Histone deacetylation is required for progression through mitosis in tobacco cells

Post-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase-anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phase-specific manner, and is important for progression through mitosis.     

Human CD34+ hematopoietic progenitor cells hyperacetylate core histones in response to sodium butyrate,but not trichostatin A

Cells positive for the cell surface marker CD34 from bone marrow or umbilical cord blood form a subset of quiescent, hematopoetic precursors that can establish human hematopoesis in immunodeficient mice and can progress down various differentiation pathways in vitro. They provide a valuable model system in which progression from quiescent to cycling to differentiated states can be linked to changes in chromatin and histone modification. We have used the deacetylase inhibitor sodium butyrate to show that turnover of histone H4 acetates is rapid and comparable in quiescent and cycling CD34+ cells from human umbilical cord blood (CD34+ UBC). Surprisingly, the widely used inhibitor trichostatin A (TSA) had little (cycling cells) or no (quiescent cells) effect on H4 acetylation in CD34+ UBC. Among five cell types examined, CD34+ UBC were unique in expressing all (putative) deacetylases tested (HDAC1, -2, -3, -4, -6, -7, and -8 and SIRT1-4), but no single deacetylase correlated with their TSA resistance. Also, HDAC1, -2, -3, and -6 complexes isolated from CD34+ UBC by immunoprecipitation were all inhibited by TSA in vitro. Thus, TSA resistance of CD34+ UBC is not due to acquired or intrinsic TSA resistance of their deacetylases and may reflect an enhanced ability to process the drug.     

Microarray analysis of murine palatogenesis: temporal expression of genes during normal palate development

The mammalian face is assembled in utero in a series of complex and interdependent molecular, cell and tissue processes. The orofacial complex appears to be exquisitely sensitive to genetic and environmental influence and this explains why clefts of the lip and palate are the most common congenital anomaly in humans (one in 700 live births). In this study, microarray technology was used to identify genes that may play pivotal roles in normal murine palatogenesis. mRNA was isolated from murine embryonic palatal shelves oriented vertically (before elevation), horizontally (following elevation, before contact), and following fusion. Changes in gene expression between the three different stages were analyzed with GeneChip microarrays. A number of genes were upregulated or downregulated, and large changes were seen in the expression of loricrin, glutamate decarboxylase, gamma-amino butyric acid type A receptor beta3 subunit, frizzled, Wnt-5a, metallothionein, annexin VIII, LIM proteins, Sox1, plakophilin1, cathepsin K and creatine kinase. In this paper, the changes in genetic profile of the developing murine palate are presented, and the possible role individual genes/proteins may play during normal palate development are discussed. Candidate genes with a putative role in cleft palate are also highlighted.     

Microarray analysis of antimicrobial resistance genes in Salmonella enterica from preharvest poultry environment

Aims:  To detect antimicrobial resistance genes in Salmonella isolates from turkey flocks using the microarray technology.
Methods and Results:  A 775 gene probe oligonucleotide microarray was used to detect antimicrobial resistance genes in 34 isolates. All tetracycline-resistant Salmonella harboured tet(A) , tet(C) or tet(R) , with the exception of one Salmonella serotype Heidelberg isolate. The sul1 gene was detected in 11 of 16 sulfisoxazole-resistant isolates. The aadA , aadA1 , aadA2 , strA or strB genes were found in aminoglycoside-resistant isolates of Salm. Heidelberg, Salmonella serotype Senftenberg and untypeable Salmonella . The prevalence of mobile genetic elements, such as class I integron and transposon genes, in drug-resistant Salmonella isolates suggested that these elements may contribute to the dissemination of antimicrobial resistance genes in the preharvest poultry environment. Hierarchical clustering analysis demonstrated a close relationship between drug-resistant phenotypes and the corresponding antimicrobial resistance gene profiles.
Conclusions:  Salmonella serotypes isolated from the poultry environment carry multiple genes that can render them resistant to several antimicrobials used in poultry and humans.
Significance and Impact of the Study:  Multiple antimicrobial resistance genes in environmental Salmonella isolates could be identified efficiently by microarray analysis. Hierarchical clustering analysis of the data was also found to be a useful tool for analysing emerging patterns of drug resistance.     

Ligand-induced transrepression by VDR through association of WSTF with acetylated histones

We have previously shown that the novel ATP-dependent chromatin-remodeling complex WINAC is required for the ligand-bound vitamin D receptor (VDR)-mediated transrepression of the 25(OH)D3 1alpha-hydroxylase (1alpha(OH)ase) gene. However, the molecular basis for VDR promoter association, which does not involve its binding to specific DNA sequences, remains unclear. To address this issue, we investigated the function of WSTF in terms of the association between WINAC and chromatin for ligand-induced transrepression by VDR. Results of in vitro experiments using chromatin templates showed that the association of unliganded VDR with the promoter required physical interactions between WSTF and both VDR and acetylated histones prior to VDR association with chromatin. The acetylated histone-interacting region of WSTF was mapped to the bromodomain, and a WSTF mutant lacking the bromodomain served as a dominant-negative mutant in terms of ligand-induced transrepression of the 1alpha(OH)ase gene. Thus, our findings indicate that WINAC associates with chromatin through a physical interaction between the WSTF bromodomain and acetylated his tones, which appears to be indispensable for VDR/promoter association for ligand-induced transrepression of 1alpha(OH)ase gene expression.     

烟草细胞色素P450的基因组学分析

细胞色素P450是一类含血红素的单加氧酶超基因家族, 在植物多种代谢途径中起着重要作用。为了解烟草中的P450的种类和数量, 文章将植物代表性P450蛋白质序列与烟草基因组序列比对, 在烟草基因组中鉴定了44个P450家族共263个成员。将这些烟草P450基因与烟草表达序列标签(EST)比对, 发现173个成员有EST证据。通过与拟南芥中已知的P450蛋白序列比较, 分析了部分烟草P450蛋白序列的特征和二级结构。根据烟草基因芯片数据和部分基因的RT-PCR结果, 发现73个烟草P450基因能够在不同的生长发育时期表达, 其中部分基因具有组织特异性。这些研究结果为烟草P450基因功能的深入分析奠定了基础。     

Expression profile analysis of trichostatin A in human gastric cancer cells

Trichostatin A (TSA) is an inhibitor of histone deacetylase. cDNA microarray analysis was used to identify genes modulated by TSA. A total of 93 genes were up-regulated and 27 genes down-regulated. The expressions of some of these genes were confirmed by RT-PCR analysis.     

A series of repetitive DNA sequences are associated with human core and H1 histone genes

Summary Repetitive DNA sequences, derived from the human β-globin gene cluster, were mapped within a series of human genomic DNA segments containing core (H2A, H2B, H3 and H4) and H1 histone genes. Cloned recombinant λCH4A phage with human histone gene inserts were analyzed by Southern blot analysis using the following³²P-labeled (nick translated) repetitive sequences as probes:Alu I,Kpn I and LTR-like. A cloned DNA designated RS002-5′C6 containing (i)a (TG)₁₆ simple repeat, (ii) an (ATTTT)n repeat and (iii)a 52 base pair alternating purine and pyrimidine sequence was also used as a radiolabelled hybridization probe. Analysis of 12 recombinant phage, containing 6 arrangements of core histone genes, indicated the presence ofAlu I,Kpn and RS002-5′C6 repetitive sequences. In contrast, analysis of 4 human genomic DNA segments, containing both core and H1 histone genes, indicated the presence of onlyAlu I family sequences. LTR-like sequences were not detected in association with any of the core or H1 histone genes examined. These results suggest that human histone and β-globin genes share certain aspects of sequence organization in flanking regions despite marked differences in their overall structure and pattern of expression.     

Integrated analysis of DNA methylation and mRNA expression profiling reveals candidate genes associated with cisplatin resistance in non-small cell lung cancer

DNA methylation plays a critical role during the development of acquired chemoresistance. The aim of this study was to identify candidate DNA methylation drivers of cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC). The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. Gene expression and methylation profiling were determined by high-throughput microarrays. Relationship of methylation status and DDP response was validated in primary tumor cell culture and the Cancer Genome Atlas (TCGA) samples. Cell proliferation, apoptosis, cell cycle, and response to DDP were determined in vitro and in vivo. A total of 372 genes showed hypermethylation and downregulation in A549/DDP cells, and these genes were involved in most fundamental biological processes. Ten candidate genes (S100P, GDA, WISP2, LOXL1, TIMP4, ICAM1, CLMP, HSP8, GAS1, BMP2) were selected, and exhibited varying degrees of association with DDP resistance. Low dose combination of 5-aza-2′-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) reversed drug resistance of A549/DDP cells in vitro and in vivo, along with demethylation and restoration of expression of candidate genes (GAS1, TIMP4, ICAM1 and WISP2). Forced expression of GAS1 in A549/DDP cells by gene transfection contributed to increased sensitivity to DDP, proliferation inhibition, cell cycle arrest, apoptosis enhancement, and in vivo growth retardation. Together, our study demonstrated that a panel of candidate genes downregulated by DNA methylation induced DDP resistance in NSCLC, and showed that epigenetic therapy resensitized cells to DDP.     

Identification and validation of key genes associated with non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) is one of the main causes of death induced by cancer globally. However, the molecular aberrations in NSCLC patients remain unclearly. In the present study, four messenger RNA microarray datasets (GSE18842, GSE40275, GSE43458, and GSE102287) were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) between NSCLC tissues and adjacent lung tissues were obtained from GEO2R and the overlapping DEGs were identified. Moreover, functional and pathway enrichment were performed by Funrich, while the protein–protein interaction (PPI) network construction were obtained from STRING and hub genes were visualized and identified by Cytoscape software. Furthermore, validation, overall survival (OS) and tumor staging analysis of selected hub genes were performed by GEPIA. A total of 367 DEGs (95 upregulated and 272 downregulated) were obtained through gene integration analysis. The PPI network consisted of 94 nodes and 1036 edges in the upregulated DEGs and 272 nodes and 464 edges in the downregulated DEGs, respectively. The PPI network identified 46 upregulated and 27 downregulated hub genes among the DEGs, and six (such as CENPE, NCAPH, MYH11, LRRK2, HSD17B6, and A2M) of that have not been identified to be associated with NSCLC so far. Moreover, the expression differences of the mentioned hub genes were consistent with that in lung adenocarcinoma and lung squamous cell carcinoma in the TCGA database. Further analysis showed that all the six hub genes were associated with tumor staging except MYH11, while only the upregulated DEG CENPE was associated with the worse OS of patients with NSCLC. In conclusion, the current study showed that CENPE, NCAPH, MYH11, LRRK2, HSD17B6, and A2M might be the key genes contributed to tumorigenesis or tumor progression in NSCLC, further functional study is needed to explore the involved mechanisms.     

Chromosomal locations of the genes for histones and a histone gene-binding protein family HBP-1 in common wheat

The chromosomal locations of the genes in common wheat that encode the five histones and five members of the HBP (histone gene-binding protein)-1 family were determined by hybridizing their cloned DNAs to genomic DNAs of nullitetrasomic and telosomic lines of common wheat, Triticum aestivum cv. Chinese Spring. The H1 and H2a genes are located on different sets of homoeologous chromosomes or chromosome arms, namely, 5A, 5B and 5D, and 2AS, 2BS and 2DS, respectively. Genes for the other histones, H2b, H3 and H4, are found in high copy number and are dispersed among a large number of chromosomes. The genes for all members of the HBP-1 family are present in small copy numbers. Those for HBP-1a(1) are located on six chromosome arms, 3BL, 5AL, 5DL, 6AL, 6BS and 7DL, whereas those for each HBP-1a(c14), 1a(17), 1b(c1), and 1b(c38) are on a single set of homoeologous chromosome arms; 4AS, 4BL, 4DL; 6AS, 6BS, 6DS; 3AL, 3BL, 3DL; and 3AS, 3BS, 3DS, respectively. The genes for histones H1 and H2a, and for all members of the HBP-1 family except HBP-1a(1) are assumed to have different phylogenetic origins. The genes for histone 2a and HBP-1a(17) are located in the RFLP maps of chromosomes 2B and 6A, respectively. Gene symbols are proposed for all genes whose chromosomal locations have been determined.     

Microarray analysis of genes with differential expression of m6A methylation in lung cancer

Purpose: N6-methyladenosine (m6A) is among the most abundant mRNA modifications in eukaryote. The aim of the present study was to investigate function of m6A mRNA methylation in lung cancer and the underlying mechanism.Methods: Microarray analysis was performed to detect the differences in RNA expression between cancerous and adjacent non-cancerous tissue samples. The target mRNAs were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Hierarchical clustering of RNAs was conducted to identify distinct m6A methylation or expression patterns between the samples.Results: In the present study, some differentially expressed genes (DEGs) of mRNAs were identified, including up-regulated secret phosphoprotein 1 (SPP1) and down-regulated pRB. Functional enrichment analysis revealed that while differential hypermethylation was related to cell cycle, intracellular part and protein binding, the main pathway involved herpes simplex virus 1 infection related to down-regulated AKT, Araf1 and BCL2A1. In the meantime, sexual reproduction, cohesin complex and protein C-terminus binding was functionally linked to differential hypomethylation, while fluid shear stress and atherosclerosis were identified as the main pathways related to up-regulated GST and CNP.Conclusions: We showed that lung cancer development involved differential expression of SPP1 and pRB mRNA, as well as m6A mRNA methylation in AKT, APAF1, BCL2A1, GST and CNP genes.     

Integrated analysis of DNA methylation and mRNA expression profiling reveals candidate genes associated with cisplatin resistance in non-small cell lung cancer

DNA methylation plays a critical role during the development of acquired chemoresistance. The aim of this study was to identify candidate DNA methylation drivers of cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC). The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. Gene expression and methylation profiling were determined by high-throughput microarrays. Relationship of methylation status and DDP response was validated in primary tumor cell culture and the Cancer Genome Atlas (TCGA) samples. Cell proliferation, apoptosis, cell cycle, and response to DDP were determined in vitro and in vivo. A total of 372 genes showed hypermethylation and downregulation in A549/DDP cells, and these genes were involved in most fundamental biological processes. Ten candidate genes (S100P, GDA, WISP2, LOXL1, TIMP4, ICAM1, CLMP, HSP8, GAS1, BMP2) were selected, and exhibited varying degrees of association with DDP resistance. Low dose combination of 5-aza-2′-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) reversed drug resistance of A549/DDP cells in vitro and in vivo, along with demethylation and restoration of expression of candidate genes (GAS1, TIMP4, ICAM1 and WISP2). Forced expression of GAS1 in A549/DDP cells by gene transfection contributed to increased sensitivity to DDP, proliferation inhibition, cell cycle arrest, apoptosis enhancement, and in vivo growth retardation. Together, our study demonstrated that a panel of candidate genes downregulated by DNA methylation induced DDP resistance in NSCLC, and showed that epigenetic therapy resensitized cells to DDP.     

Molecular characterization and expression of a tobacco histone H1 cDNA

We have isolated a 1104 bp tobacco cDNA clone (H1c12) which includes an 846 bp open reading frame. This encodes a polypeptide of 282 amino acid residues and represents the largest plant H1 histone identified so far. The structure of the deduced protein shows the classical tripartite organization of the H1-type linker histones. The expression of the tobacco H1 histone gene(s) corresponding to the H1c12 cDNA clone was examined during different developmental stages. We found that, at the level of steadystate mRNA, expression of gene(s) encoding this H1 histone was rapidly induced in germinating seeds. The H1 gene was expressed in all tissues examined. However, its expression was higher in tissues known to contain meristematic cells. Furthermore, in the leaves of mature plants accumulation of the H1 mRNA exhibits a very characteristic oscillation. This latter finding indicates that, at least in fully developed plants, the expression of this type of H1 histone gene(s) is modulated by a diurnal cycle.