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1.
Genome Res ; 30(7): 1060-1072, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32718982

RESUMO

Long noncoding RNAs (lncRNAs) constitute the majority of transcripts in the mammalian genomes, and yet, their functions remain largely unknown. As part of the FANTOM6 project, we systematically knocked down the expression of 285 lncRNAs in human dermal fibroblasts and quantified cellular growth, morphological changes, and transcriptomic responses using Capped Analysis of Gene Expression (CAGE). Antisense oligonucleotides targeting the same lncRNAs exhibited global concordance, and the molecular phenotype, measured by CAGE, recapitulated the observed cellular phenotypes while providing additional insights on the affected genes and pathways. Here, we disseminate the largest-to-date lncRNA knockdown data set with molecular phenotyping (over 1000 CAGE deep-sequencing libraries) for further exploration and highlight functional roles for ZNF213-AS1 and lnc-KHDC3L-2.


Assuntos
RNA Longo não Codificante/fisiologia , Processos de Crescimento Celular/genética , Movimento Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Canais de Potássio KCNQ/metabolismo , Anotação de Sequência Molecular , Oligonucleotídeos Antissenso , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno
2.
RNA Biol ; 20(1): 1523-1539, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-37743644

RESUMO

HOTAIRM1 is unlike most long non-coding RNAs in that its sequence is highly conserved across mammals. Such evolutionary conservation points to it having a role in key cellular processes. We previously reported that HOTAIRM1 is required to curb premature activation of downstream HOXA genes in a cell model recapitulating their sequential induction during development. We found that it regulates 3' HOXA gene expression by a mechanism involving epigenetic and three-dimensional chromatin changes. Here we show that HOTAIRM1 participates in proper progression through the early stages of neuronal differentiation. We found that it can associate with the HOXA1 transcription factor and contributes to its downstream transcriptional program. Particularly, HOTAIRM1 affects the NANOG/POU5F1/SOX2 core pluripotency network maintaining an undifferentiated cell state. HOXA1 depletion similarly perturbed expression of these pluripotent factors, suggesting that HOTAIRM1 is a modulator of this transcription factor pathway. Also, given that binding of HOTAIRM1 to HOXA1 was observed in different cell types and species, our results point to this ribonucleoprotein complex as an integral part of a conserved HOTAIRM1-HOXA1 regulatory axis modulating the transition from a pluripotent to a differentiated neuronal state.


Assuntos
MicroRNAs , Animais , MicroRNAs/genética , Diferenciação Celular/genética , Fatores de Transcrição/genética , Mamíferos/genética
3.
Nature ; 543(7646): 519-524, 2017 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-28273065

RESUMO

The organization of the genome in the nucleus and the interactions of genes with their regulatory elements are key features of transcriptional control and their disruption can cause disease. Here we report a genome-wide method, genome architecture mapping (GAM), for measuring chromatin contacts and other features of three-dimensional chromatin topology on the basis of sequencing DNA from a large collection of thin nuclear sections. We apply GAM to mouse embryonic stem cells and identify enrichment for specific interactions between active genes and enhancers across very large genomic distances using a mathematical model termed SLICE (statistical inference of co-segregation). GAM also reveals an abundance of three-way contacts across the genome, especially between regions that are highly transcribed or contain super-enhancers, providing a level of insight into genome architecture that, owing to the technical limitations of current technologies, has previously remained unattainable. Furthermore, GAM highlights a role for gene-expression-specific contacts in organizing the genome in mammalian nuclei.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Mapeamento Cromossômico , Elementos Facilitadores Genéticos/genética , Genoma/genética , Animais , Cromatina/química , Epigênese Genética , Masculino , Camundongos , Modelos Genéticos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Análise de Sequência de DNA , Transcrição Gênica/genética
4.
Genes Dev ; 28(24): 2778-91, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25512564

RESUMO

Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods.


Assuntos
Cromatina/química , Cromatina/metabolismo , Genoma/genética , Hibridização in Situ Fluorescente/normas , Coloração e Rotulagem/normas , Animais , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/citologia , Técnicas Genéticas/normas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Mutação , Proteínas do Grupo Polycomb/genética , Estrutura Terciária de Proteína
5.
Genes Dev ; 27(23): 2602-14, 2013 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-24298059

RESUMO

RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel "all-in-one" lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an "all-in-one" system to track disrupted Trp53 in chemoresistant lymphomas in the Eµ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.


Assuntos
Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Técnicas Genéticas , Animais , Proteínas Associadas a CRISPR/metabolismo , Feminino , Marcação de Genes , Genes p53/genética , Genoma/genética , Mutação INDEL/genética , Estimativa de Kaplan-Meier , Lentivirus/genética , Linfoma/genética , Linfoma/mortalidade , Linfoma/terapia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Reprodutibilidade dos Testes
6.
BMC Genomics ; 20(1): 162, 2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30819105

RESUMO

BACKGROUND: Understanding how transcription occurs requires the integration of genome-wide and locus-specific information gleaned from robust technologies. Chromatin immunoprecipitation (ChIP) is a staple in gene expression studies, and while genome-wide methods are available, high-throughput approaches to analyze defined regions are lacking. RESULTS: Here, we present carbon copy-ChIP (2C-ChIP), a versatile, inexpensive, and high-throughput technique to quantitatively measure the abundance of DNA sequences in ChIP samples. This method combines ChIP with ligation-mediated amplification (LMA) and deep sequencing to probe large genomic regions of interest. 2C-ChIP recapitulates results from benchmark ChIP approaches. We applied 2C-ChIP to the HOXA cluster to find that a region where H3K27me3 and SUZ12 linger encodes HOXA-AS2, a long non-coding RNA that enhances gene expression during cellular differentiation. CONCLUSIONS: 2C-ChIP fills the need for a robust molecular biology tool designed to probe dedicated genomic regions in a high-throughput setting. The flexible nature of the 2C-ChIP approach allows rapid changes in experimental design at relatively low cost, making it a highly efficient method for chromatin analysis.


Assuntos
Imunoprecipitação da Cromatina/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Diferenciação Celular/genética , Células Cultivadas , Epigênese Genética , Expressão Gênica , Genes Homeobox , Genômica , Humanos , RNA Longo não Codificante/fisiologia , Reação em Cadeia da Polimerase em Tempo Real
7.
Nucleic Acids Res ; 45(3): 1091-1104, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28180285

RESUMO

Thousands of long non-coding RNAs (lncRNAs) have been identified in mammals, many of which represent important regulators of gene expression. However, the mechanisms used by lncRNAs to control transcription remain largely uncharacterized. Here, we report on HOTAIRM1, a promising lncRNA biomarker in leukemia and solid tumors. We find that HOTAIRM1 contributes to three-dimensional chromatin organization changes required for the temporal collinear activation of HOXA genes. We show that distinct HOTAIRM1 variants preferentially associate with either UTX/MLL or PRC2 complexes to modulate the levels of activating and silencing marks at the bivalent domain. HOTAIRM1 contributes to physical dissociation of chromatin loops at the cluster proximal end, which delays recruitment of the histone demethylase UTX and transcription of central HOXA genes. Interestingly, we find overall proximal HOXA gene activation without chromatin conformation changes by HOTAIRM1 in a different cell type. Our results reveal a previously unappreciated relationship between chromatin structure, architecture and lncRNA function.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Genes Homeobox , Proteínas de Homeodomínio/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , MicroRNAs/antagonistas & inibidores , Modelos Genéticos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Interferência de RNA , Ativação Transcricional/efeitos dos fármacos , Tretinoína/farmacologia
8.
BMC Genomics ; 19(1): 515, 2018 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-29986647

RESUMO

BACKGROUND: Cis-regulatory elements control gene expression over large distances through the formation of chromatin loops, which allow contact between enhancers and gene promoters. Alterations in cis-acting regulatory systems could be linked to human genetic diseases. Here, we analyse the spatial organization of a large region spanning the polycystic kidney disease 2 (PKD2) gene, one of the genes responsible of autosomal dominant polycystic kidney disease (ADPKD). RESULTS: By using chromosome conformation capture carbon copy (5C) technology in primary human renal cyst epithelial cells, we identify novel contacts of the PKD2 promoter with chromatin regions, which display characteristics of regulatory elements. In parallel, by using functional analysis with a reporter assay, we demonstrate that three DNAse I hypersensitive sites regions are involved in the regulation of PKD2 gene expression. CONCLUSIONS: Finally, through alignment of CCCTC-binding factor (CTCF) sites, we suggest that these novel enhancer elements are brought to the PKD2 promoter by chromatin looping via the recruitment of CTCF.


Assuntos
Cromatina/metabolismo , Rim Policístico Autossômico Dominante/genética , Canais de Cátion TRPP/genética , Células A549 , Cromatina/química , Desoxirribonuclease I/metabolismo , Elementos Facilitadores Genéticos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Expressão Gênica , Humanos , Rim/citologia , Rim Policístico Autossômico Dominante/patologia , Regiões Promotoras Genéticas , Canais de Cátion TRPP/metabolismo
9.
Nucleic Acids Res ; 44(6): 2564-76, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26615198

RESUMO

A mechanism by which control DNA elements regulate transcription over large linear genomic distances is by achieving close physical proximity with genes, and looping of the intervening chromatin paths. Alterations of such regulatory 'chromatin looping' systems are likely to play a critical role in human genetic disease at large. Here, we studied the spatial organization of a ≈790 kb locus encompassing the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Dysregulation of CFTR is responsible for cystic fibrosis, which is the most common lethal genetic disorder in Caucasian populations. CFTR is a relatively large gene of 189 kb with a rather complex tissue-specific and temporal expression profile. We used chromatin conformation at the CFTR locus to identify new DNA sequences that regulate its transcription. By comparing 5C chromatin interaction maps of the CFTR locus in expressing and non-expressing human primary cells, we identified several new contact points between the CFTR promoter and its surroundings, in addition to regions featuring previously described regulatory elements. We demonstrate that two of these novel interacting regions cooperatively increase CFTR expression, and suggest that the new enhancer elements located on either side of the gene are brought together through chromatin looping via CTCF.


Assuntos
Cromatina/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Cromatina/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Loci Gênicos , Voluntários Saudáveis , Humanos , Cavidade Nasal/citologia , Cavidade Nasal/metabolismo , Cultura Primária de Células , Pele/citologia , Pele/patologia , Transcrição Gênica
10.
Mol Syst Biol ; 11(12): 852, 2015 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-26700852

RESUMO

Mammalian chromosomes fold into arrays of megabase-sized topologically associating domains (TADs), which are arranged into compartments spanning multiple megabases of genomic DNA. TADs have internal substructures that are often cell type specific, but their higher-order organization remains elusive. Here, we investigate TAD higher-order interactions with Hi-C through neuronal differentiation and show that they form a hierarchy of domains-within-domains (metaTADs) extending across genomic scales up to the range of entire chromosomes. We find that TAD interactions are well captured by tree-like, hierarchical structures irrespective of cell type. metaTAD tree structures correlate with genetic, epigenomic and expression features, and structural tree rearrangements during differentiation are linked to transcriptional state changes. Using polymer modelling, we demonstrate that hierarchical folding promotes efficient chromatin packaging without the loss of contact specificity, highlighting a role far beyond the simple need for packing efficiency.


Assuntos
Cromatina/química , Cromossomos/química , Células-Tronco Embrionárias Murinas/citologia , Neurônios/citologia , Transcrição Gênica , Animais , Diferenciação Celular , Células Cultivadas , Montagem e Desmontagem da Cromatina , Epigênese Genética , Regulação da Expressão Gênica , Camundongos
11.
Nucleic Acids Res ; 42(3): 1524-40, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24174538

RESUMO

Three-dimensional genome organization is an important higher order transcription regulation mechanism that can be studied with the chromosome conformation capture techniques. Here, we combined chromatin organization analysis by chromosome conformation capture-carbon copy, computational modeling and epigenomics to achieve the first integrated view, through time, of a connection between chromatin state and its architecture. We used this approach to examine the chromatin dynamics of the HoxA cluster in a human myeloid leukemia cell line at various stages of differentiation. We found that cellular differentiation involves a transient activation of the 5'-end HoxA genes coinciding with a loss of contacts throughout the cluster, and by specific silencing at the 3'-end with H3K27 methylation. The 3D modeling of the data revealed an extensive reorganization of the cluster between the two previously reported topologically associated domains in differentiated cells. Our results support a model whereby silencing by polycomb group proteins and reconfiguration of CTCF interactions at a topologically associated domain boundary participate in changing the HoxA cluster topology, which compartmentalizes the genes following differentiation.


Assuntos
Diferenciação Celular/genética , Cromatina/química , Proteínas de Homeodomínio/genética , Família Multigênica , Sítios de Ligação , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Cromatina/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Lactente , Elementos Isolantes , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Proteínas Repressoras/metabolismo , Ativação Transcricional
12.
PLoS Genet ; 9(12): e1004018, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24385922

RESUMO

HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet "DNA loops". Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.


Assuntos
Elementos Facilitadores Genéticos , Extremidades/crescimento & desenvolvimento , Proteínas de Homeodomínio/genética , Transcrição Gênica , Animais , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Vertebrados/genética , Vertebrados/crescimento & desenvolvimento
13.
Proc Natl Acad Sci U S A ; 109(40): 16173-8, 2012 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-22988072

RESUMO

Chromatin has a complex spatial organization in the cell nucleus that serves vital functional purposes. A variety of chromatin folding conformations has been detected by single-cell imaging and chromosome conformation capture-based approaches. However, a unified quantitative framework describing spatial chromatin organization is still lacking. Here, we explore the "strings and binders switch" model to explain the origin and variety of chromatin behaviors that coexist and dynamically change within living cells. This simple polymer model recapitulates the scaling properties of chromatin folding reported experimentally in different cellular systems, the fractal state of chromatin, the processes of domain formation, and looping out. Additionally, the strings and binders switch model reproduces the recently proposed "fractal-globule" model, but only as one of many possible transient conformations.


Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/química , Regulação da Expressão Gênica/genética , Genoma/genética , Modelos Químicos , Simulação por Computador , Hibridização in Situ Fluorescente , Método de Monte Carlo
15.
PLoS One ; 19(5): e0295971, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38709794

RESUMO

The human genome is pervasively transcribed and produces a wide variety of long non-coding RNAs (lncRNAs), constituting the majority of transcripts across human cell types. Some specific nuclear lncRNAs have been shown to be important regulatory components acting locally. As RNA-chromatin interaction and Hi-C chromatin conformation data showed that chromatin interactions of nuclear lncRNAs are determined by the local chromatin 3D conformation, we used Hi-C data to identify potential target genes of lncRNAs. RNA-protein interaction data suggested that nuclear lncRNAs act as scaffolds to recruit regulatory proteins to target promoters and enhancers. Nuclear lncRNAs may therefore play a role in directing regulatory factors to locations spatially close to the lncRNA gene. We provide the analysis results through an interactive visualization web portal at https://fantom.gsc.riken.jp/zenbu/reports/#F6_3D_lncRNA.


Assuntos
Cromatina , RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Cromatina/metabolismo , Cromatina/genética , Humanos , Anotação de Sequência Molecular , Núcleo Celular/metabolismo , Núcleo Celular/genética , Genoma Humano , Regiões Promotoras Genéticas
16.
Biochim Biophys Acta ; 1819(5): 401-10, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22207201

RESUMO

It has been known for some time that eukaryotic genomic DNA is packaged in the form of highly organized chromatin in vivo. This organization is important not only to reduce the length of chromosomes during interphase but also because it represents a type of higher-order genome regulation mechanism. Indeed, spatial chromatin architecture is known to be important for transcription, DNA replication and repair. Chromosome structure can be observed at different scales and studied with a variety of complementary techniques. For example, microscopy can provide single cell information while technologies such as the chromosome conformation capture (3C) method and its derivatives can yield higher-resolution data from cell populations. In this review, we report on the biological questions addressed with 3C and 3C-related techniques and what has been uncovered to date. We also explore what these methods may further reveal about the regulation of genomic DNA activities.


Assuntos
Cromatina , Cromossomos , Microscopia/métodos , Análise de Célula Única/métodos , Cromatina/metabolismo , Cromatina/ultraestrutura , Cromossomos/metabolismo , Cromossomos/ultraestrutura , DNA , Reparo do DNA/genética , Replicação do DNA/genética , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Regulação da Expressão Gênica , Genoma , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura
17.
Biochem Soc Trans ; 41(2): 508-12, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23514144

RESUMO

In the cell nucleus, chromosomes have a complex spatial organization, spanning several length scales, which serves vital functional purposes. It is unknown, however, how their three-dimensional architecture is orchestrated. In the present article, we review the application of a model based on classical polymer physics, the strings and binders switch model, to explain the molecular mechanisms of chromatin self-organization. We explore the scenario where chromatin architecture is shaped and regulated by the interactions of chromosomes with diffusing DNA-binding factors via thermodynamics mechanisms and compare it with available experimental data.


Assuntos
Cromatina/metabolismo , Modelos Biológicos , Animais , Humanos
18.
Methods ; 58(3): 255-67, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23137922

RESUMO

In eukaryotes, genome organization can be observed on many levels and at different scales. This organization is important not only to reduce chromosome length but also for the proper execution of various biological processes. High-resolution mapping of spatial chromatin structure was made possible by the development of the chromosome conformation capture (3C) technique. 3C uses chemical cross-linking followed by proximity-based ligation of fragmented DNA to capture frequently interacting chromatin segments in cell populations. Several 3C-related methods capable of higher chromosome conformation mapping throughput were reported afterwards. These techniques include the 3C-carbon copy (5C) approach, which offers the advantage of being highly quantitative and reproducible. We provide here an updated reference protocol for the production of 5C libraries analyzed by next-generation sequencing or onto microarrays. A procedure used to verify that 3C library templates bear the high quality required to produce superior 5C libraries is also described. We believe that this detailed protocol will help guide researchers in probing spatial genome organization and its role in various biological processes.


Assuntos
Cromatina/genética , Mapeamento Cromossômico/métodos , Animais , Sequência de Bases , Reagentes de Ligações Cruzadas/química , DNA/química , DNA/genética , DNA/isolamento & purificação , Primers do DNA/genética , Formaldeído/química , Biblioteca Gênica , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Fixação de Tecidos , Titulometria
19.
Cancers (Basel) ; 15(13)2023 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-37444543

RESUMO

As a group of diseases characterized by uncontrollable cell growth, cancer is highly multifaceted in how it overrides checkpoints controlling proliferation. Amongst the regulators of these checkpoints, long non-coding RNAs (lncRNAs) can have key roles in why natural biological processes go haywire. LncRNAs represent a large class of regulatory transcripts that can localize anywhere in cells. They were found to affect gene expression on many levels from transcription to mRNA translation and even protein stability. LncRNA participation in such control mechanisms can depend on cell context, with given transcripts sometimes acting as oncogenes or tumor suppressors. Importantly, the tissue-specificity and low expression levels of lncRNAs make them attractive therapeutic targets or biomarkers. Here, we review the various cellular processes affected by lncRNAs and outline molecular strategies they use to control gene expression, particularly in cancer and in relation to transcription factors.

20.
Nucleic Acids Res ; 38(21): 7472-84, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20660483

RESUMO

Spatial chromatin organization is emerging as an important mechanism to regulate the expression of genes. However, very little is known about genome architecture at high-resolution in vivo. Here, we mapped the three-dimensional organization of the human Hox clusters with chromosome conformation capture (3C) technology. We show that computational modeling of 3C data sets can identify candidate regulatory proteins of chromatin architecture and gene expression. Hox genes encode evolutionarily conserved master regulators of development which strict control has fascinated biologists for over 25 years. Proper transcriptional silencing is key to Hox function since premature expression can lead to developmental defects or human disease. We now show that the HoxA cluster is organized into multiple chromatin loops that are dependent on transcription activity. Long-range contacts were found in all four silent clusters but looping patterns were specific to each cluster. In contrast to the Drosophila homeotic bithorax complex (BX-C), we found that Polycomb proteins are only modestly required for human cluster looping and silencing. However, computational three-dimensional Hox cluster modeling identified the insulator-binding protein CTCF as a likely candidate mediating DNA loops in all clusters. Our data suggest that Hox cluster looping may represent an evolutionarily conserved structural mechanism of transcription regulation.


Assuntos
Cromatina/química , Inativação Gênica , Proteínas de Homeodomínio/genética , Família Multigênica , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Humanos , Masculino , Modelos Moleculares , Proteínas Repressoras/química , Proteínas Repressoras/fisiologia , Transcrição Gênica , Adulto Jovem
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