Cancer Biology

The basic unit of chromatin is the nucleosome, a histone octamer with 147 base pairs of DNA wrapped around it. Positions of nucleosomes relative to each other and to DNA elements have a strong impact on chromatin structure and gene activity and are tightly regulated at multiple levels, i.e., DNA sequence, transcription factor binding, histone modifications and variants, and chromatin remodeling enzymes (Bell et al., 2011; Hughes and Rando, 2014). Nucleosome positions in cells or isolated nuclei can be detected by partial nuclease digestion of native or cross-linked chromatin followed by ligation-mediated polymerase chain reaction (LM-PCR) (McPherson et al., 1993; Soutoglou and Talianidis, 2002). This protocol describes a nucleosome positioning assay using Micrococcal Nuclease (MNase) digestion of formaldehyde-fixed chromatin followed by LM-PCR. We exemplify the nucleosome positioning assay for the promoter of genes encoding ribosomal RNA (rRNA genes or rDNA) in mice, which has two mutually exclusive configurations. The rDNA promoter harbors either an upstream nucleosome (NucU) covering nucleotides -157 to -2 relative to the transcription start site, or a downstream nucleosome (NucD) at position -132 to +22 (Li et al., 2006; Xie et al., 2012). Radioactive labeling of LM-PCR products followed by denaturing urea-polyacrylamide gel electrophoresis allows resolution and relative quantification of both configurations. As depicted in the diagram in Figure 1, the nucleosome positioning assay is a versatile low to medium throughput method to map discrete nucleosome positions with high precision in a semi-quantitative manner.


Figure 1. Flow chart depicting the nucleosome positioning assay. The diagram shows how the assay is used to detect the ratio between upstream (NucU) and downstream (NucD) nucleosome positions at the mouse rDNA promoter. After all steps have been performed, the LM-PCR yields two radiolabeled products that differ in size and correspond to NucU and NucD. Signal intensities of the bands reflect the relative abundance of each nucleosome position in the original sample.

The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The following document describes the protocol to realize a neutral comet assay. This assay can be applied to different cell types and has been useful for numerous applications in fields of toxicology or DNA damage and repair.

Many environmental agents induce double-strand breaks (DSBs) in DNA. Unrepaired or improperly repaired DSBs can lead to cell death or cancer. Nonhomologous end joining is the primary DNA double-strand break repair pathway in eukaryotes. During NHEJ pathway, several proteins recognize and bind DNA ends, bring the ends in a synaptic complex and, finally, process and ligate the ends.

Briefly, NHEJ starts with Ku protein. Ku binds the broken DNA ends and recruits the catalytic subunit of DNA dependent protein kinase (DNA-PKcs) forming DNA-PK. After processing, the XRCC4/Ligase IV complex executes the final ligation stimulated by Cernunnos-XLF.

Here, we describe an end-synapsis assay. This assay can be used in order to delineate which proteins are necessary to bring the DNA ends in a stable synaptic complex during NHEJ. Briefly, NHEJ competent extracts from human cells were incubated with both a double-stranded DNA fragment bound to streptavidin-coated magnetic beads and the same soluble radio-labeled fragment. The beads were then washed in mild salt buffer and the radioactivity recovered with the beads was measured by scintillation counting. Control experiments without extracts or with DNA-free beads were run in parallel to determine unspecific background.

8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) is among the most common reactive oxygen species-induced DNA lesions and can be used as a biomarker for oxidative stress. The lesion has been linked to several biological processes and diseases, including colorectal cancer, Huntington’s disease, estrogen-induced gene expression, and thymine dimer repair (reviewed in Delaney et al., 2012). The following assay is used to quantify 8-oxo-dGuo levels in DNA as described in Sousa et al. (2013).

Detection of low copies of methylated DNA targets in clinical specimens is challenging. The quantitative Methylation-Specific PCR (qMSP) assays were designed to specifically amplify bisulphite-converted methylated DNA target sequences in the presence of an excess of unmethylated counterpart sequences. These qMSP assays are real-time PCR assays utilizing, sequence-specific primers and an intervening, also sequence specific, Taqman probe to cover an amplicon of approximately 100 bp in length. The use of Taqman probes bearing a minor groove binding (MGB) allow for the use of shorter probes and therefore facilitate design and significantly increases the analytical specificity of the reaction. In the context of the biomarker discovery program of the Liverpool Lung Project (LLP), ten gene promoters were selected. qMSP assays were developed, validated and used to screen 655 bronchial washings from patients with lung cancer and age/sex matched controls with non malignant lung disease (Nikolaidis et al., 2012).