DNA Combing Research Articles

Competing effects of Kerr nonlinearity and K-photon absorptions on continuous-wave laser inscriptions

Material processing with laser offers a reliable tool in a large variety of micromachining technology, ranging from on-disc inscriptions to cell ablations through DNA combing and imprinting for fabrications of micro and nanofluidic devices. In these applications lasers are designed to operate in specific regimes characterized by their powers and wavelengths, so understanding characteristic properties of the distinct possible laser operation regimes turns out to be a relevant step toward optimization of their uses as well as improvement of the technology. In this work we examine the stability of a model of femtosecond laser intended for laser inscriptions in nonlinear transparent media, taking into consideration the laser-induced material damage and multiphoton ionization. The model approximates the laser dynamics by a complex Ginzburg–Landau equation with a Kerr plus a high-order nonlinear term accounting for K-photon ionization, coupled to a Drude equation for time evolution of the electron plasma density. Following the Benjamin–Feir instability theory it is shown that depending on characteristic parameters of the model, multiphoton ionization processes can stabilize continuous-wave or pulse regimes. These parameters include the group-delay dispersion, the linear and nonlinear gains, the phase shift per roundtrip and the plasma ionization rate.

Micromachining of Polymeric Microfluidic Micro/Nanoelectroporation Device.

Micro/nanochannel electroporation can deliver gene/drug into single cell with precise dosage control and much higher cell viability compared to traditional bulk electroporation. However, single cell micro/nanochannel electroporation has the problems of low efficiency and complicated operation. By integrating microfluidic with micro/nanochannel electroporation, a large number of cells can be processed within a short time. In this chapter, we provide a detailed protocol of fabrication microfluidic nanochannel electroporation devices. The fabrication of this microfluidic nanochannel electroporation device integrates soft lithography, DNA combing and imprinting, and micromilling. This device is appropriate for gene/drug delivery to a batch of cells. It has the advantages of both the single cell nanochannel electroporation and microfluidic based cell manipulation. The procedures of device fabrication, holder fabrication, cell trapping, and electroporation are included in this protocol.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Microfluidic DNA combing for parallel single-molecule analysis

DNA combing is a widely used method for stretching and immobilising DNA molecules on a surface. Fluorescent labelling of genomic information enables high-resolution optical analysis of DNA at the single-molecule level. Despite its simplicity, the application of DNA combing in diagnostic workflows is still limited, mainly due to difficulties in analysing multiple small-volume DNA samples in parallel. Here, we report a simple and versatile microfluidic DNA combing technology (μDC), which allows manipulating, stretching and imaging of multiple, microliter scale DNA samples by employing a manifold of parallel microfluidic channels. Using DNA molecules with repetitive units as molecular rulers, we demonstrate that the μDC technology allows uniform stretching of DNA molecules. The stretching ratio remains consistent along individual molecules as well as between different molecules in the various channels, allowing simultaneous quantitative analysis of different samples loaded into parallel channels. Furthermore, we demonstrate the application of μDC to characterise UVB-induced DNA damage levels in human embryonic kidney cells and the spatial correlation between DNA damage sites. Our results point out the potential application of μDC for quantitative and comparative single-molecule studies of genomic features. The extremely simple design of μDC makes it suitable for integration into other microfluidic platforms to facilitate high-throughput DNA analysis in biological research and medical point-of-care applications.

Budding yeast Rif1 binds to replication origins and protects DNA at blocked replication forks

Despite its evolutionarily conserved function in controlling DNA replication, the chromosomal binding sites of the budding yeast Rif1 protein are not well understood. Here, we analyse genome‐wide binding of budding yeast Rif1 by chromatin immunoprecipitation, during G1 phase and in S phase with replication progressing normally or blocked by hydroxyurea. Rif1 associates strongly with telomeres through interaction with Rap1. By comparing genomic binding of wild‐type Rif1 and truncated Rif1 lacking the Rap1‐interaction domain, we identify hundreds of Rap1‐dependent and Rap1‐independent chromosome interaction sites. Rif1 binds to centromeres, highly transcribed genes and replication origins in a Rap1‐independent manner, associating with both early and late‐initiating origins. Interestingly, Rif1 also binds around activated origins when replication progression is blocked by hydroxyurea, suggesting association with blocked forks. Using nascent DNA labelling and DNA combing techniques, we find that in cells treated with hydroxyurea, yeast Rif1 stabilises recently synthesised DNA. Our results indicate that, in addition to controlling DNA replication initiation, budding yeast Rif1 plays an ongoing role after initiation and controls events at blocked replication forks.

Abstract 912: uPA as a topoisomerase-1 protective agent in lung fibroblast cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, with approximately 53,000 people in the U.S. diagnosed with the disease every year. The Gondi lab has previously shown that poor survival rates in pancreatic cancer are correlated with increased expression of urokinase plasminogen activator (uPA), a serine protease. Our previous data has shown for the first time that uPA binds to topoisomerase-1 (TOPO-1), in addition to the interaction of uPA with various transcription factors. Our current research is aimed at elucidating the mechanism of the interaction between uPA and TOPO-1, with the goal of demonstrating a protective role for uPA in the regulation of TOPO-1 function in the presence of TOPO-1 inhibitors. Methods: Two knock-out lung fibroblast cell lines were used. One cell line was a uPA knock-out line, while the other was a uPA knock-out line with a uPA expression vector transfected into the cells. Transfection was performed using Chariot transfection vector. The cell lines were treated with topoisomerase inhibitors TX1111 and SN38. DNA replication activity was measured using flow cytometry as a surrogate for TOPO-1 activity. Controls included untreated cells, cells treated with empty Chariot, and cells treated with Chariot containing TXC peptide. Results and Discussion: In the presence of topoisomerase inhibitors TX1111 and SN38, uPA knock-out lines demonstrated incomplete cell division relative to controls. In uPA-expressing lines, cell division activity was retained in the presence of topoisomerase inhibitors. Our data demonstrates for the first time that uPA serves a protective role in preventing the inhibition of TOPO-1 by topoisomerase inhibitors. This indicates uPA as a potential new drug target in the treatment of chemoresistant pancreatic cancer. Further studies to be performed include visualizing the degree of DNA-replication using DNA combing, and subsequently expanding this study to pancreatic cancer cells. Citation Format: Liang Ge, Manu Gnanamony, Victoria Stepanova, Dzung H. Dinh, Christopher S. Gondi. uPA as a topoisomerase-1 protective agent in lung fibroblast cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 912.

A simple and novel DNA combing methodology for Fiber-FISH and optical mapping

Single molecule analysis can help us study genomics efficiently. It involves studying single DNA molecules for genomic studies. DNA combing is one of such techniques which allowed us to study single DNA molecules for multiple uses. DNA combing technology can be used to perform Fiber-FISH and optical mapping. Physical mapping of genomes can be studied by restriction digestion of combed DNA on glass slides. Restriction fragments can be arranged into optical maps by gathering fluorescent intensity data by CCD camera and image analysis by softwares. Physical mapping and DNA segment rearrangements can be studied by Fiber-FISH which involves application of probes on genomic DNA combed over glass slides. We developed a novel methodology involving combing solution optimization, denatured combed DNA and performed restriction digestion of combed DNA. Thus we provided an efficient and robust combing platform for its application in Fiber-FISH and optical mapping.

Blocking Buffer for DNA Combing

Blocking Buffer for DNA Combing

DNA Combing Plug Solution

DNA Combing Plug Solution

PBST for DNA Combing

PBST for DNA Combing

PREP1 tumor suppressor protects the late-replicating DNA by controlling its replication timing and symmetry

The synthesis of middle-to-late-replicating DNA can be affected independently of the rest of the genome by down-regulating the tumor suppressor PREP1 (PKNOX1). Indeed, DNA combing shows that PREP1 down-regulation affects DNA replication rate, increases the number of simultaneously firing origins and the asymmetry of DNA replication, leading to DNA damage. Genome-wide analysis of replication timing by Repli-seq shows that, upon PREP1 down-regulation, 25% of the genome is replicated earlier in the S-phase. The targeted DNA sequences correspond to Lamin-Associated Domains (LADs), and include late-replicating (LRRs) and temporal transition regions (TTRs). Notably, the distribution of PREP1 DNA binding sites and of its target genes indicates that DNA replication defects are independent of the overall PREP1 transcriptional activity. Finally, PREP1 down-regulation causes a substantial decrease in Lamin B1 levels. This suggests that DNA is released from the nuclear lamina earlier than in the control cells and is available for replication, thus explaining timing defects and DNA damage.This is the first evidence that the replication timing of a specific fraction of the human genome is affected by PREP1 tumor suppressor. This previously unknown function might significantly contribute to the genomic instability observed in human tumors.

Analysis of Fission Yeast Single DNA Molecules on the Megabase Scale Using DNA Combing.

DNA combing enables the quantitative analysis of DNA replication, DNA recombination, DNA-protein interaction, and DNA methylation along genomic single DNA molecules at 1kb resolution. However, DNA combing has been restricted to short 200-500kb long DNA fragments, which introduces significant bias in data analysis. An improved DNA combing methodology that allows to routinely image Mb-scale single DNA molecules and occasionally up to full-length fission yeast chromosomes is presented in this chapter. DNA combing of Mb-scale single DNA molecules can be applied to accurately measure the dynamic properties of DNA replication such as the rate of origin firing, replication fork velocity, fork directionality and the frequency of fork blockage. In addition, Mb-scale single DNA molecules enable the quantitative analysis of complex genomic rearrangements including gross chromosomal translocations, repetitive DNA sequences, large deletions, and duplications, which are difficult to investigate with deep sequencing strategies.

KDM6A addiction of cervical carcinoma cell lines is triggered by E7 and mediated by p21CIP1 suppression of replication stress.

Expression of E7 proteins encoded by carcinogenic, high-risk human papillomaviruses (HPVs) triggers increased expression of the histone H3 lysine 27 demethylase KDM6A. KDM6A expression is necessary for survival of high-risk HPV E7 expressing cells, including several cervical cancer lines. Here we show that increased KDM6A in response to high-risk HPV E7 expression causes epigenetic de-repression of the cell cycle and DNA replication inhibitor p21CIP1, and p21CIP1 expression is necessary for survival of high-risk HPV E7 expressing cells. The requirement for KDM6A and p21CIP1 expression for survival of high-risk HPV E7 expressing cells is based on p21CIP1’s ability to inhibit DNA replication through PCNA binding. We show that ectopic expression of cellular replication factors can rescue the loss of cell viability in response to p21CIP1 and KDM6A depletion. Moreover, we discovered that nucleoside supplementation will override the loss of cell viability in response to p21CIP1 depletion, suggesting that p21CIP1 depletion causes lethal replication stress. This model is further supported by increased double strand DNA breaks upon KDM6A or p21CIP1 depletion and DNA combing experiments that show aberrant re-replication upon KDM6A or p21CIP1 depletion in high-risk HPV E7 expressing cells. Therefore, KDM6A and p21CIP1 expression are essential to curb E7 induced replication stress to levels that do not markedly interfere with cell viability.

Enhanced post wash retention of combed DNA molecules by varying multiple combing parameters

Recent advances in genomics have created a need for efficient techniques for deciphering information hidden in various genomes. Single molecule analysis is one such technique to understand molecular processes at single molecule level. Fiber- FISH performed with the help of DNA combing can help us in understanding genetic rearrangements and changes in genome at single DNA molecule level. For performing Fiber-FISH we need high retention of combed DNA molecules post wash as Fiber-FISH requires profuse washing. We optimized combing process involving combing solution, method of DNA mounting on glass slides and coating of glass slides to enhance post-wash retention of DNA molecules. It was found that average number of DNA molecules observed post-wash per field of view was maximum with our optimized combing solution. APTES coated glass slides showed lesser retention than PEI surface but fluorescent intensity was higher in case of APTES coated surface. Capillary method used to mount DNA on glass slides also showed lesser retention but straight DNA molecules were observed as compared to force flow method.

Analysis of DNA Replication in Fission Yeast by Combing.

DNA replication studies based on population experiments give an average estimate of replication kinetics from many cells. This average replication profile masks the stochastic nature of origin firing in eukaryotes, which is revealed by using single-molecule techniques, such as DNA combing. The analysis of replication kinetics by DNA combing involves isolating DNA from cells that have been pulse-labeled with thymidine analogs and stretching it on a silanized coverslip. The analog-labeled patches on the stretched DNA fibers can then be detected using fluorescent antibodies against the analog. Each fiber represents a part of the genome from a single cell; therefore, it is possible to study the variation in behavior of individual origins from one cell to another. Furthermore, each DNA fiber is uniformly stretched, making it possible to measure distances accurately at kilobase resolution. It is also possible to stretch a high density of fibers on coverslips enabling quantitative data collection.

Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing.

The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart.

TET1 Regulates DNA Replication through Targeting of Minichromosome Maintenance Genes

DNA cytosine methylation is one of the best-characterized epigenetic modifications that play important roles in diverse cellular and pathological processes. The mechanism underlying the dynamic regulation of the level and distribution of 5-methylcytosine (5mC) as well as the biological consequence of DNA methylation deregulation have been interesting research topics in recent years. TET1, first identified as a fusion partner of the histone H3 Lys4 (H3K4) methyltransferase MLL (mixed-lineage leukemia) in acute myeloid leukemia (AML), is the founding member of the Ten-Eleven-Translocation (TET) family of DNA hydroxylases which are capable of converting 5mC to 5hmC (5-hydroxymethylcytosine) and lead to gene activation. Our group has previously demonstrated that TET1 plays an oncogenic role in MLL-rearranged leukemia (Huang H, et al. PNAS 2013; 110(29):11994-9). The expression of the TET1 protein and the global level of its enzymatic product, 5hmC, are significantly up-regulated in MLL-rearranged leukemia, whereas the opposite has been reported in other cancers where TET1 functions as a tumor suppressor. Therefore, a global understanding of the targets of TET1 in MLL-rearranged leukemia would greatly help to understand the role of TET1 in this specific type of AML.To this end, we performed proteomics study in parallel with RNA-seq to systematically explore the functional targets of TET1 in a genome-wide and unbiased way. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomic profiling showed that when Tet1 was knocked down in MLL-ENL-estrogen receptor inducible (ERtm) mouse myeloid leukemia cells, a total of 123 proteins were down-regulated whereas 191 were up-regulated with a fold-change cutoff of 1.2 (Fig. 1A and B), representing positively and negatively regulated targets of TET1, respectively. Most of the proteins with altered expression upon Tet1 knock-down showed a corresponding change at the mRNA level as reflected by the RNA-seq data. Interestingly, gene ontology (GO) analysis indicated enrichment on genes associated with DNA replication and cell cycle progression. Among these genes, the minichromosome maintenance complex genes, including MCM2, MCM3, MCM4, MCM5, MCM6, and MCM7, showed significant downregulation when Tet1 expression was depleted. We further conducted chromatin immunoprecipitation (ChIP) assays and demonstrated that TET1 binds directly to the CpG islands in the promoters of these MCM genes, suggesting that the regulation of the MCM genes by TET1 may occur at the transcriptional level.The six main minichromosome maintenance proteins (MCM2-7) are recruited to DNA replication origins in early G1 phase of the cell cycle and constitute the core of the replicative DNA helicase. We showed that not only the total levels of the MCM2-7 proteins, but also their binding to chromatin (Fig. 1C), were decreased by shRNAs against TET1 in human leukemia cell lines. Examination on cell cycle distribution revealed a significant decrease in the S phase population upon TET1 knockdown (Fig. 1D), which could be phenocopied by silencing of individual MCM genes. Consistently, incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into newly synthesized DNA in the S phase can be inhibited by TET1 shRNAs (Fig. 1E), indicating the inhibition on DNA replication by TET1 silencing. Furthermore, DNA combing assays suggest that TET1 knockdown inhibits new origin firing (Fig. 1F) but does not influence replication fork speed. Collectively, our findings reveal a novel role of TET1 on regulating DNA replication in MLL-rearranged leukemia through targeting of MCM genes and highlight the therapeutic implication of targeting the TET1/MCM signaling. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Global Increase in Replication Fork Speed during a p57KIP2-Regulated Erythroid Cell Fate Switch

Cell cycle regulators are increasingly implicated in cell fate decisions such as the acquisition or loss of pluripotency and self-renewal potential. The cell cycle mechanisms that regulate these cell fate decisions are largely unknown. Here we studied an S phase- dependent cell fate switch in the erythroid fetal liver, in which murine early erythroid progenitors transition in vivo from a self-renewal state into a phase of active erythroid gene transcription and concurrent maturational cell divisions. In the fetal liver, this transition corresponds to the transition from subset S0 (CD71-low, Ter119-negative) to subset S1 (CD71-high, Ter119-negative). We found that the S0 to S1 transition takes place during an S phase that is abruptly shorter (decreasing from 7 hours to 4 hours). Further, self-renewing S0 cells uniquely express the cyclin-dependent kinase (CDK) inhibitor p57KIP2 during S phase. To investigate its potential role, we studied DNA replication in vitro and in vivo in p57KIP2 -deficient fetal liver progenitors, employing a variety of techniques, including DNA combing. We found that S0 erythroid progenitors are dependent on p57KIP2-mediated slowing of replication forks for self-renewal, either in vivo, or in dexamethasone-dependent expansion cultures in vitro. The switch from self-renewal in S0 to differentiation in wild-type S1 progenitors entails rapid downregulation of p57KIP2 with a consequent global increase in replication fork speed and an abruptly shorter S phase. In the absence of p57KIP2, replication fork processivity increases prematurely in self-renewing S0 cells, prior to the activation of the erythroid transcriptional program (Figure 1), resulting in replicative stress and cell death.It is well established that differentiation leads to reprogramming of DNA replication, reflected by changes to origin usage and to the timing of replication of chromatin domains. Here we find that the replication program is fundamentally altered in additional key respects: the global processivity of replication forks, regulated by CDK activity, increases abruptly with the switch from self-renewal to differentiation, affecting DNA synthesis rates and S phase duration. Our results are also of interest since the regulation of replication kinetics was thought to be primarily via the regulation of origin firing efficiency, rather than via fork processivity. Here we found no difference in the former (there was no significant change in inter-origin distances, Figure 1). While the full significance of faster forks to the activation of the erythroid transcriptional program is yet to be understood, a recent report found that T cell help leads to faster forks and a shorter S phase in B cells (Gitlin et al., Science 349, 643-646 2015). Regulation of global fork speed may therefore be an intrinsic part of physiological developmental programs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of DNA Replication by Optical Mapping in Nanochannels.

DNA replication is essential to maintain genome integrity in S phase of the cell division cycle. Accumulation of stalled replication forks is a major source of genetic instability, and likely constitutes a key driver of tumorigenesis. The mechanisms of regulation of replication fork progression have therefore been extensively investigated, in particular with DNA combing, an optical mapping technique that allows the stretching of single molecules and the mapping of active region for DNA synthesis by fluorescence microscopy. DNA linearization in nanochannels has been successfully used to probe genomic information patterns along single chromosomes, and has been proposed to be a competitive alternative to DNA combing. Yet this conjecture remains to be confirmed experimentally. Here, two complementary techniques are established to detect the genomic distribution of tracks of newly synthesized DNA in human cells by optical mapping in nanochannels. Their respective advantages and limitations are compared, and applied them to detect deregulations of the replication program induced by the antitumor drug hydroxyurea. The developments here thus broaden the field of applications accessible to nanofluidic technologies, and can be used in the future as part for molecular diagnostics in the context of high throughput cancer drug screening.

Human Adipose-Derived Stem Cells Expanded Under Ambient Oxygen Concentration Accumulate Oxidative DNA Lesions and Experience Procarcinogenic DNA Replication Stress.

Adipose‐derived stem cells (ADSCs) have led to growing interest in cell‐based therapy because they can be easily harvested from an abundant tissue. ADSCs must be expanded in vitro before transplantation. This essential step causes concerns about the safety of adult stem cells in terms of potential transformation. Tumorigenesis is driven in its earliest step by DNA replication stress, which is characterized by the accumulation of stalled DNA replication forks and activation of the DNA damage response. Thus, to evaluate the safety of ADSCs during ex vivo expansion, we monitored DNA replication under atmospheric (21%) or physiologic (1%) oxygen concentration. Here, by combining immunofluorescence and DNA combing, we show that ADSCs cultured under 21% oxygen accumulate endogenous oxidative DNA lesions, which interfere with DNA replication by increasing fork stalling events, thereby leading to incomplete DNA replication and fork collapse. Moreover, we found by RNA sequencing (RNA‐seq) that culture of ADSCs under atmospheric oxygen concentration leads to misexpression of cell cycle and DNA replication genes, which could contribute to DNA replication stress. Finally, analysis of acquired small nucleotide polymorphism shows that expansion of ADSCs under 21% oxygen induces a mutational bias toward deleterious transversions. Overall, our results suggest that expanding ADSCs at a low oxygen concentration could reduce the risk for DNA replication stress‐associated transformation, as occurs in neoplastic tissues. Stem Cells Translational Medicine 2017;6:68–76