Erythroid-specific activator NF-E2, but not GATA-1, marking transcriptional competence of globin genes in MEL cell during mitosis
In MEL cells, DNase I sensitivity and hypersensitivity have been established on globin gene loci [14], suggesting that they have obtained potential transcription activity. An early study also showed that hypersensitive sites, once been set up, can be propagated to daughter cells (over at least 20 generations) in the absence of the original inducer [10]. So we speculated that there might be some memory molecules, possibly involving protein factors or epigenetic marks, on regulatory sequences to help the propagation of transcription competence. There are some specific binding sites of erythroid-specific activators, such as GATA-1, NF-E2 and Fog-1, on globin gene clusters [15], whose occupancies largely contribute to the activation of globin genes. To explore the protein factors involved in the inheritance of transcriptional competence, we investigated the nuclear localization of GATA-1 and the activating subunit p45 of NF-E2 in mitotic and interphase cell by in situ immunofluorescence. It was found that GATA-1 mainly localized in interchromatin regions of cell nucleus and it can not be detected in nuclei of the mitotic cells by in situ immunofluorescence until parental nucleus divided into two progeny nuclei at telophase (Fig 1), indicating that GATA-1 was not retained on mitotic chromosome as a cellular memory mark during mitosis. In contrast, NF-E2p45, which is punctately organized in both cytoplasm and nucleus of the interphase cells, persists throughout mitosis (Fig 1), implying that it may be involved in the propagation of transcription competence of globin genes during cell division.
In order to confirm this possibility, we arrested murine erythroleukemia (MEL) cells into mitosis by treating the cells with nocodazole [3]. The mitotic index of cell population after treatment was confirmed to be 92–96% by flow cytometry analysis (Fig 2). The results also showed that a proportion of ~5% mitotic cells, about 50% G0/G1-phase and 45% S-phase cells, respectively, is present in asynchronous MEL cell population (Fig 2). The two cell populations were analysed by ChIP to compare the occupancies of NF-E2p45 on distant hypersensitive sites of two mouse globin gene clusters (Fig 3A). The result showed that NF-E2p45 could be specifically recruited to its binding sites HS26 and HS2 in interphase cells (Fig 3B). In mitotic cells it is still tightly associated with HS26 and HS2 (Fig 3B), indicating that NFE2p45 can be retained on mitotic chromosomes to maintain the hypersensitivity of the localized regulatory regions of two mouse globin gene clusters. Its retention, moreover, marks the transcriptional competence of globin genes during MEL cell division.
Epigenetic memory marking transcriptionally competent globin genes
Besides protein factors, histone modification is recently identified as an efficient epigenetic factor involved in gene expression regulation through altering chromatin structure. Do the eukaryotic cells preserve some epigenetic marks on mitotic chromosomes to maintain its gene expression states? To verify the above-proposed possibility, we first observed the nuclear localization of four kinds of active histone modification including H3 acetylation, H4 acetylation, H3-K4 dimethylation and H3-K79 dimethylation in mitotic cells by in situ immunofluorescence. The results showed that all of the four modifications are retained on the mitotic chromosomes to some extent (Fig 4). We further detected the global changes of those modifications in both asynchronized and mitotic arrested MEL cell populations by western blotting. After correcting the variation in gel-loading and normalizing for the background, we found that there is a partial loss (about 20–30%) of H3 acetylation, H4 acetylation and H3-K4 dimethylation in mitotic cell extraction compared to those in asynchronous cell extraction but no obvious loss of H3-K79 dimethylation (Fig 5). This indicates that only part of histone acetylation and H3-K4 dimethylation signals can be inherited to mitotic chromatin through DNA replication-coupled chromatin assembly process. What is the detailed distribution of these preserved modifications on the mitotic chromosome? We therefore analysed the histone modification status across the distant hypersensitive sites and the adjacent promoter regions of mouse α- and β-globin genes (Fig 3A) during mitosis by the comparative ChIP analysis of the above mentioned asynchronous and mitotic arrested MEL cell populations. The results showed that HS26, HS21 and HS8 of mouse α-globin locus and HS3, HS2 and HS1 of mouse β-globin locus, as well as the promoters of α-globin and βmaj are acetylated at H3 and all of them except HS26 and HS3 are acetylated at H4 in asynchronous cells (Fig 6A, 6B). Higher H3-K4 dimethylation at HSs and α-globin promoter was noticed when compared to that at β-globin promoter(Fig 6C). HS8 and α-globin promoter are hypermethylated at H3-K79, but HS26 and HS21 show trace of signal. HS2 and HS1 of LCR are hypermethylated at H3-K79, while HS3 and βmaj promoter are just slightly methylated at H3-K79 (Fig 6D). In mitotic cells, the levels of H3 and H4 acetylation, H3-K4 dimethylation dropped at many analyzed regions, while H3-K79 dimethylation at all the analysed regions remain stable compared to those in asynchronous cells. Moreover H3-K79 dimethylation level on mitotic chromosomes is comparable to that in asynchronous cells, which consist with their chromatin states (Fig 6). The results suggested that, despite of some losses, the established active histone code at distant regulatory regions and globin gene promoters before mitotic chromatin inactivation can be stably inherited to mitotic chromosomes to mark the transcriptional competence of mouse globin genes in MEL cells.
Epigenetic memory marking the active transcription states of the genes
Can these epigenetic marks note the different transcription states of thegenes during mitosis? We next performed the comparative chromatin immunoprecipitation (ChIP) analysis for the promoter regions of the genes with different transcription states. These genes include (1) c-myc, hsp70 and β-actin, which are universally transcribed by RNA polymerase II (RNAP II); (2) an actively RNAP III-transcribed gene (7SK), and (4) nef-3 and albumin, which are tissue-specifically silenced in MEL cells. The results showed that promoter region of the actively transcribed genes like c-myc, hsp70, β-actin and 7SK are hyperacetylated, while inactive genes, nef-3 and albumin, are hypoacetylated at both H3 and H4 in asynchronous MEL cells (Fig 7A). In synchronized mitotic cells, transcriptionally active genes keep obviously higher levels of histone acetylation at both H3 and H4 than those of inactive genes,(Fig 7A). The fact that, during mitotic chromatin inactivation, the levels of remnant histone acetylation at the promoter regions of the analysed genes depend on their previous expression states strongly suggests that the localized histone acetylation at transcriptionally competent or active gene regions can be taken as the useful epigenetic memory marks to imprint the previous expression states during cell division.
To investigate whether active histone modifications-H3-K4 dimethylation and K79 dimethylation are preserved at active regions during mitosis, we performed the similarly comparative ChIP analysis. The results showed that these two kinds of modification are also mainly confined to active gene promoters (Fig 7B). In asynchronous cells, the fully activated genes-c-myc and β-actin are hyperdimethylated at H3-K4; incompletely activated gene hsp70 are moderately dimethylated at H3-K4. Although RNA polymerase III-transcribed gene 7SK is active, similar to inactive genes as nef-3 and albumin, there is tiny enrichment of H3-K4 dimethylation on the gene. However, H3-K79 dimethylation, despite the moderate level, is distributed on the promoters of all the analyzed active genes regardless of the RNA polymerase used for transcription. In mitotic cells, these two kinds of active histone modification, similar to histone acetylation, are also preserved on the previously active gene promoter regions and the preserved levels are corresponding with gene expression states. Nevertheless, there is some decrease for H3-K4 dimethylation but no obvious change for H3-K79 dimethylation (Fig 7B). These data indicate that the localized H3-K4 dimethylation and K79 dimethylation at transcriptionally active regions can also serve as molecular memory marks along with histone acetylation. The above results also confirmed that the epigenetic marks composed of active histone modification combinations and localized at active gene regions can be transmitted to mitotic chromosomes for the maintenance of active gene expression states.