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1.
Nat Commun ; 12(1): 4802, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376665

RESUMO

During meiosis, gene expression is silenced in aberrantly unsynapsed chromatin and in heterogametic sex chromosomes. Initiation of sex chromosome silencing is disrupted in meiocytes with sex chromosome-autosome translocations. To determine whether this is due to aberrant synapsis or loss of continuity of sex chromosomes, we engineered Caenorhabditis elegans nematodes with non-translocated, bisected X chromosomes. In early meiocytes of mutant males and hermaphrodites, X segments are enriched with euchromatin assembly markers and active RNA polymerase II staining, indicating active transcription. Analysis of RNA-seq data showed that genes from the X chromosome are upregulated in gonads of mutant worms. Contrary to previous models, which predicted that any unsynapsed chromatin is silenced during meiosis, our data indicate that unsynapsed X segments are transcribed. Therefore, our results suggest that sex chromosome chromatin has a unique character that facilitates its meiotic expression when its continuity is lost, regardless of whether or not it is synapsed.


Assuntos
Caenorhabditis elegans/genética , Inativação Gênica , Meiose/genética , Cromossomo X/genética , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Pareamento Cromossômico/genética , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Histonas/metabolismo , Hibridização in Situ Fluorescente , Masculino , Microscopia de Fluorescência , Transcrição Genética
2.
J Dermatol Sci ; 103(2): 93-100, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281744

RESUMO

BACKGROUND: Numerous alterations in gene expression have been described in psoriatic lesions compared to uninvolved or healthy skin. However, the mechanisms which induce this altered expression remain unclear. Epigenetic modifications play a key role in regulating genes' expression. Only three studies compared the whole-genome DNA methylation of psoriasis versus healthy skin. The present is the first study of genome-wide comparison of histone modifications between psoriatic to healthy skins. OBJECTIVE: Our objective was to explore the pattern of H3K27Ac modifications in psoriatic lesions compared to uninvolved psoriatic and healthy skin, in order to identify new genes involved in the pathogenesis of psoriasis. METHOD: Using ChIP-seq with anti H3K27Ac we compared the acetylation of lysine 27 on histone 3 (H3K27Ac) modification between psoriatic to healthy skins, combined with mRNA array. RESULTS: We found a differential H3K27Ac pattern between psoriatic compared to uninvolved or healthy skins. We found that many of the overexpressed and H3K27Ac enriched genes in psoriasis, harbor a putative GRHL transcription factor-binding site. CONCLUSIONS: In the most overexpressed genes in psoriasis, there is an enrichment of H3K27Ac. However, the loss of H3K27 acetylation modification does not correlate with decreased gene expression. GRHL appears to play an important role in the pathogenesis of psoriasis and therefore, might be a new target for psoriasis therapeutics.

3.
Dev Cell ; 56(12): 1804-1817.e7, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34010629

RESUMO

Single-cell assays have revealed the importance of heterogeneity in many biological systems. However, limited sensitivity is a major hurdle for uncovering cellular variation. To overcome it, we developed CloneSeq, combining clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing (RNA-seq). We show that clonal cells maintain similar transcriptional profiles and cell states. CloneSeq of lung cancer cells revealed cancer-specific subpopulations, including cancer stem-like cells, that were not revealed by scRNA-seq. Clonal expansion within 3D soft microenvironments supported cellular stemness of embryonic stem cells (ESCs) even without pluripotent media, and it improved epigenetic reprogramming efficiency of mouse embryonic fibroblasts. CloneSeq of ESCs revealed that the differentiation decision is made early during Oct4 downregulation and is maintained during early clonal expansion. Together, we show CloneSeq can be adapted to different biological systems to discover rare subpopulations by leveraging the enhanced sensitivity within clones.

4.
Proc Natl Acad Sci U S A ; 118(19)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33941688

RESUMO

Mesenchymal stromal/stem cells (MSCs) form a heterogeneous population of multipotent progenitors that contribute to tissue regeneration and homeostasis. MSCs assess extracellular elasticity by probing resistance to applied forces via adhesion, cytoskeletal, and nuclear mechanotransducers that direct differentiation toward soft or stiff tissue lineages. Even under controlled culture conditions, MSC differentiation exhibits substantial cell-to-cell variation that remains poorly characterized. By single-cell transcriptional profiling of nonconditioned, matrix-conditioned, and early differentiating cells, we identified distinct MSC subpopulations with distinct mechanosensitivities, differentiation capacities, and cell cycling. We show that soft matrices support adipogenesis of multipotent cells and early endochondral ossification of nonadipogenic cells, whereas intramembranous ossification and preosteoblast proliferation are directed by stiff matrices. Using diffusion pseudotime mapping, we outline hierarchical matrix-directed differentiation and perform whole-genome screening of mechanoresponsive genes. Specifically, top-ranked tropomyosin-1 is highly sensitive to stiffness cues both at RNA and protein levels, and changes in TPM1 expression determine the differentiation toward soft versus stiff tissue lineage. Consistent with actin stress fiber stabilization, tropomyosin-1 overexpression maintains YAP1 nuclear localization, activates YAP1 target genes, and directs osteogenic differentiation. Knockdown of tropomyosin-1 reversed YAP1 nuclear localization consistent with relaxation of cellular contractility, suppressed osteogenesis, activated early endochondral ossification genes after 3 d of culture in induction medium, and facilitated adipogenic differentiation after 1 wk. Our results delineate cell-to-cell variation of matrix-directed MSC differentiation and highlight tropomyosin-mediated matrix sensing.

5.
PLoS Genet ; 17(4): e1009498, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33844685

RESUMO

In mammals, cellular identity is defined through strict regulation of chromatin modifications and DNA methylation that control gene expression. Methylation of cytosines at CpG sites in the genome is mainly associated with suppression; however, the reason for enhancer-specific methylation is not fully understood. We used sequential ChIP-bisulfite-sequencing for H3K4me1 and H3K27ac histone marks. By collecting data from the same genomic region, we identified enhancers differentially methylated between these two marks. We observed a global gain of CpG methylation primarily in H3K4me1-marked nucleosomes during mouse embryonic stem cell differentiation. This gain occurred largely in enhancer regions that regulate genes critical for differentiation. The higher levels of DNA methylation in H3K4me1- versus H3K27ac-marked enhancers, despite it being the same genomic region, indicates cellular heterogeneity of enhancer states. Analysis of single-cell RNA-seq profiles demonstrated that this heterogeneity correlates with gene expression during differentiation. Furthermore, heterogeneity of enhancer methylation correlates with transcription start site methylation. Our results provide insights into enhancer-based functional variation in complex biological systems.


Assuntos
Diferenciação Celular/genética , Cromatina/genética , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Animais , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , Nucleossomos/genética , Regiões Promotoras Genéticas/genética , RNA-Seq , Análise de Célula Única , Sítio de Iniciação de Transcrição
6.
Front Microbiol ; 11: 1844, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32849441

RESUMO

ATP-independent chaperones are widespread across all domains of life and serve as the first line of defense during protein unfolding stresses. One of the known crucial chaperones for bacterial survival in a hostile environment (e.g., heat and oxidative stress) is the highly conserved, redox-regulated ATP-independent bacterial chaperone Hsp33. Using a bioinformatic analysis, we describe novel eukaryotic homologs of Hsp33 identified in eukaryotic pathogens belonging to the kinetoplastids, a family responsible for lethal human diseases such as Chagas disease as caused by Trypanosoma cruzi, African sleeping sickness caused by Trypanosoma brucei spp., and leishmaniasis pathologies delivered by various Leishmania species. During their pathogenic life cycle, kinetoplastids need to cope with elevated temperatures and oxidative stress, the same conditions which convert Hsp33 into a powerful chaperone in bacteria, thus preventing aggregation of a wide range of misfolded proteins. Here, we focused on a functional characterization of the Hsp33 homolog in one of the members of the kinetoplastid family, T. brucei, (Tb927.6.2630), which we have named TrypOx. RNAi silencing of TrypOx led to a significant decrease in the survival of T. brucei under mild oxidative stress conditions, implying a protective role of TrypOx during the Trypanosomes growth. We then adopted a proteomics-driven approach to investigate the role of TrypOx in defining the oxidative stress response. Depletion of TrypOx significantly altered the abundance of proteins mediating redox homeostasis, linking TrypOx with the antioxidant system. Using biochemical approaches, we identified the redox-switch domain of TrypOx, showing its modularity and oxidation-dependent structural plasticity. Kinetoplastid parasites such as T. brucei need to cope with high levels of oxidants produced by the innate immune system, such that parasite-specific antioxidant proteins like TrypOx - which are depleted in mammals - are highly promising candidates for drug targeting.

7.
Nat Commun ; 11(1): 3128, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561732

RESUMO

Whole-cell cross-linking coupled to mass spectrometry is one of the few tools that can probe protein-protein interactions in intact cells. A very attractive reagent for this purpose is formaldehyde, a small molecule which is known to rapidly penetrate into all cellular compartments and to preserve the protein structure. In light of these benefits, it is surprising that identification of formaldehyde cross-links by mass spectrometry has so far been unsuccessful. Here we report mass spectrometry data that reveal formaldehyde cross-links to be the dimerization product of two formaldehyde-induced amino acid modifications. By integrating the revised mechanism into a customized search algorithm, we identify hundreds of cross-links from in situ formaldehyde fixation of human cells. Interestingly, many of the cross-links could not be mapped onto known atomic structures, and thus provide new structural insights. These findings enhance the use of formaldehyde cross-linking and mass spectrometry for structural studies.


Assuntos
Reagentes para Ligações Cruzadas/química , Formaldeído/química , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Aminoácidos/química , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas , Simulação de Acoplamento Molecular , Proteínas/metabolismo
8.
Biophys J ; 118(8): 2015-2026, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32101712

RESUMO

Transcription factor (TF) recognition is dictated by the underlying DNA motif sequence specific for each TF. Here, we reveal that DNA sequence repeat symmetry plays a central role in defining TF-DNA-binding preferences. In particular, we find that different TFs bind similar symmetry patterns in the context of different developmental layers. Most TFs possess dominant preferences for similar DNA repeat symmetry types. However, in some cases, preferences of specific TFs are changed during differentiation, suggesting the importance of information encoded outside of known motif regions. Histone modifications also exhibit strong preferences for similar DNA repeat symmetry patterns unique to each type of modification. Next, using an in vivo reporter assay, we show that gene expression in embryonic stem cells can be positively modulated by the presence of genomic and computationally designed DNA oligonucleotides containing identified nonconsensus-repetitive sequence elements. This supports the hypothesis that certain nonconsensus-repetitive patterns possess a functional ability to regulate gene expression. We also performed a solution NMR experiment to probe the stability of double-stranded DNA via imino proton resonances for several double-stranded DNA sequences characterized by different repetitive patterns. We suggest that such local stability might play a key role in determining TF-DNA binding preferences. Overall, our findings show that despite the enormous sequence complexity of the TF-DNA binding landscape in differentiating embryonic stem cells, this landscape can be quantitatively characterized in simple terms using the notion of DNA sequence repeat symmetry.


Assuntos
Células-Tronco Embrionárias , Fatores de Transcrição , Sequência de Bases , Sítios de Ligação , Células-Tronco Embrionárias/metabolismo , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Cell Stem Cell ; 24(6): 983-994.e7, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31031139

RESUMO

Following fertilization, totipotent cells undergo asymmetric cell divisions, resulting in three distinct cell types in the late pre-implantation blastocyst: epiblast (Epi), primitive endoderm (PrE), and trophectoderm (TE). Here, we aim to understand whether these three cell types can be induced from fibroblasts by one combination of transcription factors. By utilizing a sophisticated fluorescent knockin reporter system, we identified a combination of five transcription factors, Gata3, Eomes, Tfap2c, Myc, and Esrrb, that can reprogram fibroblasts into induced pluripotent stem cells (iPSCs), induced trophoblast stem cells (iTSCs), and induced extraembryonic endoderm stem cells (iXENs), concomitantly. In-depth transcriptomic, chromatin, and epigenetic analyses provide insights into the molecular mechanisms that underlie the reprogramming process toward the three cell types. Mechanistically, we show that the interplay between Esrrb and Eomes during the reprogramming process determines cell fate, where high levels of Esrrb induce a XEN-like state that drives pluripotency and high levels of Eomes drive trophectodermal fate.


Assuntos
Blastocisto/fisiologia , Endoderma/fisiologia , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Trofoblastos/fisiologia , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Reprogramação Celular , Implantação do Embrião , Camundongos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismo
10.
Genetics ; 210(4): 1239-1252, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30396881

RESUMO

Pathological mutations involving noncoding microsatellite repeats are typically located near promoters in CpG islands and are coupled with extensive repeat instability when sufficiently long. What causes these regions to be prone to repeat instability is not fully understood. There is a general consensus that instability results from the induction of unusual structures in the DNA by the repeats as a consequence of mispairing between complementary strands. In addition, there is some evidence that repeat instability is mediated by RNA transcription through the formation of three-stranded nucleic structures composed of persistent DNA:RNA hybrids, concomitant with single-strand DNA displacements (R-loops). Using human embryonic stem cells with wild-type and repeat expanded alleles in the FMR1 (CGGs) and C9orf72 (GGGGCCs) genes, we show that these loci constitute preferential sites (hotspots) for DNA unpairing. When R-loops are formed, DNA unpairing is more extensive, and is coupled with the interruptions of double-strand structures by the nontranscribing (G-rich) DNA strand. These interruptions are likely to reflect unusual structures in the DNA that drive repeat instability when the G-rich repeats considerably expand. Further, we demonstrate that when the CGGs in FMR1 are hyper-methylated and transcriptionally inactive, local DNA unpairing is abolished. Our study thus takes one more step toward the identification of dynamic, unconventional DNA structures across the G-rich repeats at FMR1 and C9orf72 disease-associated loci.


Assuntos
Proteína C9orf72/genética , Metilação de DNA/genética , Expansão das Repetições de DNA/genética , Proteína do X Frágil de Retardo Mental/genética , Alelos , Ilhas de CpG/genética , DNA de Cadeia Simples/genética , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Repetições de Microssatélites/genética , Mutação/genética
11.
Cell Rep ; 22(10): 2797-2807, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29514105

RESUMO

Transcription factor (TF) binding to DNA is crucial for transcriptional regulation. There are multiple methods for mapping such binding. These methods balance between input requirements, spatial resolution, and compatibility with high-throughput automation. Here, we describe SLIM-ChIP (short-fragment-enriched, low-input, indexed MNase ChIP), which combines enzymatic fragmentation of chromatin and on-bead indexing to address these desiderata. SLIM-ChIP reproduces a high-resolution binding map of yeast Reb1 comparable with existing methods, yet with less input material and full compatibility with high-throughput procedures. We demonstrate the robustness and flexibility of SLIM-ChIP by probing additional factors in yeast and mouse. Finally, we show that SLIM-ChIP provides information on the chromatin landscape surrounding the bound transcription factor. We identify a class of Reb1 sites where the proximal -1 nucleosome tightly interacts with Reb1 and maintains unidirectional transcription. SLIM-ChIP is an attractive solution for mapping DNA binding proteins and charting the surrounding chromatin occupancy landscape at a single-cell level.


Assuntos
Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Imunoprecipitação da Cromatina , Genoma , Camundongos , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Iniciação da Transcrição Genética
12.
Science ; 352(6286): 717-21, 2016 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-27151869

RESUMO

Different combinations of histone modifications have been proposed to signal distinct gene regulatory functions, but this area is poorly addressed by existing technologies. We applied high-throughput single-molecule imaging to decode combinatorial modifications on millions of individual nucleosomes from pluripotent stem cells and lineage-committed cells. We identified definitively bivalent nucleosomes with concomitant repressive and activating marks, as well as other combinatorial modification states whose prevalence varies with developmental potency. We showed that genetic and chemical perturbations of chromatin enzymes preferentially affect nucleosomes harboring specific modification states. Last, we combined this proteomic platform with single-molecule DNA sequencing technology to simultaneously determine the modification states and genomic positions of individual nucleosomes. This single-molecule technology has the potential to address fundamental questions in chromatin biology and epigenetic regulation.


Assuntos
Epigênese Genética , Histonas/metabolismo , Nucleossomos/química , Nucleossomos/genética , Proteômica/métodos , Animais , Linhagem Celular Tumoral , Linhagem da Célula , Células Cultivadas , Cromatina/enzimologia , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Camundongos , Imagem Molecular/métodos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de DNA/métodos
13.
Mol Cell ; 61(1): 170-80, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26687680

RESUMO

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here, we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of p300, EZH2, or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions, and drug treatments.


Assuntos
Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina/métodos , Cromatina/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Histonas/metabolismo , Leucemia/metabolismo , Reação em Cadeia da Polimerase Multiplex/métodos , Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Código de Barras de DNA Taxonômico , Epigênese Genética/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Histonas/genética , Humanos , Células K562 , Leucemia/genética , Mutação
14.
Nat Biotechnol ; 33(11): 1165-72, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26458175

RESUMO

Chromatin profiling provides a versatile means to investigate functional genomic elements and their regulation. However, current methods yield ensemble profiles that are insensitive to cell-to-cell variation. Here we combine microfluidics, DNA barcoding and sequencing to collect chromatin data at single-cell resolution. We demonstrate the utility of the technology by assaying thousands of individual cells and using the data to deconvolute a mixture of ES cells, fibroblasts and hematopoietic progenitors into high-quality chromatin state maps for each cell type. The data from each single cell are sparse, comprising on the order of 1,000 unique reads. However, by assaying thousands of ES cells, we identify a spectrum of subpopulations defined by differences in chromatin signatures of pluripotency and differentiation priming. We corroborate these findings by comparison to orthogonal single-cell gene expression data. Our method for single-cell analysis reveals aspects of epigenetic heterogeneity not captured by transcriptional analysis alone.


Assuntos
Imunoprecipitação da Cromatina/métodos , Células-Tronco Embrionárias/classificação , Células-Tronco Embrionárias/citologia , Análise de Célula Única/métodos , Animais , Cromatina/genética , Biologia Computacional , Código de Barras de DNA Taxonômico , Humanos , Camundongos , Técnicas Analíticas Microfluídicas , Análise de Sequência de DNA
15.
PLoS One ; 10(5): e0116328, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26000628

RESUMO

The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops. Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells.


Assuntos
Microfluídica/métodos , DNA Complementar/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de RNA/métodos
16.
Nat Biotechnol ; 31(12): 1133-6, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24013198

RESUMO

Mammalian gene regulation is dependent on tissue-specific enhancers that can act across large distances to influence transcriptional activity. Mapping experiments have identified hundreds of thousands of putative enhancers whose functionality is supported by cell type-specific chromatin signatures and striking enrichments for disease-associated sequence variants. However, these studies did not address the in vivo functions of the putative elements or their chromatin states and did not determine which genes, if any, a given enhancer regulates. Here we present a strategy to investigate endogenous regulatory elements by selectively altering their chromatin state using programmable reagents. Transcription activator-like (TAL) effector repeat domains fused to the LSD1 histone demethylase efficiently remove enhancer-associated chromatin modifications from target loci, without affecting control regions. We find that inactivation of enhancer chromatin by these fusion proteins frequently causes downregulation of proximal genes, revealing enhancer target genes. Our study demonstrates the potential of epigenome editing tools to characterize an important class of functional genomic elements.


Assuntos
Cromatina/genética , Elementos Facilitadores Genéticos/genética , Histonas/genética , Mutagênese Sítio-Dirigida/métodos , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Elementos Reguladores de Transcrição/genética
17.
Cell ; 151(6): 1185-99, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23217706

RESUMO

Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, whereas transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. By using a conditional SIRT6 allele, we show that SIRT6 deletion in vivo increases the number, size, and aggressiveness of tumors. SIRT6 also functions as a regulator of ribosome metabolism by corepressing MYC transcriptional activity. Lastly, Sirt6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism.


Assuntos
Neoplasias/metabolismo , Sirtuínas/metabolismo , Animais , Proliferação de Células , Regulação para Baixo , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Glicólise , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sirtuínas/genética , Transcrição Genética , Transplante Heterólogo , Proteínas Supressoras de Tumor/genética
18.
Mol Cell ; 47(5): 810-22, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22940246

RESUMO

Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene-expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction.


Assuntos
Imunoprecipitação da Cromatina/métodos , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Animais , DNA/genética , DNA/metabolismo , Camundongos , Fatores de Transcrição/metabolismo
19.
Cell ; 147(7): 1628-39, 2011 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-22196736

RESUMO

Hundreds of chromatin regulators (CRs) control chromatin structure and function by catalyzing and binding histone modifications, yet the rules governing these key processes remain obscure. Here, we present a systematic approach to infer CR function. We developed ChIP-string, a meso-scale assay that combines chromatin immunoprecipitation with a signature readout of 487 representative loci. We applied ChIP-string to screen 145 antibodies, thereby identifying effective reagents, which we used to map the genome-wide binding of 29 CRs in two cell types. We found that specific combinations of CRs colocalize in characteristic patterns at distinct chromatin environments, at genes of coherent functions, and at distal regulatory elements. When comparing between cell types, CRs redistribute to different loci but maintain their modular and combinatorial associations. Our work provides a multiplex method that substantially enhances the ability to monitor CR binding, presents a large resource of CR maps, and reveals common principles for combinatorial CR function.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/metabolismo , Genômica/métodos , Código das Histonas , Cromatina/química , Montagem e Desmontagem da Cromatina , Células-Tronco Embrionárias , Genoma , Humanos , Células K562
20.
Nucleic Acids Res ; 38(10): 3318-27, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20110253

RESUMO

Regulation of splicing in eukaryotes occurs through the coordinated action of multiple splicing factors. Exons and introns contain numerous putative binding sites for splicing regulatory proteins. Regulation of splicing is presumably achieved by the combinatorial output of the binding of splicing factors to the corresponding binding sites. Although putative regulatory sites often overlap, no extensive study has examined whether overlapping regulatory sequences provide yet another dimension to splicing regulation. Here we analyzed experimentally-identified splicing regulatory sequences using a computational method based on the natural distribution of nucleotides and splicing regulatory sequences. We uncovered positive and negative interplay between overlapping regulatory sequences. Examination of these overlapping motifs revealed a unique spatial distribution, especially near splice donor sites of exons with weak splice donor sites. The positively selected overlapping splicing regulatory motifs were highly conserved among different species, implying functionality. Overall, these results suggest that overlap of two splicing regulatory binding sites is an evolutionary conserved widespread mechanism of splicing regulation. Finally, over-abundant motif overlaps were experimentally tested in a reporting minigene revealing that overlaps may facilitate a mode of splicing that did not occur in the presence of only one of the two regulatory sequences that comprise it.


Assuntos
Splicing de RNA , Sequências Reguladoras de Ácido Ribonucleico , Animais , Sequência de Bases , Sítios de Ligação , Biologia Computacional/métodos , Sequência Conservada , Éxons , Humanos , Sítios de Splice de RNA , Proteínas de Ligação a RNA/metabolismo
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