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. Here we identified epiC as a dual post-translational modification on the same histone H4 tail, which was immunodetected for the first time. We show that the antibody selectively recognized a synthetic peptide of the histone H4 region K12-L22 containing acetylated K16 and dimethylated K20 (H4K16ac-K20me2) at 4 degrees C, but not at RT. Moreover, we show that the peptide containing acetylated K16 and either unmodified or monomethylated K20 was recognized by this antibody at both temperatures. The present and previous results together indicate that, by acetylation of histone H4 K16 during S-phase, the early replicating chromatin domains acquire the H4K16ac-K20me2 epigenetic label that persists on the chromatin throughout mitosis and become deacetylated during early G1-phase of the next cell cycle.
- MeSH
- acetylace MeSH
- buněčné linie MeSH
- buněčný cyklus fyziologie genetika MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- epigeneze genetická fyziologie genetika MeSH
- epitopy chemie imunologie MeSH
- histony imunologie metabolismus MeSH
- imunoblotting MeSH
- lidé MeSH
- metylace MeSH
- peptidy chemická syntéza chemie imunologie MeSH
- posttranslační úpravy proteinů MeSH
- teplota MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
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. The photoconvertible histone H4-Dendra2 was used to label such regions in transfected HepG2 cells, and its position was followed up to next interphase. The distribution of labeled chromatin in daughter cells exhibited a non-random character. However, its distribution in a vast majority of daughter cells extensively differed from the original ones and the labeled nucleolus-associated chromatin differently located into the vicinity of different nucleoli. Therefore, our results were not consistent with a concept of preservation chromatin position. This conclusion was supported by the finding that the numbers of nucleoli significantly differed between the two daughter cells. Our results support a view that while the transfected daughter HepG2 cells maintain some features of the parental cell chromosome organization, there is also a significant stochastic component associated with reassortment of chromosome territories/chromatin that results in their positional rearrangements.
- MeSH
- buněčné jadérko metabolismus MeSH
- buněčné jádro metabolismus MeSH
- buněčné linie MeSH
- časové faktory MeSH
- chromatin chemie metabolismus MeSH
- chromozomy ultrastruktura MeSH
- fibroblasty metabolismus MeSH
- financování organizované MeSH
- fluorescenční mikroskopie metody MeSH
- histony metabolismus MeSH
- lidé MeSH
- počítačové zpracování obrazu MeSH
- Check Tag
- lidé MeSH
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. Using CCF analysis of model images simulating either random initiation of new replication foci, or the firing of new foci in close proximity to completed ones, we were able to ascribe the changes in the early S replication foci distribution to the latter mechanism. In contrast to the data published previously, we monitored the dynamics of all replication foci for up to 3 h. In addition, we showed that the replication foci dynamics is well described by random walk model, so that the average de-localization of individual foci is proportional to square root of the applied chase.
- MeSH
- finanční podpora výzkumu jako téma MeSH
- lidé MeSH
- replikace DNA imunologie MeSH
- S fáze imunologie MeSH
- Check Tag
- lidé MeSH
- MeSH
- aktiny imunologie MeSH
- chromatin chemie MeSH
- finanční podpora výzkumu jako téma MeSH
- lidé MeSH
- replikace DNA MeSH
- Check Tag
- lidé MeSH
