RESUMO
The heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation sites make mapping the location and timing of replication initiation in human cells difficult. To address this challenge, we have developed optical replication mapping (ORM), a high-throughput single-molecule approach, and used it to map early-initiation events in human cells. The single-molecule nature of our data and a total of >2,500-fold coverage of the human genome on 27 million fibers averaging â¼300 kb in length allow us to identify initiation sites and their firing probability with high confidence. We find that the distribution of human replication initiation is consistent with inefficient, stochastic activation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.
Assuntos
Replicação do DNA/genética , Células Eucarióticas/fisiologia , Genoma Humano/genética , Linhagem Celular Tumoral , Cromatina/genética , Período de Replicação do DNA/genética , Genoma Fúngico/genética , Estudo de Associação Genômica Ampla/métodos , Células HeLa , Humanos , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Sítio de Iniciação de Transcrição/fisiologiaRESUMO
Guided by the extensive knowledge of CRISPR-Cas9 molecular mechanisms, protein engineering can be an effective method in improving CRISPR-Cas9 toward desired traits different from those of their natural forms. Here, we describe a directed protein evolution method that enables selection of catalytically enhanced CRISPR-Cas9 variants (CECas9) by targeting a shortened protospacer within a toxic gene. We demonstrate the effectiveness of this method with a previously characterized Type II-C Cas9 from Acidothermus cellulolyticus (AceCas9) and show by enzyme kinetics an up to fourfold improvement of the in vitro catalytic efficiency by AceCECas9. We further evolved the more widely used Streptococcus pyogenes Cas9 (SpyCas9) and demonstrated a noticeable improvement in the SpyCECas9-facilitated homology directed repair-based gene insertion in human colon cancer cells.
Assuntos
Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Engenharia de Proteínas , Actinobacteria/enzimologia , Actinobacteria/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias do Colo , Edição de Genes/métodos , Células HCT116 , Humanos , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genéticaRESUMO
Cohesin plays an essential role in chromatin loop extrusion, but its impact on a compartmentalized nuclear architecture, linked to nuclear functions, is less well understood. Using live-cell and super-resolved 3D microscopy, here we find that cohesin depletion in a human colon cancer derived cell line results in endomitosis and a single multilobulated nucleus with chromosome territories pervaded by interchromatin channels. Chromosome territories contain chromatin domain clusters with a zonal organization of repressed chromatin domains in the interior and transcriptionally competent domains located at the periphery. These clusters form microscopically defined, active and inactive compartments, which likely correspond to A/B compartments, which are detected with ensemble Hi-C. Splicing speckles are observed nearby within the lining channel system. We further observe that the multilobulated nuclei, despite continuous absence of cohesin, pass through S-phase with typical spatio-temporal patterns of replication domains. Evidence for structural changes of these domains compared to controls suggests that cohesin is required for their full integrity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Mitose , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Humanos , Fase S , CoesinasRESUMO
Human B-cell precursor acute lymphoid leukemias (BCP-ALLs) comprise a group of genetically and clinically distinct disease entities with features of differentiation arrest at known stages of normal B-lineage differentiation. We previously showed that BCP-ALL cells display unique and clonally heritable, stable DNA replication timing (RT) programs (ie, programs describing the variable order of replication and subnuclear 3D architecture of megabase-scale chromosomal units of DNA in different cell types). To determine the extent to which BCP-ALL RT programs mirror or deviate from specific stages of normal human B-cell differentiation, we transplanted immunodeficient mice with quiescent normal human CD34+ cord blood cells and obtained RT signatures of the regenerating B-lineage populations. We then compared these with RT signatures for leukemic cells from a large cohort of BCP-ALL patients with varied genetic subtypes and outcomes. The results identify BCP-ALL subtype-specific features that resemble specific stages of B-cell differentiation and features that seem to be associated with relapse. These results suggest that the genesis of BCP-ALL involves alterations in RT that reflect biologically significant and potentially clinically relevant leukemia-specific epigenetic changes.