Exploring The Memory Mechanisms Of Active Chromatin State And Transcription States During Cell Division

Higher eukaryote contains several hundreds of cell types, each with a distinctive set of gene expression profile. These gene expression profiles are set up and propagated during cell differentiation and ontogeny. To maintain the products of cell differentiation and ontogeny, higher eukaryote must have evolved some elegant mechanisms to remember the cell phenotypes. Every cell maintain the same genetic material-genome DNA, which is replicated during S phase and transmitted to their daughter cells through mitosis. So the epigenetic information such as nucleosome structure, histone modification, non-histone proteins, interactions between DNA and protein, protein and protein etc., which is closely related to gene expression regulation, may play an important role in the maintenance of gene expression states during cell division. Exploring the roles of epigenetic information in the maintenance of gene expression states during cell division is an significant task to elucidate the mechanisms of differentiation and development of higher organisms.Employing the immunofluorescence assay, we analyzed the changes of four active histone modifications in mitotic MEL (murine erythroleukemia) cells by using the antibodies against H3 and H4 acetylation, H3-K4 dimethylation and H3-K79 dimethylation. In interphase cell nucleus, H3 and H4 acetylation, H3-K4 dimethylation concentrat on the euchromatin compartment; H3-K79 dimethylation is mainly localized in euchromatin but some are in heterochromatin compartment. Notablely, four active histone modifications remain on mitotic chromosomes even though chromtin is compacted into higher order chromosomes. Their localizations are restored coupling the appearance of nuclear envolope and the division of daughter cell nuclei at mitotic telophase. It has reported that most of transcriptions mediated by three RNA polymerase are ceased accompanying the chromatin condensation and the ceased transcriptions are resumed accompanying chromatin decondensation at mitotic exit. To further explore wether these active histone modifications mediate transcriptional memory during mitotic chromatin inactivation, we synchronized MEL cells into mitosis by treatment with nocodazole and then comparatively analyzed the changes of them in asynchronous and synchronized mitotic MEL cell populations. The more than 95% cells in synchronized MEL population were synchronized into G2/M phase after 16h treatment with nocodazole. There are about 50% G0/G1, 45% S and less than 5% G2/M cells in asynchronous MEL cell population. The total histone proteins were extracted from asynchronous and synchronous MEL cells and were blotted by antibodies against active histone modifications. The results indicated that the global amounts of H3 and H4 acetylation, H3-K4 dimethylation decreased 20-30% in mitotic cell population compared to asynchronous cell population but H3-K79 dimethylation is stable in two populations. Next we performed the comparative chromatin immunoprecipitation (CHIP) assay and analyzed the levels of four active histone modifications at the promoters of genes with different transcription states. The results indicated that these genes with different transcription states are marked by the different histone modification patterens and these modifications are reserved at gene promoters during mitosis. The reserved levels depend on the previous gene expression states even though some modifications are lowered in mitotic cells compared to those in asynchronous interphase cells. These results suggested that the preserved active histone modifications can function as epigenetic memory marks to maintain the previous gene expression states.The distal regulatory sequences, which is a unique and significant regulatory factor in higher eukaryotes, control the expression of many tissue- or development-specific genes through programming their chromatin accessibility and binding the specific activators or repressors. By taking the well-studied mouseα- andβ-globin gene clusters as a model, we compared active histone modifications at the distant hypersensitive sites (HSs) of globin gene clusters in asynchronous and mitotic cells. The results demonstrated that these distant regulatory elements are marked by the different histone modification combinations and also these modifications persist during mitosis in spite of some decreases. The preserved levels are also corresponding to their previous levels. The above results strongly suggested that during mitotic chromatin inactivation, active histone modifications can function as epigenetic memory marks to maintain the active chromatin state and gene expression states.Two erythroid-specific transcriptional activators GATA-1 and NF-E2p45, which play important roles in globin gene expressions, were taken as models to probe their roles in transcription memory. As is known, RNA polⅡis displaced from chromosomes during mitosis. Immunofluorescence analysis showed that RNA polⅡare not colocalized with chromosomes in mitotic cells. The signals of GATA-1 are disappeared in mitotic cells, indicating that GATA-1 may be abrogated during mitosis. However, some signals of NF-E2p45 remain at chromosomes compartments. The further ChIP analysis showed that NF-E2p45 specifically binds to the distant HS26 and HS2 of globin gene clusters and are still retained at its binding sites during mitosis, suggesting that certain specific protein factor can also serve as molecular memory mark in favor of the maintenance of transcription state and transcription reactivation.The deposition and removal of H3 variant H3.3 is closely related to transcription activation. To elucidate the relationship between the distribution of H3.3 at chromatin and transcription memory, we analyzed the changes of H3.3 protein in mitoic cells using the antibodies against the endogenous H3.3. The immunofluorescence analysis showed that during mitosis some H3.3 proteins are dispersed at periphery of cell membrane in conglobation and some remain at chromosomes compartments. The comparative ChIP analysis indicated that H3.3 variants are distributed at the promoters of the active and inactive genes and reserved during mitosis. However, it needs analyzing more genes and heterochromatin sites to disclose the correlation between its distribution and gene expression and its possible roles in transcription memory.Conclusions: (1) the preserved active histone modifications at mitotic chromosomes can imprint the previous gene expression states in favor of the transcriptional resumption of a large scale genes at mitoic exit. (2) The preserved active histone modification combinations at mitotic chromosomes can mark the active chromatin states of distant regulatory sequences and the transcriptional state of its controlled genes to maintain their previous chromatin activity. (3) The reserved histone modifications at mitotic chromosomes is a universal and efficient epigenetic memory mechanism to maintain active chromatin state and gene expression states. (4) Some specific transcription factors can function as molecular memory marks during mitosis to help the rapid restoration of active chromaton state at mitotic exit. (5) It needs further investigation to verify the relationship between the distribution of H3.3 variant and transcription memory.