RESUMEN
It is largely unclear whether genes that are naturally embedded in lamina-associated domains (LADs) are inactive due to their chromatin environment or whether LADs are merely secondary to the lack of transcription. We show that hundreds of human promoters become active when moved from their native LAD position to a neutral context in the same cells, indicating that LADs form a repressive environment. Another set of promoters inside LADs is able to "escape" repression, although their transcription elongation is attenuated. By inserting reporters into thousands of genomic locations, we demonstrate that escaper promoters are intrinsically less sensitive to LAD repression. This is not simply explained by promoter strength but by the interplay between promoter sequence and local chromatin features that vary strongly across LADs. Enhancers also differ in their sensitivity to LAD chromatin. This work provides a general framework for the systematic understanding of gene regulation by repressive chromatin.
Asunto(s)
Regulación de la Expresión Génica/genética , Lámina Nuclear/genética , Regiones Promotoras Genéticas/genética , Cromatina/genética , Cromatina/metabolismo , Expresión Génica/genética , Genoma Humano/genética , Genómica , Humanos , Células K562RESUMEN
In metazoan cell nuclei, hundreds of large chromatin domains are in close contact with the nuclear lamina. Such lamina-associated domains (LADs) are thought to help organize chromosomes inside the nucleus and have been associated with gene repression. Here, we discuss the properties of LADs, the molecular mechanisms that determine their association with the nuclear lamina, their dynamic links with other nuclear compartments, and their proposed roles in gene regulation.
Asunto(s)
Núcleo Celular/química , Cromatina/química , Animales , Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Heterocromatina , Humanos , Laminas/metabolismo , Lámina Nuclear/química , Poro Nuclear/metabolismoRESUMEN
The spatial organization of chromosomes influences many nuclear processes including gene expression. The cohesin complex shapes the 3D genome by looping together CTCF sites along chromosomes. We show here that chromatin loop size can be increased and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. Cohesin's DNA release factor WAPL restricts this loop extension and also prevents looping between incorrectly oriented CTCF sites. We reveal that the SCC2/SCC4 complex promotes the extension of chromatin loops and the formation of topologically associated domains (TADs). Our data support the model that cohesin structures chromosomes through the processive enlargement of loops and that TADs reflect polyclonal collections of loops in the making. Finally, we find that whereas cohesin promotes chromosomal looping, it rather limits nuclear compartmentalization. We conclude that the balanced activity of SCC2/SCC4 and WAPL enables cohesin to correctly structure chromosomes.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Acetiltransferasas/metabolismo , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN , Elongasas de Ácidos Grasos , Edición Génica , Humanos , Complejos Multiproteicos/metabolismo , Proteínas Represoras/metabolismo , CohesinasRESUMEN
The spatial organization of the genome into compartments and topologically associated domains can have an important role in the regulation of gene expression. But could gene expression conversely regulate genome organization? Here, we review recent studies that assessed the requirement of transcription and/or the transcription machinery for the establishment or maintenance of genome topology. The results reveal different requirements at different genomic scales. Transcription is generally not required for higher-level genome compartmentalization, has only moderate effects on domain organization and is not sufficient to create new domain boundaries. However, on a finer scale, transcripts or transcription does seem to have a role in the formation of subcompartments and subdomains and in stabilizing enhancer-promoter interactions. Recent evidence suggests a dynamic, reciprocal interplay between fine-scale genome organization and transcription, in which each is able to modulate or reinforce the activity of the other.
Asunto(s)
Cromatina/metabolismo , Elementos de Facilitación Genéticos , Genoma Humano , Regiones Promotoras Genéticas , Transcripción Genética , Animales , Cromatina/genética , HumanosRESUMEN
Mammalian interphase chromosomes interact with the nuclear lamina (NL) through hundreds of large lamina-associated domains (LADs). We report a method to map NL contacts genome-wide in single human cells. Analysis of nearly 400 maps reveals a core architecture consisting of gene-poor LADs that contact the NL with high cell-to-cell consistency, interspersed by LADs with more variable NL interactions. The variable contacts tend to be cell-type specific and are more sensitive to changes in genome ploidy than the consistent contacts. Single-cell maps indicate that NL contacts involve multivalent interactions over hundreds of kilobases. Moreover, we observe extensive intra-chromosomal coordination of NL contacts, even over tens of megabases. Such coordinated loci exhibit preferential interactions as detected by Hi-C. Finally, the consistency of NL contacts is inversely linked to gene activity in single cells and correlates positively with the heterochromatic histone modification H3K9me3. These results highlight fundamental principles of single-cell chromatin organization. VIDEO ABSTRACT.
Asunto(s)
Cromatina/metabolismo , Lámina Nuclear/metabolismo , Análisis de la Célula Individual/métodos , Línea Celular Tumoral , Cromatina/química , Cromosomas/química , Cromosomas/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Hibridación Fluorescente in Situ , InterfaseRESUMEN
Gene expression is in part controlled by cis-regulatory elements (CREs) such as enhancers and repressive elements. Anecdotal evidence has indicated that a CRE and a promoter need to be biochemically compatible for promoter regulation to occur, but this compatibility has remained poorly characterized in mammalian cells. We used high-throughput combinatorial reporter assays to test thousands of CRE-promoter pairs from three Mb-sized genomic regions in mouse cells. This revealed that CREs vary substantially in their promoter compatibility, ranging from striking specificity to broad promiscuity. More than half of the tested CREs exhibit significant promoter selectivity. Housekeeping promoters tend to have similar CRE preferences, but other promoters exhibit a wide diversity of compatibilities. Higher-order transcription factors (TF) motif combinations may account for compatibility. CRE-promoter selectivity does not correlate with looping interactions in the native genomic context, suggesting that chromatin folding and compatibility are two orthogonal mechanisms that confer specificity to gene regulation.
Asunto(s)
Elementos de Facilitación Genéticos , Genoma , Regiones Promotoras Genéticas , Factores de Transcripción , Animales , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Genoma/genética , Genómica , Mamíferos/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Transcriptional heterogeneity due to plasticity of the epigenetic state of chromatin contributes to tumour evolution, metastasis and drug resistance1-3. However, the mechanisms that cause this epigenetic variation are incompletely understood. Here we identify micronuclei and chromosome bridges, aberrations in the nucleus common in cancer4,5, as sources of heritable transcriptional suppression. Using a combination of approaches, including long-term live-cell imaging and same-cell single-cell RNA sequencing (Look-Seq2), we identified reductions in gene expression in chromosomes from micronuclei. With heterogeneous penetrance, these changes in gene expression can be heritable even after the chromosome from the micronucleus has been re-incorporated into a normal daughter cell nucleus. Concomitantly, micronuclear chromosomes acquire aberrant epigenetic chromatin marks. These defects may persist as variably reduced chromatin accessibility and reduced gene expression after clonal expansion from single cells. Persistent transcriptional repression is strongly associated with, and may be explained by, markedly long-lived DNA damage. Epigenetic alterations in transcription may therefore be inherently coupled to chromosomal instability and aberrations in nuclear architecture.
Asunto(s)
Inestabilidad Cromosómica , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Micronúcleos con Defecto Cromosómico , Neoplasias , Transcripción Genética , Humanos , Cromatina/genética , Cromatina/metabolismo , Cromosomas/genética , Células Clonales/metabolismo , Daño del ADN/genética , Neoplasias/genética , Neoplasias/patología , Análisis de Expresión Génica de una Sola CélulaRESUMEN
DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN , Secuencia de Bases , Reparación del ADN por Unión de Extremidades , Eucromatina/metabolismo , Reordenamiento Génico , Genoma Humano , Heterocromatina/metabolismo , Humanos , Mutación INDEL/genética , Células K562 , Cinética , Unión Proteica , Reproducibilidad de los ResultadosRESUMEN
The architecture of interphase chromosomes is important for the regulation of gene expression and genome maintenance. Chromosomes are linearly segmented into hundreds of domains with different protein compositions. Furthermore, the spatial organization of chromosomes is nonrandom and is characterized by many local and long-range contacts among genes and other sequence elements. A variety of genome-wide mapping techniques have made it possible to chart these properties at high resolution. Combined with microscopy and computational modeling, the results begin to yield a more coherent picture that integrates linear and three-dimensional (3D) views of chromosome organization in relation to gene regulation and other nuclear functions.
Asunto(s)
Núcleo Celular/genética , Cromosomas/química , Interfase , Animales , Nucléolo Celular/metabolismo , Estructuras Cromosómicas , Cromosomas/metabolismo , Humanos , Lámina Nuclear/metabolismoRESUMEN
Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here, we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two different promoters, show â¼1,000-fold variation in expression levels. Data analysis indicates that lamina-associated domains act as attenuators of transcription, likely by reducing access of transcription factors to binding sites. Furthermore, chromatin compaction is predictive of reporter activity. We also found evidence for crosstalk between neighboring genes and estimate that enhancers can influence gene expression on average over â¼20 kb. The multiplexed reporter assay is highly flexible in design and can be modified to query a wide range of aspects of gene regulation.
Asunto(s)
Efectos de la Posición Cromosómica , Técnicas Genéticas , Animales , Cromatina/metabolismo , Células Madre Embrionarias/metabolismo , Genes Reporteros , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Regiones Promotoras GenéticasRESUMEN
The nuclear lamina (NL) interacts with hundreds of large genomic regions termed lamina associated domains (LADs). The dynamics of these interactions and the relation to epigenetic modifications are poorly understood. We visualized the fate of LADs in single cells using a "molecular contact memory" approach. In each nucleus, only ~30% of LADs are positioned at the periphery; these LADs are in intermittent molecular contact with the NL but remain constrained to the periphery. Upon mitosis, LAD positioning is not detectably inherited but instead is stochastically reshuffled. Contact of individual LADs with the NL is linked to transcriptional repression and H3K9 dimethylation in single cells. Furthermore, we identify the H3K9 methyltransferase G9a as a regulator of NL contacts. Collectively, these results highlight principles of the dynamic spatial architecture of chromosomes in relation to gene regulation.
Asunto(s)
Cromosomas/metabolismo , Regulación de la Expresión Génica , Lámina Nuclear/química , Análisis de la Célula Individual/métodos , Adenina/metabolismo , Línea Celular Tumoral , Metilación de ADN , Genoma , Heterocromatina/metabolismo , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Mitosis , Lámina Nuclear/metabolismoRESUMEN
Chromosome structure in mammals is thought to regulate transcription by modulating three-dimensional interactions between enhancers and promoters, notably through CTCF-mediated loops and topologically associating domains (TADs)1-4. However, how chromosome interactions are actually translated into transcriptional outputs remains unclear. Here, to address this question, we use an assay to position an enhancer at large numbers of densely spaced chromosomal locations relative to a fixed promoter, and measure promoter output and interactions within a genomic region with minimal regulatory and structural complexity. A quantitative analysis of hundreds of cell lines reveals that the transcriptional effect of an enhancer depends on its contact probabilities with the promoter through a nonlinear relationship. Mathematical modelling suggests that nonlinearity might arise from transient enhancer-promoter interactions being translated into slower promoter bursting dynamics in individual cells, therefore uncoupling the temporal dynamics of interactions from those of transcription. This uncovers a potential mechanism of how distal enhancers act from large genomic distances, and of how topologically associating domain boundaries block distal enhancers. Finally, we show that enhancer strength also determines absolute transcription levels as well as the sensitivity of a promoter to CTCF-mediated transcriptional insulation. Our measurements establish general principles for the context-dependent role of chromosome structure in long-range transcriptional regulation.
Asunto(s)
Cromosomas , Elementos de Facilitación Genéticos , Animales , Cromatina/genética , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Genómica , Mamíferos/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
Some Ts in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (ß-D-glucosyl-hydroxymethyluracil). In Leishmania, about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA polymerase II termination sites. This internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J in JBP2 knockout cells is accompanied by massive readthrough at RNA polymerase II termination sites. The readthrough varies between transcription units but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive readthrough of transcriptional stops.
Asunto(s)
Glucósidos/metabolismo , Leishmania/genética , Leishmania/metabolismo , Transcripción Genética , Uracilo/análogos & derivados , Técnicas de Inactivación de Genes , ARN Polimerasa II/metabolismo , ARN Bicatenario/metabolismo , Uracilo/metabolismoRESUMEN
The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to â¼10 hr. Furthermore, repair of the DSBs tends to be error prone. Both classical and microhomology-mediated end joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci and indicates that Cas9-induced DSBs are repaired in an unusual manner.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Edición Génica/métodos , Proteína 9 Asociada a CRISPR/metabolismo , Humanos , Mutación INDEL , Células K562 , Cinética , Modelos GenéticosRESUMEN
The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break in different chromatin environments. We then measured Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 58 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g. PCI-24781), while others were only effective in euchromatin and H3K27me3-marked regions (e.g. apicidin). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a resource to improve Cas9 editing more selectively at the desired location.
RESUMEN
Chromatin is important for the regulation of transcription and other functions, yet the diversity of chromatin composition and the distribution along chromosomes are still poorly characterized. By integrative analysis of genome-wide binding maps of 53 broadly selected chromatin components in Drosophila cells, we show that the genome is segmented into five principal chromatin types that are defined by unique yet overlapping combinations of proteins and form domains that can extend over > 100 kb. We identify a repressive chromatin type that covers about half of the genome and lacks classic heterochromatin markers. Furthermore, transcriptionally active euchromatin consists of two types that differ in molecular organization and H3K36 methylation and regulate distinct classes of genes. Finally, we provide evidence that the different chromatin types help to target DNA-binding factors to specific genomic regions. These results provide a global view of chromatin diversity and domain organization in a metazoan cell.
Asunto(s)
Cromatina/clasificación , Proteínas de Unión al ADN/análisis , Proteínas de Drosophila/análisis , Drosophila melanogaster/genética , Animales , Línea Celular , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Eucromatina/metabolismo , Heterocromatina/metabolismo , Histonas/metabolismo , Análisis de Componente PrincipalRESUMEN
In this issue of Molecular Cell, Xie et al. (2017) introduce Mosaic-seq, a powerful technology that combines CRISPRi and single-cell RNA-seq. This method enables the high-throughput assessment of contributions of enhancers to gene regulation.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , ARN/genéticaRESUMEN
TP53 is a transcription factor that controls multiple cellular processes, including cell cycle arrest, DNA repair and apoptosis. The relation between TP53 binding site architecture and transcriptional output is still not fully understood. Here, we systematically examined in three different cell lines the effects of binding site affinity and copy number on TP53-dependent transcriptional output, and also probed the impact of spacer length and sequence between adjacent binding sites, and of core promoter identity. Paradoxically, we found that high-affinity TP53 binding sites are less potent than medium-affinity sites. TP53 achieves supra-additive transcriptional activation through optimally spaced adjacent binding sites, suggesting a cooperative mechanism. Optimally spaced adjacent binding sites have a â¼10-bp periodicity, suggesting a role for spatial orientation along the DNA double helix. We leveraged these insights to construct a log-linear model that explains activity from sequence features, and to identify new highly active and sensitive TP53 reporters.
RESUMEN
Classic promoter mutagenesis strategies can be used to study how proximal promoter regions regulate the expression of particular genes of interest. This is a laborious process, in which the smallest sub-region of the promoter still capable of recapitulating expression in an ectopic setting is first identified, followed by targeted mutation of putative transcription factor binding sites. Massively parallel reporter assays such as survey of regulatory elements (SuRE) provide an alternative way to study millions of promoter fragments in parallel. Here we show how a generalized linear model (GLM) can be used to transform genome-scale SuRE data into a high-resolution genomic track that quantifies the contribution of local sequence to promoter activity. This coefficient track helps identify regulatory elements and can be used to predict promoter activity of any sub-region in the genome. It thus allows in silico dissection of any promoter in the human genome to be performed. We developed a web application, available at cissector.nki.nl, that lets researchers easily perform this analysis as a starting point for their research into any promoter of interest.
Asunto(s)
Regiones Promotoras Genéticas , Programas Informáticos , Humanos , Sitios de Unión , Genoma Humano/genética , Unión Proteica , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Transcriptionally inactive genes are often positioned at the nuclear lamina (NL), as part of large lamina-associated domains (LADs). Activation of such genes is often accompanied by repositioning toward the nuclear interior. How this process works and how it impacts flanking chromosomal regions are poorly understood. We addressed these questions by systematic activation or inactivation of individual genes, followed by detailed genome-wide analysis of NL interactions, replication timing, and transcription patterns. Gene activation inside LADs typically causes NL detachment of the entire transcription unit, but rarely more than 50-100 kb of flanking DNA, even when multiple neighboring genes are activated. The degree of detachment depends on the expression level and the length of the activated gene. Loss of NL interactions coincides with a switch from late to early replication timing, but the latter can involve longer stretches of DNA. Inactivation of active genes can lead to increased NL contacts. These extensive datasets are a resource for the analysis of LAD rewiring by transcription and reveal a remarkable flexibility of interphase chromosomes.