An epichromatin epitope: Persistence in the cell cycle and conservation in evolution

Abstract

NucleusVolume 2 Issue 1 U2OS and HL-60/S4 cells by deconvolution immunofluorescence microscopy with the aim of searching for chromatin substructures. One anti-nucleosome antibody (PL2-6) specifically stained a chromatin compartment at the periphery of interphase nuclei, as well as staining the surface of mitotic chromosomes. We suggest that this compartment represents a unique surface chromatin conformation, to which we assign the name "epichromatin". Furthermore, we formulate an "epichromatin hypothesis", suggesting that this chromatin may play a crucial role in organizing interphase nuclear architecture, justifying its conservation in the evolution of cell structure. Results

Retrotransposon Alu is enriched in the epichromatin of HL-60 cells

“…In the present study, we continued our characterization of interphase epichromatin regions (i.e., chromatin proximal to the NE) 8,9 in undifferentiated and differentiated HL-60/ S4 cells, using a modified chromatin immunoprecipitation (ChIP) protocol. Monoclonal antibodies PL2-6 and 1H6 were used to ChIP chromatin, followed by purification of epichromatin-associated DNA, "deep" sequencing, annotation of the sequences and mapping the enriched regions on to the human chromosomes.…”

“…It is important to emphasize that the two mouse monoclonal antibodies employed in this ChIP-Seq study are not specific for Alu-containing chromatin. Both antibodies can immunostain epichromatin in cells from various species that do not possess Alu repetitive elements (e.g., PL2-6 stains interphase epichromatin in Drosophila and tobacco cells 8 ; 1H6, interphase epichromatin in mouse and Drosophila cells 9 ). The conserved epitopes probably reflect a conserved nucleosome structure or conformation exposed at the surface of chromatin.…”

“…3C-E) and imaging with the DeltaVision deconvolution microscope has been described earlier. 8,9 Electrophoretic mobility shift assay (EMSA) For the titration experiments, stock solutions included: Hela mononucleosomes (1.2 µM) in 0.1 M NaCl plus TE buffer (pH 7.5); PL2-6 (8.38 µM, based upon A280 measurements) in PBS; Table S6). Epichromatin regions were extended up to 1 kb to investigate proximity relationships to chromatin features.…”

“…[5][6][7] Beside the likely role of inner nuclear membrane proteins (e.g., lamin B receptor, LBR) in nuclear reformation, it has been suggested that properties of the NE-associated chromatin may facilitate the post-mitotic reconstruction of nuclear architecture. 8,9 The surface of chromatin facing the nuclear envelope in an interphase nucleus ("epichromatin") reveals a "rim" staining pattern with specific antibodies against histone H2A/H2B/DNA (mAb PL2-6) and phosphatidylserine (mAb 1H6) epitopes. 8,9 High-resolution 3D-SIM images of PL2-6 and 1H6 immunostaining at the surface of interphase nuclei reveal a meshwork of chromatin fibers running parallel to the NE in tangential "grazing" views ( Fig.…”

“…8,9 The surface of chromatin facing the nuclear envelope in an interphase nucleus ("epichromatin") reveals a "rim" staining pattern with specific antibodies against histone H2A/H2B/DNA (mAb PL2-6) and phosphatidylserine (mAb 1H6) epitopes. 8,9 High-resolution 3D-SIM images of PL2-6 and 1H6 immunostaining at the surface of interphase nuclei reveal a meshwork of chromatin fibers running parallel to the NE in tangential "grazing" views ( Fig. 1).…”