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As a Nucleus Enters a Small Pore, Chromatin Stretches and Maintains Integrity, Even with DNA Breaks.

ABSTRACT: As a cell pushes or pulls its nucleus through a small constriction, the chromatin must distort and somehow maintain genomic stability despite ever-present double-strand breaks in the DNA. Here we visualize within a living cell the pore-size dependent deformation of a specific locus engineered into chromosome-1 and cleaved. An mCherry-tagged nuclease targets the submicron locus, causing DNA cleavage and recruiting repair factors such as GFP-53BP1 to a large region around the locus. Aspiration of a cell and its nucleus into a micropipette shows that chromatin aligns and stretches parallel to the pore. Extension is largest in small pores, increasing >10-fold but remaining 30-fold shorter than the DNA contour length in the locus. Brochard and de Gennes' blob model for tube geometry fits the data, with a simple modification for chromatin crowding. Continuity of the highly extended, cleaved chromatin is also maintained, consistent with folding and cross bridging of the DNA. Surprisingly, extensional integrity is unaffected by an inhibitor of the DNA repair scaffold.

SUBMITTER: Irianto J

PROVIDER: S-EPMC5300774 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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As a Nucleus Enters a Small Pore, Chromatin Stretches and Maintains Integrity, Even with DNA Breaks.

Irianto Jerome J Xia Yuntao Y Pfeifer Charlotte R CR Greenberg Roger A RA Discher Dennis E DE

Biophysical journal 20161027 3

As a cell pushes or pulls its nucleus through a small constriction, the chromatin must distort and somehow maintain genomic stability despite ever-present double-strand breaks in the DNA. Here we visualize within a living cell the pore-size dependent deformation of a specific locus engineered into chromosome-1 and cleaved. An mCherry-tagged nuclease targets the submicron locus, causing DNA cleavage and recruiting repair factors such as GFP-53BP1 to a large region around the locus. Aspiration of ...[more]

PMID: 28341535

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Project description:BACKGROUND: Gene expression studies can be used to help identify disease-associated genes by comparing the levels of expressed transcripts between cases and controls, and to identify functional genetic variants (expression quantitative loci or eQTLs) by comparing expression levels between individuals with different genotypes. While many of these studies are performed in blood or lymphoblastoid cell lines due to tissue accessibility, the relevance of expression differences in tissues that are not the primary site of disease is unclear. Further, many eQTLs are tissue specific. Thus, there is a clear and compelling need to conduct gene expression studies in tissues that are specifically relevant to the disease of interest. One major technical concern about using autopsy-derived tissue is how representative it is of physiologic conditions, given the effect of postmortem interval on tissue degradation. RESULTS: In this study, we monitored the gene expression of 13 tissue samples harvested from a rapid autopsy heart (non-failed heart) and 7 from a cardiac explant (failed heart) through 24 hours of autolysis. The 24 hour autopsy simulation was designed to reflect a typical autopsy scenario where a body may begin cooling to ambient temperature for ~12 hours, before transportation and storage in a refrigerated room in a morgue. In addition, we also simulated a scenario wherein the body was left at room temperature for up to 24 hours before being found. A small fraction (< 2.5%) of genes showed fluctuations in expression over the 24 hr period and largely belong to immune and signal response and energy metabolism-related processes. Global expression analysis suggests that RNA expression is reproducible over 24 hours of autolysis with 95% genes showing < 1.2 fold change. Comparing the rapid autopsy to the failed heart identified 480 differentially expressed genes, including several types of collagens, lumican (LUM), natriuretic peptide A (NPPA) and connective tissue growth factor (CTGF), which allows for the clear separation between failing and non-failing heart based on gene expression profiles. CONCLUSIONS: Our results demonstrate that RNA from autopsy-derived tissue, even up to 24 hours of autolysis, can be used to identify biologically relevant expression pattern differences, thus serving as a practical source for gene expression experiments.

Dataset Information

As a Nucleus Enters a Small Pore, Chromatin Stretches and Maintains Integrity, Even with DNA Breaks.

Publications

As a Nucleus Enters a Small Pore, Chromatin Stretches and Maintains Integrity, Even with DNA Breaks.

Similar Datasets

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