 2/biossíntese , Regeneração/fisiologia , Animais , Animais Recém-Nascidos , Coração/crescimento & desenvolvimento , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Complexo Repressor Polycomb 2/deficiênciaRESUMO
Aminopeptidases are widely used for creating protein hydrolysates and peptide sequencing. The ywaD gene from a new Bacillus isolate, named Bacillus subtilis subsp. subtilis str. BSP1, was cloned into the yeast expression vector pHBM905A and expressed and secreted by Pichia pastoris strain GS115. The deduced amino acid sequence of the aminopeptidase encoded by the ywaD gene shared up to 98% identity with aminopeptidases from B. subtilis strains 168 and zj016. The yield (3.81 g/l) and specific activity (788 U/mg) of recombinant YwaD in high-density fermentation were extremely high. And 829.83 mg of the purified enzyme (4089.72 U/mg) were harvested. YwaD was glycosylated, and its activity decreased after deglycosylation, which was similar to that of the aminopeptidase from B. subtilis strain zj016. YwaD was most active toward l-arginine-4-nitroanilide. Moreover, it exhibited high resistance to carbamide, which was not true for aminopeptidases from B. subtilis strains 168 and zj016, which could simplify the purification of YwaD. Moreover, the expression and parts of characterization of the aminopeptidase from B. subtilis strain 168 in Pichia pastoris were added as supplementary material. The sequence and other characteristics of YwaD were compared with those of aminopeptidases from B. subtilis strains 168 and zj016, and they will provide a solid foundation for further research on the influence of amino acid mutations on the function of aminopeptidases.
Assuntos
Aminopeptidases/genética , Aminopeptidases/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Clonagem Molecular/métodos , Pichia/genética , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/isolamento & purificação , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Fermentação , Glicosilação , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Proteinase K is widely used in scientific research and industries. This report was aimed to achieve high-level expression of proteinase K using Pichia pastoris GS115 as the host strain. The coding sequence of a variant of proteinase K that has higher activity than the wild type protein was chosen and optimized based on the codon usage preference of P. pastoris. The novel open reading frame was synthesized and a series of multi-copy expression vectors were constructed based on the pHBM905BDM plasmid, allowing for the tandem integration of multiple copies of the target gene into the genome of P. pastoris with a single recombination. These strains were used to study the correlation between the gene copy number and the expression level of proteinase K. The results of quantitative polymerase chain reaction (qPCR) indicated that the tandem expression cassettes were integrated into the host genome stably. Meanwhile, the results of qPCR and enzyme activity assays indicated that the mRNA and protein expression levels of the target gene increased as the gene copy number increased. Moreover, the effect of gene dosage on the expression level of the recombinant protein was more obvious using high-density fermentation. The maximum expression level and enzyme activity of proteinase K, which were obtained from the recombinant yeast strain bearing 5 copies of the target gene after an 84-h induction, were approximately 8.069 mg/mL and 108,295 U/mL, respectively. The recombinant proteinase was purified and characterized. The optimum pH and temperature for the activity of this protease were approximately pH 11 and 55 °C, respectively.
Assuntos
Ascomicetos/enzimologia , Clonagem Molecular/métodos , Endopeptidase K/genética , Pichia/genética , Ascomicetos/genética , Ascomicetos/metabolismo , Endopeptidase K/isolamento & purificação , Endopeptidase K/metabolismo , Fermentação , Dosagem de Genes , Fases de Leitura Aberta , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Recombinação GenéticaRESUMO
BACKGROUND: L-ornithine (L-Orn), is an intermediate metabolite in the urea cycle that plays a significant role in humans. L-Orn can be obtained from the catalysis of L-arginine (L-Arg) by arginase. The Pichia pastoris expression system offers the possibility of generating a large amount of recombinant protein. The immobilized enzyme technology can overcome the difficulties in recovery, recycling and long-term stability that result from the use of free enzyme. METHODS: The recombinant human arginase I (ARG I) was obtained using an optimized method with the Pichia pastoris GS115 as the host strain. Chitosan paticles were cross-linked with glutaraldehyde and rinsed exhaustively. Then the expressed ARG I was immobilized on the crosslinked chitosan particles, and the enzymatic properties of both the free and immobilized enzymes were evaluated. At last, the immobilized ARG I was employed to catalyze L-Arg to L-Orn. RESULTS: The results indicated that these two states both exhibited optimal activity under the same condition of pH10 at 40 °C. However, the immobilized ARG I exhibited the remarkable thermal and long-term stability as well as broad adaptability to pH, suggesting its potential for wide application in future industry. After a careful analysis of its catalytic conditions, immobilized ARG I was employed to catalyze the conversion of L-Arg to L-Orn under optimal condition of 1 % glutaraldehyde, 1 mM Mn(2+), 40 °C, pH10 and an L-arginine (L-Arg) concentration of 200 g/L, achieving a highly converted content of 149.g/L L-Orn. CONCLUSIONS: In this work, ARG Ι was abundantly expressed, and an efficient, facile and repeatable method was developed to synthesize high-quality L-Orn. This method not only solved the problem of obtaining a large amount of arginase, but also provided a promising alternative for the future industrial production of L-Orn.
Assuntos
Arginase/biossíntese , Ornitina/biossíntese , Proteínas Recombinantes/biossíntese , Arginase/genética , Arginina/metabolismo , Quitosana/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/genética , Regulação Enzimológica da Expressão Gênica , Humanos , Pichia/genética , Proteínas Recombinantes/genéticaRESUMO
Carboxypeptidase Y is widely used in peptide sequencing and mass spectrometry. PRC1 coding for proteinase C from Saccharomyces cerevisiae was expressed in Pichia pastoris GS115 as procarboxypeptidase Y with a yield of ~605 mg/l in shake-flasks after 168 h induction with 1 % (v/v) methanol. This precursor of carboxypeptidase Y was cleaved by endogenous proteinases of P. pastoris and released into the fermentation broth as active carboxypeptidase Y within 2 weeks at 10 °C, which facilitated the preparation of mature carboxypeptidase Y. The recombinant enzyme was purified. It was optimally active at 30 °C and pH 6.0, with an optimal activity of ~305 U/mg using benzyloxycarbonyl-L-phenylalanyl-L-leucine as substrate. This is the first report about high-level expression and activation of carboxypeptidase Y in P. pastoris.
Assuntos
Catepsina A/química , Catepsina A/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Catepsina A/genética , Catepsina A/isolamento & purificação , Estabilidade Enzimática , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/enzimologiaRESUMO
In the present study, recombinant organophosphorus hydrolase OPHC2 was successfully produced by Yarrowia lipolytica and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses showed a major polypeptide band of 36 kDa. The purified enzyme was optimally active at 65°C and pH 8.5 and also displayed good thermal and pH stability using methyl parathion (O,O-dimethyl-O-4-p-nitrophenyl phosphorothioate) as a substrate. Moreover, as Y. lipolytica is a non-pathogenic, generally regarded as safe (GRAS) yeast, the cell culture supernatant can be used directly on vegetables and fruits that are contaminated by organophosphorus pesticides.
Assuntos
Arildialquilfosfatase/genética , Arildialquilfosfatase/metabolismo , Metil Paration/metabolismo , Yarrowia/enzimologia , Yarrowia/genética , Eletroforese em Gel de Poliacrilamida , Recuperação e Remediação AmbientalRESUMO
Mapping of the holistic cell behaviours sculpting the four-chambered mammalian heart has been a goal or previous studies, but so far only success in transparent invertebrates and lower vertebrates with two-chambered hearts has been achieved. Using a live-imaging system comprising a customized vertical light-sheet microscope equipped with a mouse embryo culture module, a heartbeat-gated imaging strategy and a digital image processing framework, we realized volumetric imaging of developing mouse hearts at single-cell resolution and with uninterrupted cell lineages for up to 1.5 d. Four-dimensional landscapes of Nppa+ cardiomyocyte cell behaviours revealed a blueprint for ventricle chamber formation by which biased outward migration of the outermost cardiomyocytes is coupled with cell intercalation and horizontal division. The inner-muscle architecture of trabeculae was developed through dual mechanisms: early fate segregation and transmural cell arrangement involving both oriented cell division and directional migration. Thus, live-imaging reconstruction of uninterrupted cell lineages affords a transformative means for deciphering mammalian organogenesis.
Assuntos
Ventrículos do Coração/citologia , Ventrículos do Coração/embriologia , Processamento de Imagem Assistida por Computador/métodos , Miócitos Cardíacos/citologia , Animais , Divisão Celular , Linhagem da Célula , Movimento Celular , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Coração/embriologia , Camundongos , Microscopia , Morfogênese , Miocárdio/citologia , Análise de Célula Única , Técnicas de Cultura de TecidosRESUMO
Single-cell measurement of chromatin states, including histone modifications and non-histone protein binding, remains challenging. Here, we present a low-cost, efficient, simultaneous indexing and tagmentation-based ChIP-seq (itChIP-seq) method, compatible with both low cellular input and single cells for profiling chromatin states. itChIP combines chromatin opening, simultaneous cellular indexing and chromatin tagmentation within a single tube, enabling the processing of samples from tens of single cells to, more commonly, thousands of single cells per assay. We demonstrate that single-cell itChIP-seq (sc-itChIP-seq) yields ~9,000 unique reads per cell. Using sc-itChIP-seq to profile H3K27ac, we sufficiently capture the earliest epigenetic priming event during the cell fate transition from naive to primed pluripotency, and reveal the basis for cell-type specific enhancer usage during the differentiation of bipotent cardiac progenitor cells into endothelial cells and cardiomyocytes. Our results demonstrate that itChIP is a widely applicable technology for single-cell chromatin profiling of epigenetically heterogeneous cell populations in many biological processes.
Assuntos
Cromatina/metabolismo , Células Endoteliais/metabolismo , Processamento de Proteína Pós-Traducional/genética , Análise de Sequência de DNA , Animais , Sítios de Ligação , Imunoprecipitação da Cromatina/métodos , Epigenômica/métodos , Histonas/metabolismo , Camundongos Transgênicos , Análise de Sequência de DNA/métodosRESUMO
In proliferating cells, where most Polycomb repressive complex 2 (PRC2) studies have been performed, gene repression is associated with PRC2 trimethylation of H3K27 (H3K27me3). However, it is uncertain whether PRC2 writing of H3K27me3 is mechanistically required for gene silencing. Here, we studied PRC2 function in postnatal mouse cardiomyocytes, where the paucity of cell division obviates bulk H3K27me3 rewriting after each cell cycle. EED (embryonic ectoderm development) inactivation in the postnatal heart (EedCKO) caused lethal dilated cardiomyopathy. Surprisingly, gene upregulation in EedCKO was not coupled with loss of H3K27me3. Rather, the activating histone mark H3K27ac increased. EED interacted with histone deacetylases (HDACs) and enhanced their catalytic activity. HDAC overexpression normalized EedCKO heart function and expression of derepressed genes. Our results uncovered a non-canonical, H3K27me3-independent EED repressive mechanism that is essential for normal heart function. Our results further illustrate that organ dysfunction due to epigenetic dysregulation can be corrected by epigenetic rewiring.
Assuntos
Repressão Epigenética , Coração/embriologia , Histona Desacetilases/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Células Cultivadas , Metilação , Camundongos , Camundongos Knockout , Miócitos Cardíacos/fisiologiaRESUMO
To express recombinant carboxypeptidase from Thermus aquaticus (Cpase Taq) in Pichia pastosis, the open reading frame coding thermostable Cpase Taq was optimized based on the preference of P. pastoris codon usage and synthesized in vitro. The novel gene was cloned into P. pastoris expression vector pHBM905A and the sequence coding 6xHis tag was fused with the ORF of Cpase Taq gene. The recombinant plasmid was named pHBM905A-Cpase Taq and transformed into P. pastoris GS 115. Transformants were induced with 1% methanol for 72 h until the enzyme yield reached 0.1 mg/ml. The enzyme was purified and its enzymatic properties were analyzed. The results showed that the specific enzyme activity reached maximum at 75 °C and pH 7.5, which was about 80 U/mg. It was the first report about the secretory expression of Cpase Taq in P. pastoris GS115. Because of its large-scale preparation, this enzyme may be applied in industrial hydrolysis of peptides into amino acids in the future.