Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay

Long Interspersed Elements (LINE-1s, L1s) are the most active mobile elements in the human genome and account for a significant fraction of its mass. The propagation of L1 in the human genome requires disruption and repair of DNA at the site of integration. As Barbara McClintock first hypothesized, genotoxic stress may contribute to the mobilization of transposable elements, and conversely, element mobility may contribute to genotoxic stress. We tested the ability of genotoxic agents to increase L1 retrotransposition in a cultured cell assay. We observed that cells exposed to gamma radiation exhibited increased levels of L1 retrotransposition. The L1 retrotransposition frequency was proportional to the number of phosphorylated H2AX foci, an indicator of genotoxic stress. To explore the role of the L1 endonuclease in this context, endonuclease-deficient tagged L1 constructs were produced and tested for their activity in irradiated cells. The activity of the endonuclease-deficient L1 was very low in irradiated cells, suggesting that most L1 insertions in irradiated cells still use the L1 endonuclease. Consistent with this interpretation, DNA sequences that flank L1 insertions in irradiated cells harbored target site duplications. These results suggest that increased L1 retrotransposition in irradiated cells is endonuclease dependent. The mobilization of L1 in irradiated cells potentially contributes to genomic instability and could be a driving force for secondary mutations in patients undergoing radiation therapy

The effects of brain death and ischemia on tolerance induction are organ‐specific

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143776/1/ajt14674_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143776/2/ajt14674.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143776/3/ajt14674-sup-0001-FigS1-S2.pd

Comparative Analysis of DNA Replication Timing Reveals Conserved Large-Scale Chromosomal Architecture

Recent evidence suggests that the timing of DNA replication is coordinated across megabase-scale domains in metazoan genomes, yet the importance of this aspect of genome organization is unclear. Here we show that replication timing is remarkably conserved between human and mouse, uncovering large regions that may have been governed by similar replication dynamics since these species have diverged. This conservation is both tissue-specific and independent of the genomic G+C content conservation. Moreover, we show that time of replication is globally conserved despite numerous large-scale genome rearrangements. We systematically identify rearrangement fusion points and demonstrate that replication time can be locally diverged at these loci. Conversely, rearrangements are shown to be correlated with early replication and physical chromosomal proximity. These results suggest that large chromosomal domains of coordinated replication are shuffled by evolution while conserving the large-scale nuclear architecture of the genome

CpG Islands: Starting Blocks for Replication and Transcription

International audienc

Healing of fracture nonunions treated with low-intensity pulsed ultrasound (LIPUS): A systematic review and meta-analysis

Introduction: Bone fractures fail to heal and form nonunions in roughly 5% of cases, with little expectation of spontaneous healing thereafter. We present a systematic review and meta-analysis of published papers that describe nonunions treated with low-intensity pulsed ultrasound (LIPUS). Methods: Articles in PubMed, Ovid MEDLINE, CINAHL, AMED, EMBASE, Cochrane Library, and Scopus databases were searched, using an approach recommended by the Methodological Index for Non-Randomized Studies (MINORS), with a Level of Evidence rating by two reviewers independently. Studies are included here if they reported fractures older than 3 months, presented new data with a sample N ≥ 12, and reported fracture outcome (Heal/Fail). Results: Thirteen eligible papers reporting LIPUS treatment of 1,441 nonunions were evaluated. The pooled estimate of effect size for heal rate was 82% (95% CI: 77-87%), for any anatomical site and fracture age of at least 3 months, with statistical heterogeneity detected across all primary studies (Q = 41.2 (df = 12), p < 0.001, Tau2 = 0.006, I2 = 71). With a stricter definition of nonunion as fracture age of at least 8 months duration, the pooled estimate of effect size was 84% (95% CI: 77% − 91.6%; heterogeneity present: Q = 21 (df = 8), p < 0.001, Tau2 = 0.007, I2 = 62). Hypertrophic nonunions benefitted more than biologically inactive atrophic nonunions. An interval without surgery of <6 months prior to LIPUS was associated with a more favorable result. Stratification of nonunions by anatomical site revealed no statistically significant differences between upper and lower extremity long bone nonunions. Conclusions: LIPUS treatment can be an alternative to surgery for established nonunions. Given that no spontaneous healing of established nonunions is expected, and that it is challenging to test the efficacy of LIPUS for nonunion by randomized clinical trial, findings are compelling. LIPUS may be most useful in patients for whom surgery is high risk, including elderly patients at risk of delirium, or patients with dementia, extreme hypertension, extensive soft-tissue trauma, mechanical ventilation, metabolic acidosis, multiple organ failure, or coma. With an overall average success rate for LIPUS >80% this is comparable to the success of surgical treatment of non-infected nonunions

Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics

Somatic expression of LINE-1 elements in human tissues

LINE-1 expression damages host DNA via insertions and endonuclease-dependent DNA double-strand breaks (DSBs) that are highly toxic and mutagenic. The predominant tissue of LINE-1 expression has been considered to be the germ line. We show that both full-length and processed L1 transcripts are widespread in human somatic tissues and transformed cells, with significant variation in both L1 expression and L1 mRNA processing. This is the first demonstration that RNA processing is a major regulator of L1 activity. Many tissues also produce translatable spliced transcript (SpORF2). An Alu retrotransposition assay, COMET assays and 53BP1 foci staining show that the SpORF2 product can support functional ORF2 protein expression and can induce DNA damage in normal cells. Tests of the senescence-associated β-galactosidase expression suggest that expression of exogenous full-length L1, or the SpORF2 mRNA alone in human fibroblasts and adult stem cells triggers a senescence-like phenotype, which is one of the reported responses to DNA damage. In contrast to previous assumptions that L1 expression is germ line specific, the increased spectrum of tissues exposed to L1-associated damage suggests a role for L1 as an endogenous mutagen in somatic tissues. These findings have potential consequences for the whole organism in the form of cancer and mammalian aging

Replication Timing: A Fingerprint for Cell Identity and Pluripotency

Many types of epigenetic profiling have been used to classify stem cells, stages of cellular differentiation, and cancer subtypes. Existing methods focus on local chromatin features such as DNA methylation and histone modifications that require extensive analysis for genome-wide coverage. Replication timing has emerged as a highly stable cell type-specific epigenetic feature that is regulated at the megabase-level and is easily and comprehensively analyzed genome-wide. Here, we describe a cell classification method using 67 individual replication profiles from 34 mouse and human cell lines and stem cell-derived tissues, including new data for mesendoderm, definitive endoderm, mesoderm and smooth muscle. Using a Monte-Carlo approach for selecting features of replication profiles conserved in each cell type, we identify “replication timing fingerprints” unique to each cell type and apply a k nearest neighbor approach to predict known and unknown cell types. Our method correctly classifies 67/67 independent replication-timing profiles, including those derived from closely related intermediate stages. We also apply this method to derive fingerprints for pluripotency in human and mouse cells. Interestingly, the mouse pluripotency fingerprint overlaps almost completely with previously identified genomic segments that switch from early to late replication as pluripotency is lost. Thereafter, replication timing and transcription within these regions become difficult to reprogram back to pluripotency, suggesting these regions highlight an epigenetic barrier to reprogramming. In addition, the major histone cluster Hist1 consistently becomes later replicating in committed cell types, and several histone H1 genes in this cluster are downregulated during differentiation, suggesting a possible instrument for the chromatin compaction observed during differentiation. Finally, we demonstrate that unknown samples can be classified independently using site-specific PCR against fingerprint regions. In sum, replication fingerprints provide a comprehensive means for cell characterization and are a promising tool for identifying regions with cell type-specific organization

Preferential Localization of Human Origins of DNA Replication at the 5′-Ends of Expressed Genes and at Evolutionarily Conserved DNA Sequences

Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7–1.5 kb obtained from the breast cancer cell line MCF–7 hybridized to a custom tiling array containing 50–60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5′ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors

Replication Fork Polarity Gradients Revealed by Megabase-Sized U-Shaped Replication Timing Domains in Human Cell Lines

In higher eukaryotes, replication program specification in different cell types remains to be fully understood. We show for seven human cell lines that about half of the genome is divided in domains that display a characteristic U-shaped replication timing profile with early initiation zones at borders and late replication at centers. Significant overlap is observed between U-domains of different cell lines and also with germline replication domains exhibiting a N-shaped nucleotide compositional skew. From the demonstration that the average fork polarity is directly reflected by both the compositional skew and the derivative of the replication timing profile, we argue that the fact that this derivative displays a N-shape in U-domains sustains the existence of large-scale gradients of replication fork polarity in somatic and germline cells. Analysis of chromatin interaction (Hi-C) and chromatin marker data reveals that U-domains correspond to high-order chromatin structural units. We discuss possible models for replication origin activation within U/N-domains. The compartmentalization of the genome into replication U/N-domains provides new insights on the organization of the replication program in the human genome