An open chromatin architecture devoid of compact chromatin is thought to be associated with pluripotency in embryonic stem cells. Establishing this distinct epigenetic state may also be required for somatic cell reprogramming. However, there has been little direct examination of global structural domains of chromatin during the founding and loss of pluripotency that occurs in preimplantation mouse development. Here, we used electron spectroscopic imaging to examine large-scale chromatin structural changes during the transition from one-cell to early postimplantation stage embryos. In one-cell embryos chromatin was extensively dispersed with no noticeable accumulation at the nuclear envelope. Major changes were observed from one-cell to two-cell stage embryos, where chromatin became confined to discrete blocks of compaction and with an increased concentration at the nuclear envelope. In eight-cell embryos and pluripotent epiblast cells, chromatin was primarily distributed as an extended meshwork of uncompacted fibres and was indistinguishable from chromatin organization in embryonic stem cells. In contrast, lineage-committed trophectoderm and primitive endoderm cells, and the stem cell lines derived from these tissues, displayed higher levels of chromatin compaction, suggesting an association between developmental potential and chromatin organisation. We examined this association in vivo and found that deletion of Oct4, a factor required for pluripotency, caused the formation of large blocks of compact chromatin in putative epiblast cells. Together, these studies show that an open chromatin architecture is established in the embryonic lineages during development and is sufficient to distinguish pluripotent cells from tissue-restricted progenitor cells.

Although the mechanism of chromosomal segregation is well known, it is unclear how other nuclear compartments such as promyelocytic leukemia (PML) nuclear bodies partition during mitosis and re-form in G1. We demonstrate that PML nuclear bodies partition via mitotic accumulations of PML protein (MAPPs), which are distinct from PML nuclear bodies in their dynamics, biochemistry and structure. During mitosis PML nuclear bodies lose biochemical components such as SUMO-1 and Sp100. We demonstrate that MAPPs are also devoid of Daxx and these biochemical changes occur prior to chromatin condensation and coincide with the loss of nuclear membrane integrity. MAPPs are highly mobile, yet do not readily exchange PML protein as demonstrated by fluorescence recovery after photo-bleaching (FRAP). A subset of MAPPs remains associated with mitotic chromosomes, providing a possible nucleation site for PML nuclear body formation in G1. As the nuclear envelope reforms in late anaphase, these nascent PML nuclear bodies accumulate components sequentially, for example Sp100 and SUMO-1 before Daxx. After cytokinesis, MAPPs remain in the cytoplasm long after the reincorporation of splicing components and their disappearance coincides with new PML nuclear body formation even in the absence of new protein synthesis. The PML protein within MAPPs is not degraded during mitosis but is recycled to contribute to the formation of new PML nuclear bodies in daughter nuclei. The recycling of PML protein from one cell cycle to the next via mitotic accumulations may represent a common mechanism for the partitioning of other nuclear bodies during mitosis.

Promyelocytic leukemia (PML) nuclear bodies have been implicated in a variety of cellular processes including apoptosis, tumour suppression, anti-viral response, DNA repair and transcriptional regulation. PML nuclear bodies are both positionally and structurally stable over extended periods of interphase. As demonstrated in this study, the structural stability is lost as cells enter S phase, evidenced both by distortions in shape and by fission and fusion events. At the end of this period of structural instability, the number of PML nuclear bodies has increased by a factor of twofold. Association of the fission products with chromatin implies that the PML nuclear bodies respond to changes in chromatin organisation or topology, and thus could play a role in monitoring genome integrity during DNA synthesis or in the continued maintenance of functional chromosomal domains prior to mitosis.

Aflatoxins (AFs) are highly hazardous mycotoxins with potent carcinogenic, mutagenic and immune disregulatory properties. Cytochrome P450 (CYP) isoforms are central for enhanced AFB₁ toxicity in situ. It remains to be seen whether and how these AFB₁ activators work in human leukocytes. To investigate the involvement of CYP isoforms in AFB₁ toxicity of circulating mononuclear cells, we examined the impact of environmentally relevant levels of AFB1 on lymphocytes and monocytes. Very low and moderate doses of AFB₁ with/without CYP inducers on transcription of key CYP isoforms and toll-like receptor 4 (TLR4) were examined in human lymphocytes, monocytes and HepG2 cells; cell cycle distribution and viability were also analyzed in AFB₁-exposed lymphocytes and monocytes. Only CYP1A1, CYP1B1, CYP3A4, CYP3A5 and CYP3A7 expressed in lymphocytes and monocytes. TLR4 much more expressed in monocytes than in lymphocytes, but HepG2 showed little TLR4 transcription. While CYP1A1, CYP1B1 and CYP3A4 were highly induced by AFB₁ in monocytes, in lymphocytes only CYP1A1 was induced. Among CYP1A1, CYP1B1 and CYP3A4 only CYP1A1 responded to low and moderate levels of AFB₁. Enhanced transcripts of CYPs by AFB₁ yielded little synergies on TLR4 transcription in lymphocytes and monocytes. Cell cycle arrest and necrosis were also detected in AFB₁-exposed lymphocytes and monocytes. Our novel findings indicate that AFB₁ more intensively stimulates CYP genes expression in monocytes than in lymphocytes. Mechanistically, this could explain a more pronounced immunotoxicity of AFB₁ in myeloid than in lymphoid lineage cells in vitro/situ/vivo.