In situ detection of methylated DNA by histo endonuclease-linked detection of methylated DNA sites: a new principle of analysis of DNA methylation

Abstract

For a better understanding of epigenetic regulation of cell differentiation, it is important to analyze DNA methylation at a specific site. Although previous studies described methylation of isolated DNA extracted from cells and tissues using a combination of appropriate restriction endonucleases, no application to tissue cell level has been reported. Here, we report a new method, named histo endonuclease-linked detection of methylation sites of DNA (HELMET), designed to detect methylation sites of DNA with a specific sequences in a tissue section. In this study, we examined changes in the methylation level of CCGG sites during spermatogenesis in paraffin-embedded sections of mouse testis. In principle, the 3'-OH ends of DNA strand breaks in a section were firstly labeled with a mixture of dideoxynucleotides by terminal deoxynucleotidyl transferase (TdT), not to be further elongated by TdT. Then the section was digested with Hpa II, resulting in cutting the center portion of non-methylated CCGG. The cutting sites were labeled with biotin-16-dUTP by TdT. Next, the section was treated with Msp I, which can cut the CCGG sequence irrespective of the presence or absence of methylation of the second cytosine, and the cutting sites were labeled with digoxigenin-11-dUTP by TdT.Finally, both biotin and digoxigenin were visualized by enzyme-or fluorescence-immunohistochemistry. Using this method, we found hypermethylation of CCGG sites in most of the germ cells although non-methylated CCGG were colocalized in elongated spermatids. Interestingly, some TUNEL-positive germ cells, which are frequent in mammalian spermatogenesis, became markedly Hpa II-reactive, indicating that the CCGG sites may be demethylated during apoptosis. Koji T et al, Page 3

5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming

“…S4a). Following McrBC in situ digestion the DNA ends are marked by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labelling, modified HELMET assay 16 , see Supplementary Methods). McrBC TUNEL label increased in developing paternal pronuclei compared with maternal ones, whereas TUNEL signals were equal for both at early PN2 stages (see Supplementary Fig.…”

High expression of trimethylated histone H3 at lysine 27 predicts better prognosis in non-small cell lung cancer

“…To evaluate the DNA methylation level of pathological slides of NSCLC at CCGG sites, histo-endonuclease-linked detection of methylation sites of DNA (HELMET) was performed (30). Paraffin sections were dewaxed and digested with 10 µg/ml of proteinase K in PBS at 37˚C for 15 min.…”

“…We examined the correlation between these parameters and the level of DNA methylation at CCGG sites, detected by histo-endonuclease-linked detection of methylation sites of DNA (HELMET) (30). Since there had been several reports to indicate that H3K27me3 and EZH2 were involved in the early stage of various cancers, we focused on stage Ⅰ NSCLCs in this study.…”

Efficacy of aerosolized liposomal amphotericin B against murine invasive pulmonary mucormycosis

“…Immunohistochemical detection of AMB was performed on 5-μm frozen sections of mouse lung, as described previously [16]. …”