The G-rich strand of human telomeric DNA can fold into a four-stranded structure called G-quadruplex and inhibit telomerase activity that is expressed in 85-90% tumor cells. For this reason, telomere quadruplex is emerging as a potential therapeutic target for cancer. Information on the structure of the quadruplex in the physiological environment is important for structure-based drug design targeting the quadruplex. Recent studies have raised significant controversy regarding the exact structure of the quadruplex formed by human telomeric DNA in a physiological relevant environment. Studies on the crystal prepared in K+ solution revealed a distinct propeller-shaped parallel-stranded conformation. However, many later works failed to confirm such structure in physiological K+ solution but rather led to the identification of a different hybrid-type mixed parallel/antiparallel quadruplex. Here we demonstrate that human telomere DNA adopts a parallel-stranded conformation in physiological K+ solution under molecular crowding conditions created by PEG. At the concentration of 40% (w/v), PEG induced complete structural conversion to a parallel-stranded G-quadruplex. We also show that the quadruplex formed under such a condition has unusual stability and significant negative impact on telomerase processivity. Since the environment inside cells is molecularly crowded, our results obtained under the cell mimicking condition suggest that the parallel-stranded quadruplex may be the more favored structure under physiological conditions, and drug design targeting the human telomeric quadruplex should take this into consideration.

Yeast contains heterochromatin at telomeres and the silent mating-type loci (HML/HMR). Genes positioned within the telomeric heterochromatin of Saccharomyces cerevisiae switch stochastically between epigenetically bistable ON and OFF expression states. Important aspects of the mechanism of variegated gene expression, including the chromatin structure of the natural ON state and the mechanism by which it is maintained, are unknown. To address this issue, we developed approaches to select cells in the ON and OFF states. We found by chromatin immunoprecipitation (ChIP) that natural ON telomeres are associated with Rap1 binding and, surprisingly, also contain known characteristics of OFF telomeres, including significant amounts of Sir3 and H4K16 deacetylated nucleosomes. Moreover, we found that H3K79 methylation (H3K79me), H3K4me, and H3K36me, which are depleted from OFF telomeres, are enriched at ON telomeres. We demonstrate in vitro that H3K79me, but not H3K4me or H3K36me, disrupts transcriptional silencing. Importantly, H3K79me does not significantly reduce Sir complex binding in vivo or in vitro. Finally, we show that maintenance of H3K79me at ON telomeres is dependent on transcription. Therefore, although Sir proteins are required for silencing, we propose that epigenetic variegation of telomeric gene expression is due to the bistable enrichment/depletion of H3K79me and not the fluctuation in the amount of Sir protein binding to nucleosomes.

Eukaryotic histone H3-H4 tetramers contain a putative copper (Cu2+) binding site at the H3-H3′ dimerization interface with unknown function. The coincident emergence of eukaryotes with global oxygenation, which challenged cellular copper utilization, raised the possibility that histones may function in cellular copper homeostasis. We report that the recombinant Xenopus laevis H3-H4 tetramer is an oxidoreductase enzyme that binds Cu2+ and catalyzes its reduction to Cu1+ in vitro. Loss- and gain-of-function mutations of the putative active site residues correspondingly altered copper binding and the enzymatic activity, as well as intracellular Cu1+ abundance and copper-dependent mitochondrial respiration and Sod1 function in the yeast Saccharomyces cerevisiae. The histone H3-H4 tetramer, therefore, has a role other than chromatin compaction or epigenetic regulation and generates biousable Cu1+ ions in eukaryotes.