Super-Resolution Localization Microscopy of γ-H2AX and Heterochromatin after Folate Deficiency

Abstract

Folate is an essential water-soluble vitamin in food and nutrition supplements. As a one-carbon source, it is involved in many central regulatory processes, such as DNA, RNA, and protein methylation as well as DNA synthesis and repair. Deficiency in folate is considered to be associated with an increased incidence of several malignancies, including cervical cancer that is etiologically linked to an infection with “high-risk” human papilloma viruses (HPV). However, it is still not known how a recommended increase in dietary folate after its deprivation affects the physiological status of cells. To study the impact of folate depletion and its subsequent reconstitution in single cells, we used quantitative chromatin conformation measurements obtained by super-resolution fluorescence microscopy, i.e., single molecule localization microscopy (SMLM). As a read-out, we examined the levels and the (re)positioning of γ-H2AX tags and histone H3K9me3 heterochromatin tags after immunostaining in three-dimensional (3D)-conserved cell nuclei. As model, we used HPV16 positive immortalized human keratinocytes that were cultivated under normal, folate deficient, and reconstituted conditions for different periods of time. The results were compared to cells continuously cultivated in standard folate medium. After 13 weeks in low folate, an increase in the phosphorylation of the histone H2AX was noted, indicative of an accumulation of DNA double strand breaks. DNA repair activity represented by the formation of those γ-H2AX clusters was maintained during the following 15 weeks of examination. However, the clustered arrangements of tags appeared to relax in a time-dependent manner. Parallel to the repair activity, the chromatin methylation activity increased as detected by H3K9me3 tags. The progress of DNA double strand repair was accompanied by a reduction of the detected nucleosome density around the γ-H2AX clusters, suggesting a shift from hetero- to euchromatin to allow access to the repair machinery. In conclusion, these data demonstrated a folate-dependent repair activity and chromatin re-organization on the SMLM nanoscale level. This offers new opportunities to further investigate folate-induced chromatin re-organization and the associated mechanisms.

Particles with similar LET values generate DNA breaks of different complexity and reparability: a high-resolution microscopy analysis of γH2AX/53BP1 foci

“…The biological nature of the complex γH2AX/53BP1 focus clusters observed in the present study and the precise numbers of DSBs present in γH2AX/53BP1 foci/focus clusters (in general) therefore remain uncertain and must be further studied. Several recent reports [75][76][77][78] have suggested that super-resolution localization light microscopy offers an exciting but still challenging means of analysing the same biological specimens in parallel with confocal microscopy, but with much more detailed data. This strategy could be used to obtain answers to many questions discussed in this chapter in the near future.…”

Fusobacterium nucleatumCaused DNA Damage and Promoted Cell Proliferation by theKu70/p53Pathway in Oral Cancer Cells

“…Histone H2A (H2AX) is a meiosis-specific isoform of histone H2A. gH2AX, the phosphorylated form of H2AX, is rapidly and accurately recruited when there is a DNA double-strand break (DSB), the most serious type of DNA damage, and it is applied as a DSB molecular marker (Rogakou et al, 1998;Bach et al, 2017). Once the DNA damage occurs, DSBs are repaired by homologous recombination or nonhomologous end joining (NHEJ).…”

Quantification of DNA damage induced γH2AX focus formation via super-resolution dSTORM localization microscopy

“…However, the limitation of the technique is that it shows the average of a given focus by combining the data obtained from a large population of cells. Recent applications of electron-microscopy and super-resolution light microscopy 24,30,31,55–57 have demonstrated that it is feasible to study single molecular arrangements within a repair focus. By improving the resolution of microscopy and data evaluation of structures in meso-and nano-scale level, the searching for the best-suited analysis parameters and potentially useful classification criteria of repair foci and damaged chromatin sites have become to be important.…”