A new epigenetic marker: the replication-coupled, cell cycle-dependent, dual modification of the histone H4 tail.

Recently, we described the cold-dependent detection of an epitope, epiC, that was selectively recognized by a monoclonal anti-actin antibody at 4 degrees C, but not at RT, in the early replicating chromatin domains of human fibroblast cell nuclei and chromosomes. EpiC was present in a distinct cell cycle window extending from S-phase throughout mitosis until early G1-phase of the next cell generation, indicating its possible involvement in the transfer/maintenance of epigenetic information on transcriptionally competent parts of the genome. However, the molecular nature of epiC remained unresolved.

Chromatin position in human HepG2 cells: although being non-random, significantly changed in daughter cells.

Mammalian chromosomes occupy chromosome territories within nuclear space the positions of which are generally accepted as non-random. However, it is still controversial whether position of chromosome territories/chromatin is maintained in daughter cells. We addressed this issue and investigated maintenance of various chromatin regions of unknown composition as well as nucleolus-associated chromatin, a significant part of which is composed of nucleolus organizer region-bearing chromosomes.

Dynamics of replication foci in early S phase as visualized by cross-correlation function.

To monitor gradual changes in the replication foci distribution during early S phase, different segments of newly synthesized DNA were visualized by immunocytochemical mapping of two consecutively incorporated deoxythymidine analogs in pulse-chase-pulse experiments in HeLa cells. The resulting dual-labeled fluorescence images were evaluated using cross-correlation function (CCF) analysis. General changes of CCF shape due to image deterioration caused by blur, noise, and lateral sampling (pixel size) were also discussed.

Replication-coupled modulation of early replicating chromatin domains detected by anti-actin antibody.

Evidence is presented for the reversible, cold-dependent immunofluorescence detection of the epitope (hereafter referred to as epiC), recognized by a monoclonal anti-actin antibody in diploid human fibroblast cell nuclei and mitotic chromosomes. The nuclear/chromosomal epiC was detected in a cell cycle window beginning in early S phase and extending through S phase, G(2) phase, mitosis until early G(1) phase of the subsequent daughter cells. A small but significant level of co-localization was measured between the nuclear epiC and active sites of DNA replication in early S phase.

Electron microscopy of DNA replication in 3-D: evidence for similar-sized replication foci throughout S-phase.

DNA replication sites (RS) in synchronized HeLa cells have been studied at the electron microscopic level. Using an improved method for detection following the in vivo incorporation of biotin-16-deoxyuridine triphosphate, discrete RS, or foci are observed throughout the S-phase. In particular, the much larger RS or foci typically observed by fluorescence microscopic approaches in mid- and late-S-phase, are found to be composed of smaller discrete foci that are virtually identical in size to the RS observed in early-S-phase.