Compounds in cigarette smoke induce EGR1 expression via the AHR, resulting in apoptosis and COPD.

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide, and pulmonary epithelial cell apoptosis is regarded as one of the most important factors in its pathogenesis. Here, we examined the molecular mechanisms of apoptosis caused by cigarette smoke (CS). In the normal bronchial epithelium cell line BEAS-2B, a CS extract markedly induced apoptosis together with transient early growth response 1 (EGR1) protein expression, which is activated over time via the aryl hydrocarbon receptor (AHR). The CS extract-induced apoptosis decreased cell count of BEAS-2B cells and was significantly reversed by knockdown of either EGR1 or AHR. In vivo, the CS extract caused alveolar wall destruction, mimicking COPD, 1 week after intrathoracic injection. Bronchoalveolar lavage fluid (BALF) from the CS extract-treated mice contained massive numbers of apoptotic epithelial cells. Furthermore, it was found that aminoanthracene induced EGR1 expression and cell apoptosis. By contrast, the AHR antagonist stemregenin 1 (SR1) restored apoptosis upon CS treatment. These results suggest that aryl hydrocarbons, such as aminoanthracene, induce EGR1 expression via the AHR, resulting in cell apoptosis and that this can be prevented by administration of an antagonist of AHR.

Nucleosome assembly protein 1 is a regulator of histone H1 acetylation.

Acetylation of histone H1 is generally considered to activate transcription, whereas deacetylation of H1 represses transcription. However, the precise mechanism of the acetylation is unknown. Here, using chromatography, we identified nucleosome assembly protein 1 (NAP-1) as having inhibitory activity against histone H1 acetylation by acetyltransferase p300. We found that native NAP-1 interacts with H1 in a Drosophila crude extract. We also found that it inhibits the deacetylation of histone H1 by histone deacetylase 1. The core histones in nucleosomes were acetylated in a GAL4-VP16 transcriptional activator-dependent manner in vitro. This acetylation was strongly repressed by hypoacetylated H1 but to a lesser extent by hyperacetylated H1. Consistent with these findings, a micrococcal nuclease assay indicated that hypoacetylated H1, which represses activator-dependent acetylation, was incorporated into chromatin, whereas hyperacetylated H1 was not. To determine the contribution of NAP-1 to transcriptional regulation in vivo, we compared NAP-1 knockdown (KD) with coactivator CREB-binding protein (CBP) KD using RNA sequencing in Drosophila Schneider 2 cells. Most genes were downregulated rather than upregulated by NAP-1 KD, and those downregulated genes were also downregulated by CBP KD. Our results suggest that NAP-1 plays a role in transcriptional regulation by fine-tuning the acetylation of histone H1. Graphical Abstract.

Long noncoding RNAs transcribed downstream of the human ß-globin locus regulate ß-globin gene expression.

The five ß-like globin genes (ε, Gγ, Aγ, δ and ß) at the human ß-globin gene locus are known to be expressed at specific developmental stages, although details of the underlying mechanism remain to be uncovered. Here we used an in vitro transcription assay to clarify the mechanisms that control this gene expression. We first tested nuclear RNA from HeLa cells using RT-qPCR and discovered a long noncoding RNAs (lncRNAs) within a 5.2-kb region beginning 4.4 kb downstream of the ß-globin gene coding region. We investigated nuclear RNA from K562 cells using a primer-extension assay and determined the transcription start sites (TSSs) of these lncRNAs. To clarify their functional role, we performed knockdown (KD) of these lncRNAs in K562 cells. Hydroxyurea (HU), which induces differentiation of K562 cells, increased haemoglobin peptide production, and the effect was enhanced by KD of these lncRNAs, which also enhanced upregulation of the γ-globin expression induced by HU. To confirm these results, we performed an in vitro transcription assay. Noncoding single-stranded RNAs inhibited ß-globin expression, which was upregulated by GATA1. Furthermore, lncRNAs interacted with GATA1 without sequence specificity and inhibited its binding to its target DNA response element in vitro. Our results suggest that lncRNAs downstream of the ß-globin gene locus are key factors regulating globin gene expression.

The nucleolus from a liquid droplet perspective.

The nucleolus is a membrane-less organelle sequestered from the nucleus by liquid droplet formation through a liquid-liquid phase separation (LLPS). It plays important roles in cell homoeostasis through its internal thermodynamic changes. Reversible nucleolar transitions between coalescence and dispersion are dependent on the concentrations, conformations and interactions of its molecular liquid droplet-forming components, including DNA, RNA and protein. The liquid droplet-like properties of the nucleolus enable its diverse dynamic roles. The liquid droplet formation mechanism, by which the nucleolus is sequestered from the nucleoplasm despite the absence of a membrane, explains a number of complex nucleolar functions.

Cigarette smoke, but not novel tobacco vapor products, causes epigenetic disruption and cell apoptosis.

Heat-Not-Burn (HNB) products, generating vapor without combusting tobacco leaves, have been developed with the expectation that the number and quantity of chemicals in the vapor of these products would be reduced compared with the smoke from conventional combustible cigarettes. However, whether the lower chemical levels correlate with lower toxicity remains to be determined. Here we examined differences in the biological effects of conventional cigarette smoke (CS) and two HNB products, Ploom TECH and Ploom TECH+, using the cultured cancer cell line A549 and the normal bronchial epithelium cell line BEAS-2B. The conventional CS 3R4F extract (0.5%) markedly decreased cell proliferation of both A549 and BEAS-2B cells; however, 0.5% extracts of these commercially available HNB products did not affect cell growth. To determine the cause of decreased cell proliferation, a TUNEL assay was performed, and the results indicated that apoptosis had occurred in both A549 and BEAS-2B cells at 24 h after exposure to 3R4F. To further explore the effect of CS on epigenetics, we performed western blotting to detect histone H2A phosphorylation, which is known to affect transcriptional regulation. Only the 3R4F extract decreased histone H2A phosphorylation in both A549 and BEAS-2B cells. Next, we examined alterations in gene expression after treatment of A549 cells with Ploom TECH, Ploom TECH+, or 3R4F extracts. It was found that 339, 107, and 103 genes were upregulated more than 2 fold in A549 cells treated with 3R4F, Ploom TECH, or Ploom TECH + extracts, respectively. Among the 339 genes that were upregulated in response to 3R4F, we focused on EGR1, FOS, and FOSB, since they were upregulated more than 100 fold, which was confirmed using RT-qPCR. These results suggest that CS, but not HNB products, cause epigenetic disruption and cell apoptosis, possibly by elevating transcription of genes such as EGR1.

miR-222 regulates proliferation of primary mouse hepatocytes in vitro.

It is well known that hepatocytes regenerate after liver injury, although it is difficult to reproduce this phenomenon in vitro. The goal of this research was to determine the factors that stimulate proliferation of primary mouse hepatocytes (PMHs) in vitro. We first tested knockdown (KD) of tumor protein 53 (p53) alone as well as partial hepatectomy (PH, performed 72 h prior to PMHs preparation) alone. However, neither intervention stimulated hepatocyte proliferation during the 72-h observation period in vitro. We then tested the combination of p53 KD with PH and found that these interventions together stimulated cell proliferation in vitro. Under these latter conditions we analyzed gene expression of these cells by mRNA sequencing (RNA-seq) and microRNA sequencing (miRNA-seq). TargetScan analysis, which determines the relationship between microRNAs and gene expression, found a relationship between downregulated mmu-mir-222 (miR-222) and upregulated genes such as mitogen-activated protein kinase kinase kinase 2 (Map3k2). To confirm this relationship, we performed miR-222 KD and overexpression (OE) and observed the expected changes in target gene expression. Furthermore, the finding that miR-222 KD or OE stimulates or suppresses, respectively, hepatocyte proliferation is well explained by the association between miR-222 and its target genes, which stimulate growth. Our results suggest that miR-222 is one of the key factors regulating PMH proliferation in vitro.

Defects in centromeric/pericentromeric histone H2A T120 phosphorylation by hBUB1 cause chromosome missegregation producing multinucleated cells.

Histone H2A phosphorylation plays a role both in chromatin condensation during mitosis and in transcriptional activation during the G1/S transition. Bub1 and NHK1/VRK1 have been identified as histone H2A kinases. However, little is known about the importance of histone H2A phosphorylation in chromosome segregation. Here, we expressed recombinant hBUB1 and confirmed that it phosphorylates histone H2A T120 in the in vitro-assembled nucleosome. Knockdown (KD) of BUB1 decreases bulk H2A T120 phosphorylation in HeLa cells, whereas hBUB1 is upregulated during mitosis, which corresponds with H2A T120 phosphorylation. ChIP-qPCR of the DXZ1 centromeric and γ-ALR pericentromeric region showed that BUB1 localizes to this region and increases local H2A T120 phosphorylation during M phase. BUB1 KD did not induce apoptosis but increased the M phase cell population, as detected by flow cytometry. BUB1 KD also caused an abnormal metaphase and telophase, resulting in multinucleated cells and impaired cancer cell growth both in vitro and in vivo. Over-expression of the histone H2A T120D or T120E mutations, which mimic phosphorylated threonine, decreased the number of multinucleated cells caused by BUB1 KD. These results strengthen the apparent importance of BUB1-mediated H2A T120 phosphorylation in normal mitosis.

Enhancer function regulated by combinations of transcription factors and cofactors.

Regulation of the expression of diverse genes is essential for making possible the complexity of higher organisms, and the temporal and spatial regulation of gene expression allows for the alteration of cell types and growth patterns. A critical component of this regulation is the DNA sequence-specific binding of transcription factors (TFs). However, most TFs do not independently participate in gene transcriptional regulation, because they lack an effector function. Instead, TFs are thought to work by recruiting cofactors, including Mediator complex (Mediator), chromatin-remodeling complexes (CRCs), and histone-modifying complexes (HMCs). Mediator associates with the majority of transcribed genes and acts as an integrator of multiple signals. On the other hand, CRCs and HMCs are selectively recruited by TFs. Although all the pairings between TFs and CRCs or HMCs are not fully known, there are a growing number of established TF-CRC and TF-HMC combinations. In this review, we focused on the most important of these pairings and discuss how they control gene expression.

MicroRNA-3662 expression correlates with antiviral drug resistance in adult T-cell leukemia/lymphoma cells.

Interferon regulatory factor (IRF) 4 and the proto-oncogene c-Rel cooperate in growth and antiviral drug resistance of adult T-cell leukemia/lymphoma (ATLL). To elucidate the target of IRF4 and c-Rel in ATLL, we determined the simultaneous binding sites of IRF4 and c-Rel using ChIP-seq technology. Nine genes were identified within 2 kb of binding sites, including MIR3662. Expression of miR-3662 was regulated by IRF4, and to a lesser extent by c-Rel. Cell proliferation was inhibited by knockdown of miR-3662 and expression of miR-3662 was correlated with antiviral drug resistance in ATLL cell lines. Thus, miR-3662 represents a target for therapies against ATLL.

Histone H2A T120 Phosphorylation Promotes Oncogenic Transformation via Upregulation of Cyclin D1.

How deregulation of chromatin modifiers causes malignancies is of general interest. Here, we show that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrate that this phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation.

SMARCAD1 is an ATP-dependent stimulator of nucleosomal H2A acetylation via CBP, resulting in transcriptional regulation.

Histone acetylation plays a pivotal role in transcriptional regulation, and ATP-dependent nucleosome remodeling activity is required for optimal transcription from chromatin. While these two activities have been well characterized, how they are coordinated remains to be determined. We discovered ATP-dependent histone H2A acetylation activity in Drosophila nuclear extracts. This activity was column purified and demonstrated to be composed of the enzymatic activities of CREB-binding protein (CBP) and SMARCAD1, which belongs to the Etl1 subfamily of the Snf2 family of helicase-related proteins. SMARCAD1 enhanced acetylation by CBP of H2A K5 and K8 in nucleosomes in an ATP-dependent fashion. Expression array analysis of S2 cells having ectopically expressed SMARCAD1 revealed up-regulated genes. Using native genome templates of these up-regulated genes, we found that SMARCAD1 activates their transcription in vitro. Knockdown analysis of SMARCAD1 and CBP indicated overlapping gene control, and ChIP-seq analysis of these commonly controlled genes showed that CBP is recruited to the promoter prior to SMARCAD1. Moreover, Drosophila genetic experiments demonstrated interaction between SMARCAD1/Etl1 and CBP/nej during development. The interplay between the remodeling activity of SMARCAD1 and histone acetylation by CBP sheds light on the function of chromatin and the genome-integrity network.

Dzip3 regulates developmental genes in mouse embryonic stem cells by reorganizing 3D chromatin conformation.

In mouse embryonic stem (mES) cells, ubiquitylation of histone H2A lysine 119 represses a large number of developmental genes and maintains mES cell pluripotency. It has been suggested that a number of H2A ubiquitin ligases as well as deubiquitylases and related peptide fragments contribute to a delicate balance between self-renewal and multi-lineage differentiation in mES cells. Here, we tested whether known H2A ubiquitin ligases and deubiquitylases are involved in mES cell regulation and discovered that Dzip3, the E3 ligase of H2AK119, represses differentiation-inducible genes, as does Ring1B. The two sets of target genes partially overlapped but had different spectra. We found that Dzip3 represses gene expression by orchestrating changes in 3D organization, in addition to regulating ubiquitylation of H2A. Our results shed light on the epigenetic mechanism of transcriptional regulation, which depends on 3D chromatin reorganization to regulate mES cell differentiation.

Enhancer of Acetyltransferase Chameau (EAChm) Is a Novel Transcriptional Co-Activator.

Acetylation of nucleosomal histones by diverse histone acetyltransferases (HAT) plays pivotal roles in many cellular events. Discoveries of novel HATs and HAT related factors have provided new insights to understand the roles and mechanisms of histone acetylation. In this study, we identified prominent Histone H3 acetylation activity in vitro and purified its activity, showing that it is composed of the MYST acetyltransferase Chameau and Enhancer of the Acetyltransferase Chameau (EAChm) family. EAChm is a negatively charged acidic protein retaining aspartate and glutamate. Furthermore, we identified that Chameau and EAChm stimulate transcription in vitro together with purified general transcription factors. In addition, RNA-seq analysis of Chameu KD and EAChm KD S2 cells suggest that Chameau and EAChm regulate transcription of common genes in vivo. Our results suggest that EAChm regulates gene transcription in Drosophila embryos by enhancing Acetyltransferase Chameau activity.

The USP21 short variant (USP21SV) lacking NES, located mostly in the nucleus in vivo, activates transcription by deubiquitylating ubH2A in vitro.

USP21 is a deubiquitylase that catalyzes isopeptide bond hydrolysis between ubiquitin and histone H2A. Since ubiqutylated H2A (ubH2A) represses transcription, USP21 plays a role in transcriptional activation. On the other hand, the localization of USP21 suggests it has an additional function in the cytoplasm. Here, we identified a USP21 short variant (USP21SV) lacking a nuclear export signal (NES). Differential localization of USP21SV, more in the nucleus than the USP21 long variant (USP21LV), suggests they have redundant roles in the cell. Ectopic expression of both USP21 variants decreased ubH2A in the nucleus. Furthermore, both recombinant USP21 variants activate transcription by deubiquitylating ubH2A in vitro. These data suggest multiple roles for USP21 in the ubiquitylation-deubiquitylation network in the cell.

Deubiquitylation of histone H2A activates transcriptional initiation via trans-histone cross-talk with H3K4 di- and trimethylation.

Transcriptional initiation is a key step in the control of mRNA synthesis and is intimately related to chromatin structure and histone modification. Here, we show that the ubiquitylation of H2A (ubH2A) correlates with silent chromatin and regulates transcriptional initiation. The levels of ubH2A vary during hepatocyte regeneration, and based on microarray expression data from regenerating liver, we identified USP21, a ubiquitin-specific protease that catalyzes the hydrolysis of ubH2A. When chromatin is assembled in vitro, ubH2A, but not H2A, specifically represses the di- and trimethylation of H3K4. USP21 relieves this ubH2A-specific repression. In addition, in vitro transcription analysis revealed that ubH2A represses transcriptional initiation, but not transcriptional elongation, by inhibiting H3K4 methylation. Notably, ubH2A-mediated repression was not observed when H3 Lys 4 was changed to arginine. Furthermore, overexpression of USP21 in the liver up-regulates a gene that is normally down-regulated during hepatocyte regeneration. Our studies revealed a novel mode of trans-histone cross-talk, in which H2A ubiquitylation controls the di- and trimethylation of H3K4, resulting in regulation of transcriptional initiation.

Nucleosomal histone kinase-1 phosphorylates H2A Thr 119 during mitosis in the early Drosophila embryo.

Posttranslational histone modifications are important for the regulation of many biological phenomena. Here, we show the purification and characterization of nucleosomal histone kinase-1 (NHK-1). NHK-1 has a high affinity for chromatin and phosphorylates a novel site, Thr 119, at the C terminus of H2A. Notably, NHK-1 specifically phosphorylates nucleosomal H2A, but not free H2A in solution. In Drosophila embryos, phosphorylated H2A Thr 119 is found in chromatin, but not in the soluble core histone pool. Immunostaining of NHK-1 revealed that it goes to chromatin during mitosis and is excluded from chromatin during S phase. Consistent with the shuttling of NHK-1 between chromatin and cytoplasm, H2A Thr 119 is phosphorylated during mitosis but not in S phase. These studies reveal that NHK-1-catalyzed phosphorylation of a conserved serine/threonine residue in H2A is a new component of the histone code that might be related to cell cycle progression.

Isoflavones stimulate estrogen receptor-mediated core histone acetylation.

The isoflavones genistein and daidzein and the daidzein metabolite equol have been reported to interact with estrogen receptors (ERs). Some studies indicate that they behave clinically like estrogen in some estrogen-deficiency diseases. However, the detailed molecular mechanism used by these compounds to create beneficial effects in patients with estrogen-related diseases has not been clarified. Using histone acetyltransferase (HAT) assay, we found that equol, genistein, and AglyMax had significant effects on ERalpha-mediated histone acetylation. Although 17beta-estradiol (E2)-dependent HAT activity of steroid receptor coactivators 2 (SRC2) and p300 mediated by ERbeta could be detected, it was weaker than that mediated by ERalpha. Equol, genistein, AglyMax, and daidzein all markedly stimulated ERbeta-mediated histone acetylation. On the other hand, anti-estrogenic compounds ICI 182,780 (ICI) and tamoxifen (TA) did not have an effect on HAT activity mediated by either ERalpha or ERbeta. Our data indicate that estrogenic ligands exert their effects by elevating histone acetylation and coactivator activity of ER, and suggest that the risk of estrogen-related diseases might be reduced by a sufficient amount of genistein or AglyMax supplements.

The chromatin-remodeling complex WINAC targets a nuclear receptor to promoters and is impaired in Williams syndrome.

We identified a human multiprotein complex (WINAC) that directly interacts with the vitamin D receptor (VDR) through the Williams syndrome transcription factor (WSTF). WINAC has ATP-dependent chromatin-remodeling activity and contains both SWI/SNF components and DNA replication-related factors. The latter might explain a WINAC requirement for normal S phase progression. WINAC mediates the recruitment of unliganded VDR to VDR target sites in promoters, while subsequent binding of coregulators requires ligand binding. This recruitment order exemplifies that an interaction of a sequence-specific regulator with a chromatin-remodeling complex can organize nucleosomal arrays at specific local sites in order to make promoters accessible for coregulators. Furthermore, overexpression of WSTF could restore the impaired recruitment of VDR to vitamin D regulated promoters in fibroblasts from Williams syndrome patients. This suggests that WINAC dysfunction contributes to Williams syndrome, which could therefore be considered, at least in part, a chromatin-remodeling factor disease.