The expression of the histone H1° gene in the human hepatoma cell line HepG2 is independent of the state of cell proliferation

The H1 histone subtype H1 (0) is a characteristic component of the chromatin of several mammalian tissues. Since H1 (0) is synthesized in nondividing cells upon terminal differentiation, it has been mostly considered either as a prerequisite for or as a consequence of an arrest of DNA replication during the process of differentiation. In several H1 (0)-expressing systems studied until now, inducers of differentiation or inhibitors of DNA synthesis cause an increase of the ratio between H1 (0) and the other H1 proteins. We have studied the steady-state levels of histone H1 (0) mRNA under varied growth conditions in the human hepatoma cell lines HepG2 and Hep3B, and we show in the HepG2 system that H1 (0) is not confined to resting cells, that the H1 (0) gene appears to be expressed throughout the cell cycle and that established inducers of de novo H1 (0) synthesis fail to cause a further increase of the high H1 (0) level. This constitutive expression of H1 (0) appears to reflect the chromatin structure of the liver cells, from which the HepG2 hepatoblastoma cells initially may have evolved. In contrast to the situation in nondividing adult liver cells, the H1 (0) gene is transcribed in HepG2 at a high level, and this expression is compatible with DNA replication.     

组蛋白赖氨酸甲基转移酶2D调控H9c2细胞中HIF-1α/VEGF通路

组蛋白赖氨酸甲基转移酶2D (histone-lysine N-methyltransferase 2D, KMT2D) 作为主要的组蛋白3第4位赖氨酸 (H3K4) 甲基转移酶,在调控胚胎发育、组织分化、代谢和肿瘤抑制方面发挥重要作用。在小鼠体内,敲除Kmt2d会导致严重的心脏发育缺陷最终造成胚胎期死亡。低氧诱导因子-1α (hypoxia-inducible factor 1α, HIF-1α) 作为调节细胞应对低氧的关键转录因子,能够调控多种下游基因转录。有相关研究揭示,表观遗传调控者能够调节HIF-1α的稳定性和活性。同样,作为表观遗传调控者的组蛋白甲基转移酶KMT2D是否参与低氧条件下HIF-1α对下游基因的调控,目前仍未知。在本研究中,观察在Kmt2d正常或缺乏的情况下,心肌细胞H9c2对低氧环境的应答反应。结果显示,与常氧条件相比,低氧状态下HIF-1α、组蛋白乙酰化酶P300、KMT2D及其介导的H3K4一甲基化 (H3K4 mono-methylation, H3K4me1)的蛋白质水平增加 (P<0.05);HIF-1α下游基因血管内皮生长因子 (vascular endothelial growth factor, Vegf) 的mRNA表达水平明显上调 (P<0.01)。染色质免疫共沉淀实验 (chromatin immunoprecipitation assay, ChIP-qPCR) 检测结果显示,H3K4me1和组蛋白3第27位赖氨酸乙酰化 (histone 3 lysine 27 acetylation, H3K27ac) 在Vegf基因启动子区域的结合丰度明显增加 (P<0.05)。低氧条件下沉默Kmt2d之后,H3K4me1蛋白水平和Vegf的mRNA表达下降 (P<0.05)。本研究表明,低氧条件下KMT2D参与调控HIF-1α和下游基因Vegf的表达。     

组蛋白赖氨酸甲基转移酶2D调控H9c2细胞中HIF-1α/VEGF通路

组蛋白赖氨酸甲基转移酶2D (histone-lysine N-methyltransferase 2D, KMT2D) 作为主要的组蛋白3第4位赖氨酸 (H3K4) 甲基转移酶,在调控胚胎发育、组织分化、代谢和肿瘤抑制方面发挥重要作用。在小鼠体内,敲除Kmt2d会导致严重的心脏发育缺陷最终造成胚胎期死亡。低氧诱导因子-1α (hypoxia-inducible factor 1α, HIF-1α) 作为调节细胞应对低氧的关键转录因子,能够调控多种下游基因转录。有相关研究揭示,表观遗传调控者能够调节HIF-1α的稳定性和活性。同样,作为表观遗传调控者的组蛋白甲基转移酶KMT2D是否参与低氧条件下HIF-1α对下游基因的调控,目前仍未知。在本研究中,观察在Kmt2d正常或缺乏的情况下,心肌细胞H9c2对低氧环境的应答反应。结果显示,与常氧条件相比,低氧状态下HIF-1α、组蛋白乙酰化酶P300、KMT2D及其介导的H3K4一甲基化 (H3K4 mono-methylation, H3K4me1)的蛋白质水平增加 (P<0.05);HIF-1α下游基因血管内皮生长因子 (vascular endothelial growth factor, Vegf) 的mRNA表达水平明显上调 (P<0.01)。染色质免疫共沉淀实验 (chromatin immunoprecipitation assay, ChIP-qPCR) 检测结果显示,H3K4me1和组蛋白3第27位赖氨酸乙酰化 (histone 3 lysine 27 acetylation, H3K27ac) 在Vegf基因启动子区域的结合丰度明显增加 (P<0.05)。低氧条件下沉默Kmt2d之后,H3K4me1蛋白水平和Vegf的mRNA表达下降 (P<0.05)。本研究表明,低氧条件下KMT2D参与调控HIF-1α和下游基因Vegf的表达。     

Hypoxia causes epigenetic gene regulation in macrophages by attenuating Jumonji histone demethylase activity

Epigenetic processes elicit changes in gene expression by modifying DNA bases or histone side chains without altering DNA sequences. Recently discovered Jumonji histone demethylases (JHDMs) affect gene expression by demethylating lysine residues of histone tails. JHDMs belong to a family of dioxygenases and share similarities with prolyl hydroxylases (PHDs). Therefore, we investigated the influence of hypoxia in macrophages on histone methylation. All JHDM family members JMJD1A–C and JMJD2A–D are expressed in macrophages. Thus, we analyzed the methylation status of histone H3 residues not only under hypoxia but also after treatment with the dioxygenase-inhibitors DMOG, NO and ROS. Western analysis revealed increased methylations in H3K9me2/me3 and H3K36me3 at pO₂ below 3%, DMOG, NO and ROS treatment. Chromatin immunoprecipitation (ChIP) assays demonstrated increased repressive marks H3K9me2 and H3K9me3 in specific promoter regions of the chemokine Ccl2 and the chemokine receptors Ccr1 and Ccr5, which correlated with a downregulation of their mRNA expression under hypoxic conditions. In contrasts, the hypoxia-inducible factor (HIF) target gene adrenomedullin (ADM) mRNA was upregulated and no increase in its histone modification was observed. We suggest that hypoxia and a concomitant loss of JHDM activity increases H3K9 methylation and decreases chemokine expression.     

The involvement of histone methylation in osteoblastic differentiation of human periosteum‐derived cells cultured in vitro under hypoxic conditions

Although oxygen concentrations affect the growth and function of mesenchymal stem cells (MSCs), the impact of hypoxia on osteoblastic differentiation is not understood. Likewise, the effect of hypoxia‐induced epigenetic changes on osteoblastic differentiation of MSCs is unknown. The aim of this study was to examine the in vitro hypoxic response of human periosteum‐derived cells (hPDCs). Hypoxia resulted in greater proliferation of hPDCs as compared with those cultured in normoxia. Further, hypoxic conditions yielded decreased expression of apoptosis‐ and senescence‐associated genes by hPDCs. Osteoblast phenotypes of hPDCS were suppressed by hypoxia, as suggested by alkaline phosphatase activity, alizarin red‐S‐positive mineralization, and mRNA expression of osteoblast‐related genes. Chromatin immunoprecipitation assays showed an increased presence of H3K27me3, trimethylation of lysine 27 on histone H3, on the promoter region of bone morphogenetic protein‐2. In addition, mRNA expression of histone lysine demethylase 6B (KDM6B) by hPDCs was significantly decreased in hypoxic conditions. Our results suggest that an increased level of H3K27me3 on the promoter region of bone morphogenetic protein‐2, in combination with downregulation of KDM6B activity, is involved in the suppression of osteogenic phenotypes of hPDCs cultured in hypoxic conditions. Although oxygen tension plays an important role in the viability and maintenance of MSCs in an undifferentiated state, the effect of hypoxia on osteoblastic differentiation of MSCs remains controversial. In addition, evidence regarding the importance of epigenetics in regulating MSCs has been limited. This study was to examine the role hypoxia on osteoblastic differentiation of hPDCs, and we examined whether histone methylation is involved in the observed effect of hypoxia on osteogenic differentiation of hPDCs.     

H1 subtype expression during cell proliferation and growth arrest

H1 histone subtype genes differ in their expression patterns during the different stages of the cell cycle interphase. While the group of replication-dependent H1 histone subtypes is synthesized during S phase, the replacement histone subtype H1.0 is also expressed replication-independently in non-proliferating cells. The present study is the first report about the analysis of the cell cycle-dependent expression of all five replication-dependent H1 subtypes, the replacement histone H1.0 and the ubiquitously expressed subtype H1x. The expression of these H1 histone subtypes in HeLa cells was analysed on mRNA level by quantitative real-time RT-PCR as well as on protein level by immunoblotting. We found that after arrest of HeLa cells in G1 phase by treatment with sodium butyrate, the mRNA levels of all replication-dependently expressed H1 subtypes decreased, but to very different extent. During S phase the individual replication-dependently expressed H1 subtypes show similar kinetics regarding their mRNA levels. However, the variations in their protein amounts partially differ from the respective RNA levels which especially applies to histone H1.3. In contrast, the mRNA as well as the protein level of H1x remained nearly unchanged in G1 as well as during S phase progression. The results of the present study demonstrate that the cell cycle-dependent mRNA and protein expression of various H1 subtypes is differentially regulated, supporting the hypothesis of a functional heterogeneity.     

Hypoxia reduces the expression of heme oxygenase-2 in various types of human cell lines. A possible strategy for the maintenance of intracellular heme level

Heme oxygenase consists of two structurally related isozymes, heme oxygenase-1 and and heme oxygenase-2, each of which cleaves heme to form biliverdin, iron and carbon monoxide. Expression of heme oxygenase-1 is increased or decreased depending on cellular microenvironments, whereas little is known about the regulation of heme oxygenase-2 expression. Here we show that hypoxia (1% oxygen) reduces the expression levels of heme oxygenase-2 mRNA and protein after 48 h of incubation in human cell lines, including Jurkat T-lymphocytes, YN-1 and K562 erythroleukemia, HeLa cervical cancer, and HepG2 hepatoma, as judged by northern blot and western blot analyses. In contrast, the expression level of heme oxygenase-1 mRNA varies under hypoxia, depending on the cell line; it was increased in YN-1 cells, decreased in HeLa and HepG2 cells, and remained undetectable in Jurkat and K562 cells. Moreover, heme oxygenase-1 protein was decreased in YN-1 cells under the conditions used, despite the induction of heme oxygenase-1 mRNA under hypoxia. The heme oxygenase activity was significantly decreased in YN-1, K562 and HepG2 cells after 48 h of hypoxia. To explore the mechanism for the hypoxia-mediated reduction of heme oxygenase-2 expression, we showed that hypoxia shortened the half-life of heme oxygenase-2 mRNA (from 12 h to 6 h) in YN-1 cells, without affecting the half-life of heme oxygenase-1 mRNA (9.5 h). Importantly, the heme contents were increased in YN-1, HepG2 and HeLa cells after 48 h of incubation under hypoxia. Thus, the reduced expression of heme oxygenase-2 may represent an important adaptation to hypoxia in certain cell types, which may contribute to the maintenance of the intracellular heme level.     

Cell-cycle-dependent gene expression studied by two-colour fluorescent detection of a mRNA and histone mRNA

We investigated whether a probe specific for histone H3 mRNA could be used as a marker to study cell-cycle dependency of gene expression by double-fluorescent RNA in situ hybridization (FISH). First, we showed that all S-phase cells in cell cultures having incorporated BrdU revealed histone H3 mRNA expression by RNA FISH, indicating that histone H3 expression is a reliable marker for S-phase cells. Second, we analysed whether the expression of human cytomegalovirus immediate early genes in rat 9G cells, which are known to be induced in an S-phase dependent way by cycloheximide, correlated with the expression of histone H3 mRNA. Double-hybridization experiments with a digoxigenin-labelled probe for IE mRNA and a fluoresceinated probe for histone H3 mRNA revealed that cells expressing IE mRNA also expressed histone H3 mRNA. Third, we examined the cell-cycle dependency of luciferase gene expression in X1 cells. Luciferase mRNA is heterogeneously expressed in X1 cell cultures, but cells expressing luciferase did not necessarily express histone H3 mRNA. This indicates that luciferase gene expression in X1 cells is not induced during S-phase. The results of our study show that histone H3 mRNA expression can be successfully used as a marker to establish cell-cycle dependency of gene expression by double-RNA FISH.     

Histone demethylase JMJD2B-mediated cell proliferation regulated by hypoxia and radiation in gastric cancer cell

Histone modifying factors are functional components of chromatin and play a role in gene regulation. The expression level of JMJD2B, a histone demethylase, is notably up-regulated in cancer tissues. Upregulation of JMJD2B promotes cancer cell proliferation under hypoxic conditions through target gene expression. Here, we describe the patterns of histone methylation and JMJD2B expression under various stressed conditions, such as hypoxia and radiation, in a gastric cancer cell line. JMJD2B expression in AGS cells was actively regulated by hypoxia and radiation. Chromatin immunoprecipitation experiments demonstrated that binding of JMJD2B on the cyclin A1 (CCNA1) promoter resulted in CCNA1 upregulation under hypoxic conditions. Furthermore, we confirmed that AGS cell proliferation was directly affected by JMJD2B and CCNA1 expression by performing experiments with JMJD2B depleted cells. Interestingly, the effects of JMJD2B on cell growth under hypoxia were remarkably repressed after gamma-ray irradiation. These results suggest that JMJD2B may play a central role in gastric cancer cell growth and might constitute a novel therapeutic target to overcome hypoxia-induced radio-resistance, thereby improving the efficiency of radiation therapy.