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

Abstract

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. The level of co-localization was strikingly enhanced beginning approximately 1 h after the initial labeling of early S phase replicating chromatin domains. In contrast, epiC did not co-localize with late S phase replicated chromatin either during DNA replication or at any other time in the cell cycle. We propose a replication-coupled modulation of early S phase replicated chromatin domains that is detected by the chromatin epiC positivity, persists on the chromatin domains from early S until early G(1) of the next cell generation, and may be involved in the regulation and/or coordination of replicational and transcriptional processes during the cell cycle. Further studies will be required to resolve the possible role of nuclear actin in this modulation process.

Nucleosomes in the neighborhood

“…48 Interestingly, the simultaneous presence of H4K20me2 and H4K16ac at early replicating domains in S phase persisted until it was removed in the following G 1 when preRC formation occurs. 49,50 This dual mark on the same histone H4 molecule was also detected in D. melanogaster, where H4K16ac was enriched at origins containing H4K20me2. 25,[51][52][53] This association could implicate H4K20me2 (like H4K16ac) in maintaining an open, but also inactive or poised, chromatin state with respect to origin licensing.…”

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

“…To verify that the epitope recognized by anti-actin at 4°C was not degraded during the incubation at RT, we tested the characteristic reversibility of cold-dependent epiC detection, observed previously in situ (Fidlerova et al, 2005), as described in Section 3. The results demonstrated the identical patterns of cold dependency and reversibility of anti-actin labeling of both epiC detected previously in situ (Fidlerova et al, 2005) and the epitope detected in the histone H4 band in vitro ( Fig. 1A and B).…”

“…EpiC was detected in nuclear histone H4 but not in recombinant histone H4 protein Considering the possibility that some low molecular weight histone proteins might escape detection under the PAGE/blotting conditions previously used for actin (Fidlerova et al, 2005), we tested the putative presence of epiC, the epitope recognized by anti-actin selectively at 4°C, but not at RT, in the acid-extracted fraction of nuclear proteins, which are known to contain predominantly histones (Shechter et al, 2007).…”

“…Indeed, the identification of the dual PTM on the same histone H4 tail could not be accomplished with any of the presently known antihistone Abs. Surprisingly, it was achieved with an anti-actin Ab and resulted from the discovery of a reversible, cold-dependent detection of a chromatin epitope and the description of its relevant biological features (Fidlerova et al, 2005). In the previous study, by using detailed 3D confocal microscopy and cross-correlation analysis, we demonstrated with both synchronized and non-synchronized human fibroblast cells that the commercial monoclonal anti-actin antibody (hereafter referred to as anti-actin) labeled early replicating chromatin domains at 4°C, but not at RT, in a distinct cell cycle window extending from early S, through S, G2 and M until early G1 of the next cell generation.…”

“…The unique spatio-temporal pattern of epiC appearance suggested that we had detected some replication-coupled modulation of early replicated chromatin domains that could be involved in the transfer and maintenance of epigenetic information on the transcriptionally competent part of the genome. However, the molecular nature of epiC remained unresolved (Fidlerova et al, 2005).…”