RESUMO
Stochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of four specific genomic loci. MATAC-Seq relies on preferential modification of accessible DNA by methyltransferases combined with Nanopore-Sequencing for direct readout of methylated DNA-bases. Applying MATAC-Seq to selected early-efficient and late-inefficient yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an accessible nucleosome-free region in combination with well-positioned +1 and +2 nucleosomes as a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during eukaryotic DNA replication. Consequently, our single-molecule chromatin accessibility assay will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.
Assuntos
Origem de Replicação , Saccharomyces cerevisiae , Cromatina/genética , DNA , Replicação do DNA , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Regulated intramembrane proteolysis (RIP) is a key mechanism for activating transmembrane proteins such as epithelial cell adhesion molecule (EpCAM) for cellular signaling and degradation. EpCAM is highly expressed in carcinomas and progenitor and embryonic stem cells and is involved in the regulation of cell adhesion, proliferation, and differentiation. Strictly sequential cleavage of EpCAM through RIP involves initial shedding of the extracellular domain by α-secretase (ADAM) and ß-secretase (BACE) sheddases, generating a membrane-tethered C-terminal fragment EpCTF. Subsequently, the rate-limiting γ-secretase complex catalyzes intramembrane cleavage of EpCTF, generating an extracellular EpCAM-Aß-like fragment and an intracellular EpICD fragment involved in nuclear signaling. Here, we have combined biochemical approaches with live-cell imaging of fluorescent protein tags to investigate the kinetics of γ-secretase-mediated intramembrane cleavage of EpCTF. We demonstrate that γ-secretase-mediated proteolysis of exogenously and endogenously expressed EpCTF is a slow process with a 50% protein turnover in cells ranging from 45 min to 5.5 h. The slow cleavage was dictated by γ-secretase activity and not by EpCTF species, as indicated by cross-species swapping experiments. Furthermore, both human and murine EpICDs generated from EpCTF by γ-secretase were degraded efficiently (94-99%) by the proteasome. Hence, proteolytic cleavage of EpCTF is a comparably slow process, and EpICD generation does not appear to be suited for rapidly transducing extracellular cues into nuclear signaling, but appears to provide steady signals that can be further controlled through efficient proteasomal degradation. Our approach provides an unbiased bioassay to investigate proteolytic processing of EpCTF in single living cells.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Membrana Celular/metabolismo , Molécula de Adesão da Célula Epitelial/química , Molécula de Adesão da Célula Epitelial/metabolismo , Espaço Intracelular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Animais , Linhagem Celular , Humanos , Cinética , Camundongos , Domínios ProteicosRESUMO
The effects of Photobacterium damselae ssp. piscicida (Phdp) on immune responses and intestinal ultrastructure of Artemia franciscana following infection and their amelioration by the probiotic bacteria Bacillus subtilis, Lactobacillus plantarum and Lactococcus lactis were evaluated. Pathogen growth inhibition in coculture with each probiotic and its virulence against Artemia were confirmed with an LC50 of 105 CFU mL-1. Phdp administration to Artemia at sublethal levels resulted in depletion of superoxide dismutase, glutathione reductase, glutathione transferase and phenoloxidase activities, extensive lipid peroxidation and reduced survival. Following a combined administration of each probiotic and the pathogen, enzyme activities and survival were significantly higher, while lipid peroxidation was reduced, compared to the infected group with no probiotic treatment (P < 0.05). The transmission electron microscopy study revealed that pathogen infection resulted in disarranged and fragmented microvilli, formation of empty or pathogen containing cytoplasmic vacuoles and damaged mitochondria. In the probiotic-treated and Phdp-infected series, intestinal cells showed normal appearance, except for the presence of pathogen-containing vacuoles and highly ordered but laterally stacked microvilli. The results of the present study indicate that Phdp induces cell death through an oxidative stress response and probiotics enhance Artemia immune responses to protect it against the Phdp induced damage.
RESUMO
The chromatin environment at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to known origin interactors, we find the microtubule-binding Ask1/DASH complex as an origin-regulating factor. Strikingly, tethering of Ask1 to individual origin sites advances replication timing (RT) of the targeted chromosomal domain. Targeted degradation of Ask1 globally changes RT of a subset of origins, which can be reproduced by inhibiting microtubule dynamics. Thus, our findings mechanistically connect RT and chromosomal organization via Ask1/DASH with the microtubule cytoskeleton.
Assuntos
Proteínas Associadas aos Microtúbulos , Origem de Replicação , Proteínas de Saccharomyces cerevisiae , Cromatina/metabolismo , DNA/metabolismo , Replicação do DNA , Período de Replicação do DNA , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos/metabolismo , Proteômica , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Besides the basic organization in nucleosome core particles (NCPs), eukaryotic chromatin is further packed through interactions with numerous protein complexes including transcription factors, chromatin remodeling and modifying enzymes. This nucleoprotein complex provides the template for many important biological processes, such as DNA replication, transcription, and DNA repair. Thus, to understand the molecular basis of these DNA transactions, it is critical to define individual changes of the chromatin structure at precise genomic regions where these machineries assemble and drive biological reactions. Single-molecule approaches provide the only possible solution to overcome the heterogenous nature of chromatin and monitor the behavior of individual chromatin transactions in real-time. In this review, we will give an overview of currently available single-molecule methods to obtain mechanistic insights into nucleosome positioning, histone modifications and DNA replication and transcription analysis-previously unattainable with population-based assays.