RESUMO
Cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small populations of cells within developing embryos. To understand their functions in vivo, it is important to identify TF binding sites in these cells. However, current methods cannot profile TFs genome-wide at or near the single-cell level. Here we adapt the cleavage under targets and release using nuclease (CUT&RUN) method to profile TFs in low cell numbers, including single cells and individual pre-implantation embryos. Single-cell experiments suggest that only a fraction of TF binding sites are occupied in most cells, in a manner broadly consistent with measurements of peak intensity from multi-cell studies. We further show that chromatin binding by the pluripotency TF NANOG is highly dependent on the SWI/SNF chromatin remodeling complex in individual blastocysts but not in cultured cells. Ultra-low input CUT&RUN (uliCUT&RUN) therefore enables interrogation of TF binding from rare cell populations of particular importance in development or disease.
Assuntos
Blastocisto/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fatores de Transcrição/metabolismo , Animais , Feminino , CamundongosRESUMO
Methods derived from CUT&RUN and CUT&Tag enable genome-wide mapping of the localization of proteins on chromatin from as few as one cell. These and other mapping approaches focus on one protein at a time, preventing direct measurements of co-localization of different chromatin proteins in the same cells and requiring prioritization of targets where samples are limiting. Here, we describe multi-CUT&Tag, an adaptation of CUT&Tag that overcomes these hurdles by using antibody-specific barcodes to simultaneously map multiple proteins in the same cells. Highly specific multi-CUT&Tag maps of histone marks and RNA Polymerase II uncovered sites of co-localization in the same cells, active and repressed genes, and candidate cis-regulatory elements. Single-cell multi-CUT&Tag profiling facilitated identification of distinct cell types from a mixed population and characterization of cell-type-specific chromatin architecture. In sum, multi-CUT&Tag increases the information content per cell of epigenomic maps, facilitating direct analysis of the interplay of different chromatin proteins.
Assuntos
Cromatina/química , RNA Polimerases Dirigidas por DNA/química , Animais , Imunoprecipitação da Cromatina , Mapeamento Cromossômico , Análise por Conglomerados , Células-Tronco Embrionárias/citologia , Epigênese Genética , Epigenômica , Epitopos/química , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Código das Histonas , Histonas/química , Camundongos , RNA Polimerase II/metabolismo , Sensibilidade e EspecificidadeRESUMO
Mammalian sperm show an unusual and heavily compacted genomic packaging state. In addition to its role in organizing the compact and hydrodynamic sperm head, it has been proposed that sperm chromatin architecture helps to program gene expression in the early embryo. Scores of genome-wide surveys in sperm have reported patterns of chromatin accessibility, nucleosome localization, histone modification, and chromosome folding. Here, we revisit these studies in light of recent reports that sperm obtained from the mouse epididymis are contaminated with low levels of cell-free chromatin. In the absence of proper sperm lysis, we readily recapitulate multiple prominent genome-wide surveys of sperm chromatin, suggesting that these profiles primarily reflect contaminating cell-free chromatin. Removal of cell-free DNA, and appropriate lysis conditions, are together required to reveal a sperm chromatin state distinct from most previous reports. Using ATAC-seq to explore relatively accessible genomic loci, we identify a landscape of open loci associated with early development and transcriptional control. Histone modification and chromosome folding profiles also strongly support the hypothesis that prior studies suffer from contamination, but technical challenges associated with reliably preserving the architecture of the compacted sperm head prevent us from confidently assaying true localization patterns for these epigenetic marks. Together, our studies show that our knowledge of chromosome packaging in mammalian sperm remains largely incomplete, and motivate future efforts to more accurately characterize genome organization in mature sperm.
Assuntos
Cromatina , Espermatozoides , Masculino , Animais , Camundongos , Espermatozoides/metabolismo , Cromatina/metabolismo , Cromatina/genética , Montagem e Desmontagem da Cromatina , Código das Histonas , Histonas/metabolismoRESUMO
Numerous chromatin regulators are required for embryonic stem (ES) cell self-renewal and pluripotency, but few have been studied in detail. Here, we examine the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We find that Mbd3 and Brg1 antagonistically regulate a common set of genes by regulating promoter nucleosome occupancy. Furthermore, both Mbd3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC): Mbd3 colocalizes with Tet1 and 5hmC in vivo, Mbd3 knockdown preferentially affects expression of 5hmC-marked genes, Mbd3 localization is Tet1-dependent, and Mbd3 preferentially binds to 5hmC relative to 5-methylcytosine in vitro. Finally, both Mbd3 and Brg1 are themselves required for normal levels of 5hmC in vivo. Together, our results identify an effector for 5hmC, and reveal that control of gene expression by antagonistic chromatin regulators is a surprisingly common regulatory strategy in ES cells.
Assuntos
Citosina/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Fatores de Transcrição/metabolismo , 5-Metilcitosina/análogos & derivados , Animais , Montagem e Desmontagem da Cromatina , Citosina/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Polimerase II/metabolismoRESUMO
Establishment of a healthy ovarian reserve is contingent upon numerous regulatory pathways during embryogenesis. Previously, mice lacking TBP-associated factor 4b (Taf4b) were shown to exhibit a diminished ovarian reserve. However, potential oocyte-intrinsic functions of TAF4b have not been examined. Here, we use a combination of gene expression profiling and chromatin mapping to characterize TAF4b-dependent gene regulatory networks in mouse oocytes. We find that Taf4b-deficient oocytes display inappropriate expression of meiotic, chromatin modification/organization, and X-linked genes. Furthermore, dysregulated genes in Taf4b-deficient oocytes exhibit an unexpected amount of overlap with dysregulated genes in oocytes from XO female mice, a mouse model of Turner Syndrome. Using Cleavage Under Targets and Release Using Nuclease (CUT&RUN), we observed TAF4b enrichment at genes involved in chromatin remodeling and DNA repair, some of which are differentially expressed in Taf4b-deficient oocytes. Interestingly, TAF4b target genes were enriched for Sp/Klf family and NFY target motifs rather than TATA-box motifs, suggesting an alternative mode of promoter interaction. Together, our data connect several gene regulatory nodes that contribute to the precise development of the mammalian ovarian reserve.
Assuntos
Redes Reguladoras de Genes/genética , Oogênese , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/genética , Animais , Reparo do DNA , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Meiose , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/citologia , Oócitos/metabolismo , Regiões Promotoras Genéticas , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/deficiência , Fator de Transcrição TFIID/metabolismo , Cromossomo X/genética , Cromossomo X/metabolismoRESUMO
Cells can secrete molecules that help each other's replication. In cell cultures, chemical signals might diffuse only within a cell colony or between colonies. A chemical signal's interaction length-how far apart interacting cells are-is often assumed to be some value without rigorous justifications because molecules' invisible paths and complex multicellular geometries pose challenges. Here we present an approach, combining mathematical models and experiments, for determining a chemical signal's interaction length. With murine embryonic stem (ES) cells as a testbed, we found that differentiating ES cells secrete FGF4, among others, to communicate over many millimeters in cell culture dishes and, thereby, form a spatially extended, macroscopic entity that grows only if its centimeter-scale population density is above a threshold value. With this 'macroscopic quorum sensing', an isolated macroscopic, but not isolated microscopic, colony can survive differentiation. Our integrated approach can determine chemical signals' interaction lengths in generic multicellular communities.
Assuntos
Células-Tronco Embrionárias , Percepção de Quorum , Animais , Camundongos , Diferenciação Celular , Modelos TeóricosRESUMO
Proper regulation of chromatin structure is necessary for the maintenance of cell type-specific gene expression patterns. The embryonic stem cell (ESC) expression pattern governs self-renewal and pluripotency. Here, we present an RNAi screen in mouse ESCs of 1008 loci encoding chromatin proteins. We identified 68 proteins that exhibit diverse phenotypes upon knockdown (KD), including seven subunits of the Tip60-p400 complex. Phenotypic analyses revealed that Tip60-p400 is necessary to maintain characteristic features of ESCs. We show that p400 localization to the promoters of both silent and active genes is dependent upon histone H3 lysine 4 trimethylation (H3K4me3). Furthermore, the Tip60-p400 KD gene expression profile is enriched for developmental regulators and significantly overlaps with that of the transcription factor Nanog. Depletion of Nanog reduces p400 binding to target promoters without affecting H3K4me3 levels. Together, these data indicate that Tip60-p400 integrates signals from Nanog and H3K4me3 to regulate gene expression in ESCs.
Assuntos
Canais de Cálcio/metabolismo , Células-Tronco Embrionárias/metabolismo , Histona Acetiltransferases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Lisina Acetiltransferase 5 , Camundongos , Proteína Homeobox Nanog , Regiões Promotoras Genéticas , Interferência de RNA , Transativadores , Transcrição GênicaRESUMO
Chromatin-associated RNAs have diverse roles in the nucleus. However, their mechanisms of action are poorly understood, in part because of the inability to identify proteins that specifically associate with chromatin-bound RNAs. Here, we address this problem for a subset of chromatin-associated RNAs that form R-loops-RNA-DNA hybrid structures that include a displaced strand of ssDNA. R-loops generally form cotranscriptionally and have important roles in regulation of gene expression, immunoglobulin class switching, and other processes. However, unresolved R-loops can lead to DNA damage and chromosome instability. To identify factors that may bind and regulate R-loop accumulation or mediate R-loop-dependent functions, we used a comparative immunoprecipitation/MS approach, with and without RNA-protein crosslinking, to identify a stringent set of R-loop-binding proteins in mouse embryonic stem cells. We identified 364 R-loop-interacting proteins, which were highly enriched for proteins with predicted RNA-binding functions. We characterized several R-loop-interacting proteins of the DEAD-box family of RNA helicases and found that these proteins localize to the nucleolus and, to a lesser degree, the nucleus. Consistent with their localization patterns, we found that these helicases are required for rRNA processing and regulation of gene expression. Surprisingly, depletion of these helicases resulted in misregulation of highly overlapping sets of protein-coding genes, including many genes that function in differentiation and development. We conclude that R-loop-interacting DEAD-box helicases have nonredundant roles that are critical for maintaining the normal embryonic stem cell transcriptome.
Assuntos
RNA Helicases DEAD-box/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Estruturas R-Loop , Animais , Células Cultivadas , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica , Camundongos , Proteômica/métodos , RNA/metabolismo , Processamento Pós-Transcricional do RNARESUMO
Approximately 75% of the human genome is transcribed, the majority of which does not encode protein. However, many noncoding RNAs (ncRNAs) are rapidly degraded after transcription, and relatively few have established functions, questioning the significance of this observation. Here we show that esBAF, a SWI/SNF family nucleosome remodeling factor, suppresses transcription of ncRNAs from â¼57,000 nucleosome-depleted regions (NDRs) throughout the genome of mouse embryonic stem cells (ESCs). We show that esBAF functions to both keep NDRs nucleosome-free and promote elevated nucleosome occupancy adjacent to NDRs. Reduction of adjacent nucleosome occupancy upon esBAF depletion is strongly correlated with ncRNA expression, suggesting that flanking nucleosomes form a barrier to pervasive transcription. Upon forcing nucleosome occupancy near two NDRs using a nucleosome-positioning sequence, we found that esBAF is no longer required to silence transcription. Therefore, esBAF's function to enforce nucleosome occupancy adjacent to NDRs, and not its function to maintain NDRs in a nucleosome-free state, is necessary for silencing transcription over ncDNA. Finally, we show that the ability of a strongly positioned nucleosome to repress ncRNA depends on its translational positioning. These data reveal a novel role for esBAF in suppressing pervasive transcription from open chromatin regions in ESCs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/fisiologia , RNA não Traduzido/genética , Animais , Montagem e Desmontagem da Cromatina , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Metal-regulatory transcription factor 1 (MTF1) is a conserved metal-binding transcription factor in eukaryotes that binds to conserved DNA sequence motifs, termed metal response elements. MTF1 responds to both metal excess and deprivation, protects cells from oxidative and hypoxic stresses, and is required for embryonic development in vertebrates. To examine the role for MTF1 in cell differentiation, we use multiple experimental strategies [including gene knockdown (KD) mediated by small hairpin RNA and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), immunofluorescence, chromatin immunopreciptation sequencing, subcellular fractionation, and atomic absorbance spectroscopy] and report a previously unappreciated role for MTF1 and copper (Cu) in cell differentiation. Upon initiation of myogenesis from primary myoblasts, both MTF1 expression and nuclear localization increased. Mtf1 KD impaired differentiation, whereas addition of nontoxic concentrations of Cu+-enhanced MTF1 expression and promoted myogenesis. Furthermore, we observed that Cu+ binds stoichiometrically to a C terminus tetra-cysteine of MTF1. MTF1 bound to chromatin at the promoter regions of myogenic genes, and Cu addition stimulated this binding. Of note, MTF1 formed a complex with myogenic differentiation (MYOD)1, the master transcriptional regulator of the myogenic lineage, at myogenic promoters. These findings uncover unexpected mechanisms by which Cu and MTF1 regulate gene expression during myoblast differentiation.-Tavera-Montañez, C., Hainer, S. J., Cangussu, D., Gordon, S. J. V., Xiao, Y., Reyes-Gutierrez, P., Imbalzano, A. N., Navea, J. G., Fazzio, T. G., Padilla-Benavides, T. The classic metal-sensing transcription factor MTF1 promotes myogenesis in response to copper.
Assuntos
Diferenciação Celular , Cobre/farmacologia , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Muscular , Mioblastos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Proteína MyoD/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Fator MTF-1 de TranscriçãoRESUMO
BACKGROUND: Differential accessibility of DNA to nuclear proteins underlies the regulation of numerous cellular processes. Although DNA accessibility is primarily determined by the presence or absence of nucleosomes, differences in nucleosome composition or dynamics may also regulate accessibility. Methods for mapping nucleosome positions and occupancies genome-wide (MNase-seq) have uncovered the nucleosome landscapes of many different cell types and organisms. Conversely, methods specialized for the detection of large nucleosome-free regions of chromatin (DNase-seq, FAIRE-seq) have uncovered numerous gene regulatory elements. However, these methods are less successful in measuring the accessibility of DNA sequences within nucelosome arrays. RESULTS: Here we probe the genome-wide accessibility of multiple cell types in an unbiased manner using restriction endonuclease digestion of chromatin coupled to deep sequencing (RED-seq). Using this method, we identified differences in chromatin accessibility between populations of cells, not only in nucleosome-depleted regions of the genome (e.g., enhancers and promoters), but also within the majority of the genome that is packaged into nucleosome arrays. Furthermore, we identified both large differences in chromatin accessibility in distinct cell lineages and subtle but significant changes during differentiation of mouse embryonic stem cells (ESCs). Most significantly, using RED-seq, we identified differences in accessibility among nucleosomes harboring well-studied histone variants, and show that these differences depend on factors required for their deposition. CONCLUSIONS: Using an unbiased method to probe chromatin accessibility genome-wide, we uncover unique features of chromatin structure that are not observed using more widely-utilized methods. We demonstrate that different types of nucleosomes within mammalian cells exhibit different degrees of accessibility. These findings provide significant insight into the regulation of DNA accessibility.
Assuntos
Enzimas de Restrição do DNA/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Nucleossomos/genética , Nucleossomos/metabolismo , Animais , Diferenciação Celular , DNA/genética , Células-Tronco Embrionárias/citologia , Histonas/genética , Histonas/metabolismo , Humanos , CamundongosRESUMO
GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this genetic mutation leads to neurodegeneration remains largely unknown. Using CRISPR-Cas9 technology, we deleted EXOC2, which encodes an essential exocyst subunit, in induced pluripotent stem cells (iPSCs) derived from C9ORF72-ALS/FTD patients. These cells are viable owing to the presence of truncated EXOC2, suggesting that exocyst function is partially maintained. Several disease-relevant cellular phenotypes in C9ORF72 iPSC-derived motor neurons are rescued due to, surprisingly, the decreased levels of dipeptide repeat (DPR) proteins and expanded G4C2 repeats-containing RNA. The treatment of fully differentiated C9ORF72 neurons with EXOC2 antisense oligonucleotides also decreases expanded G4C2 repeats-containing RNA and partially rescued disease phenotypes. These results indicate that EXOC2 directly or indirectly regulates the level of G4C2 repeats-containing RNA, making it a potential therapeutic target in C9ORF72-ALS/FTD.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Expansão das Repetições de DNA , Demência Frontotemporal , Células-Tronco Pluripotentes Induzidas , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Demência Frontotemporal/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Expansão das Repetições de DNA/genética , Neurônios Motores/metabolismo , Neurônios Motores/patologiaRESUMO
Neuroblastoma remains a formidable challenge in pediatric oncology, representing 15% of cancer-related mortalities in children. Despite advancements in combinatorial and targeted treatments improving survival rates, nearly 50% of patients with high-risk neuroblastoma will ultimately succumb to their disease. Dysregulation of the epithelial-mesenchymal transition (EMT) is a key mechanism of tumor cell dissemination, resulting in metastasis and poor outcomes in many cancers. Our prior work identified PRMT5 as a key regulator of EMT via methylation of AKT at arginine 15, enhancing the expression of EMT-driving transcription factors and facilitating metastasis. Here, we identify that PRMT5 directly regulates the transcription of the epidermal growth factor receptor (EGFR). PRMT5, through independent modulation of the EGFR and AKT pathways, orchestrates the activation of NFκB, resulting in the upregulation of the pro-EMT transcription factors ZEB1, SNAIL, and TWIST1. Notably, EGFR and AKT form a compensatory feedback loop, reinforcing the expression of these EMT transcription factors. Small molecule inhibition of PRMT5 methyltransferase activity disrupts EGFR/AKT signaling, suppresses EMT transcription factor expression and ablates tumor growth in vivo . Our findings underscore the pivotal role of PRMT5 in the control of the EMT program in high-risk neuroblastoma.
RESUMO
Ordered cell cycle progression is coordinated by cyclin dependent kinases (CDKs). CDKs often phosphorylate substrates at multiple sites clustered within disordered regions. However, for most substrates, it is not known which phosphosites are functionally important. We developed a high-throughput approach, Phosphosite Scanning, that tests the importance of each phosphosite within a multisite phosphorylated domain. We show that Phosphosite Scanning identifies multiple combinations of phosphosites that can regulate protein function and reveals specific phosphorylations that are required for phosphorylation at additional sites within a domain. We applied this approach to the yeast transcription factor Hcm1, a conserved regulator of mitotic genes that is critical for accurate chromosome segregation. Phosphosite Scanning revealed a complex CDK-regulatory circuit that mediates Cks1-dependent phosphorylation of key activating sites in vivo. These results illuminate the mechanism of Hcm1 activation by CDK and establish Phosphosite Scanning as a powerful tool for decoding multisite phosphorylated domains.
Assuntos
Quinases Ciclina-Dependentes , Proteínas de Saccharomyces cerevisiae , Fosforilação , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição Forkhead/metabolismoRESUMO
Methylation is a widely occurring modification that requires the methyl donor S-adenosylmethionine (SAM) and acts in regulation of gene expression and other processes. SAM is synthesized from methionine, which is imported or generated through the 1-carbon cycle (1 CC). Alterations in 1 CC function have clear effects on lifespan and stress responses, but the wide distribution of this modification has made identification of specific mechanistic links difficult. Exploiting a dynamic stress-induced transcription model, we find that two SAM synthases in Caenorhabditis elegans, SAMS-1 and SAMS-4, contribute differently to modification of H3K4me3, gene expression and survival. We find that sams-4 enhances H3K4me3 in heat shocked animals lacking sams-1, however, sams-1 cannot compensate for sams-4, which is required to survive heat stress. This suggests that the regulatory functions of SAM depend on its enzymatic source and that provisioning of SAM may be an important regulatory step linking 1 CC function to phenotypes in aging and stress.
Assuntos
Histonas , S-Adenosilmetionina , Animais , S-Adenosilmetionina/metabolismo , Histonas/metabolismo , Caenorhabditis elegans/fisiologia , Resposta ao Choque Térmico , Expressão GênicaRESUMO
Immunotherapies that produce durable responses in some malignancies have failed in pancreatic ductal adenocarcinoma (PDAC) due to rampant immune suppression and poor tumor immunogenicity. We and others have demonstrated that induction of the senescence-associated secretory phenotype (SASP) can be an effective approach to activate anti-tumor natural killer (NK) cell and T cell immunity. In the present study, we found that the pancreas tumor microenvironment suppresses NK cell and T cell surveillance after therapy-induced senescence through enhancer of zeste homolog 2 (EZH2)-mediated epigenetic repression of proinflammatory SASP genes. EZH2 blockade stimulated production of SASP chemokines CCL2 and CXCL9/10, leading to enhanced NK cell and T cell infiltration and PDAC eradication in mouse models. EZH2 activity was also associated with suppression of chemokine signaling and cytotoxic lymphocytes and reduced survival in patients with PDAC. These results demonstrate that EZH2 represses the proinflammatory SASP and that EZH2 inhibition combined with senescence-inducing therapy could be a powerful means to achieve immune-mediated tumor control in PDAC.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Camundongos , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Linhagem Celular Tumoral , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Fenótipo Secretor Associado à Senescência , Microambiente Tumoral/genéticaRESUMO
DamID maps protein-genome interactions using DNA adenine methyltransferase tethered to individual chromatin proteins. In a recent issue of Molecluar Cell, Rang et al. introduce EpiDamID, a powerful extension of DamID suitable for mapping histone marks while simultaneously measuring mRNA levels in single cells.
Assuntos
Cromatina , Código das Histonas , Cromatina/genética , Transcriptoma , Multiômica , GenomaRESUMO
Genome-wide chromatin mapping approaches typically focus on one protein at a time. We recently developed multi-CUT&Tag, which enables simultaneous mapping of multiple chromatin proteins in the same single cells or pools of cells. Using barcoded adapters loaded onto antibody-protein A-Tn5 transposase complexes, multi-CUT&Tag marks the locations of each chromatin protein and directly detects colocalization of different proteins in the same cell(s). Although slightly more laborious than CUT&Tag, multi-CUT&Tag provides a powerful option for generating multi-factor maps for epigenomic profiling. For complete details on the use and execution of this protocol, please refer to Gopalan et al. (2021).
Assuntos
Cromatina/metabolismo , Mapeamento Cromossômico/métodos , Eletroforese em Gel de Poliacrilamida , HumanosAssuntos
Diferenciação Celular/fisiologia , Montagem e Desmontagem da Cromatina/fisiologia , Células-Tronco Embrionárias/metabolismo , Nucleossomos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Acetilação , Animais , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes/citologiaRESUMO
Determining the genomic location of DNA-binding proteins is essential to understanding their function. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a powerful method for mapping protein-DNA interactions at high resolution. In CUT&RUN, a recombinant protein A-microccocal nuclease (pA-MN) fusion is recruited by an antibody targeting the chromatin protein of interest; this can be done with either uncrosslinked or formaldehyde-crosslinked cells. DNA fragments near sites of antibody binding are released from the insoluble bulk chromatin through endonucleolytic cleavage and used to build barcoded DNA-sequencing libraries that can be sequenced in pools of at least 30. Therefore, CUT&RUN provides an alternative to ChIP-seq approaches for mapping chromatin proteins, which typically have relatively high signal-to-noise ratios, while using fewer cells and at a lower cost. Here, we describe the methods for performing CUT&RUN, generating DNA-sequencing libraries, and analyzing the resulting datasets. © 2019 by John Wiley & Sons, Inc.