Histone acetylation and chromatin remodeling are required for UV-B-dependent transcriptional activation of regulated genes in maize

The nuclear proteomes of maize (Zea mays) lines that differ in UV-B tolerance were compared by two-dimensional gel electrophoresis after UV light treatment. Differential accumulation of chromatin proteins, particularly histones, constituted the largest class identified by mass spectrometry. UV-B-tolerant landraces and the B73 inbred line show twice as many protein changes as the UV-B-sensitive b, pl W23 inbred line and transgenic maize expressing RNA interference constructs directed against chromatin factors. Mass spectrometic analysis of posttranslational modifications on histone proteins demonstrates that UV-B-tolerant lines exhibit greater acetylation on N-terminal tails of histones H3 and H4 after irradiation. These acetylated histones are enriched in the promoter and transcribed regions of the two UV-B-upregulated genes examined; radiation-sensitive lines lack this enrichment. DNase I and micrococcal nuclease hypersensitivity assays indicate that chromatin adopts looser structures around the selected genes in the UV-B-tolerant samples. Chromatin immunoprecipitation experiments identified additional chromatin factor changes associated with the nfc102 test gene after UV-B treatment in radiation-tolerant lines. Chromatin remodeling is thus shown to be a key process in acclimation to UV-B, and lines deficient in this process are more sensitive to UV-B.     

Split-plot microarray design allows sensitive detection of expression differences after ultraviolet radiation in the inbred parental lines of a key maize mapping population

Gene expression levels were quantified after ultraviolet radiation treatment in the parental inbred lines of the maize mapping (IBM) population. This allows us to take advantage of natural variation between maize lines to analyse variation in gene expression. Using a statistically sound split‐plot experiment cDNAs were identified with differently regulated expression in B73 and Mo17 after UV treatment. Fewer genes were down‐regulated in B73; this global strain difference in the number of genes up‐ and down‐regulated does not appear to reflect general hybridization differences. Contrary to our expectation, there was a higher proportion of highly expressed genes (based on EST recovery) that were differently expressed by UV between lines. Genes affected by UV (but not significantly different between B73 and Mo17) include gene types proposed to function in UV acclimation and adaptation based on experiments in other species or other experiments in maize. Several new functional classes were identified as UV‐regulated, including genes encoding proteins that modulate chromatin structure.     

Molecular consequences of silencing mutant K-ras in pancreatic cancer cells: justification for K-ras-directed therapy

Mutation of the K-ras gene is an early event in the development of pancreatic adenocarcinoma and, therefore, RNA interference (RNAi) directed toward mutant K-ras could represent a novel therapy. In this study, we examine the phenotypic and molecular consequences of exposure of pancreatic tumor cells to mutant-specific K-ras small interfering RNA. Specific reduction of activated K-ras via RNAi in Panc-1 and MiaPaca-2 cells resulted in cellular changes consistent with a reduced capacity to form malignant tumors. These changes occur through distinct mechanisms but likely reflect an addiction of each cell line to oncogene stimulation. Both cell lines show reduced proliferation after K-ras RNAi, but only MiaPaca-2 cells showed increased apoptosis. Both cell lines showed reduced migration after K-ras knockdown, but changes in integrin levels were not consistent between the cell lines. Both cell lines showed alteration of the level of GLUT-1, a metabolism-associated gene that is downstream of c-myc, with Panc-1 cells demonstrating decreased GLUT-1 levels, whereas MiaPaca-2 cells showed increased levels of expression after K-ras knockdown. Furthermore, after K-ras RNAi, there was a reduction in angiogenic potential of both Panc-1 and MiaPaca-2 cells. Panc-1 cells increased the level of expression of thrombospondin-1, an endogenous inhibitor of angiogenesis, whereas MiaPaca-2 cells decreased the production of vascular endothelial growth factor, a primary stimulant of angiogenesis in pancreatic tumors. We have found that silencing mutant K-ras through RNAi results in alteration of tumor cell behavior in vitro and suggests that targeting mutant K-ras specifically might be effective against pancreatic cancer in vivo.     

Contribution of phenolic compounds to the UV-B screening capacity of developing barley primary leaves in relation to DNA damage and repair under elevated UV-B levels

Epidermally located UV-absorbing hydroxycinnamic acid conjugates and flavonoid glycosides are known to be efficient UV-B protectants in higher plants, although important biological molecules are not always fully protected. However, repair mechanisms also exist, such as repair of damaged DNA by photolyases. To distinguish between the relative importance of the phenolic compounds and of DNA repair, developing primary leaves of two barley lines, mutant ant 30-310, deficient in flavonoids, and its parent line Ca 33787, were grown under relatively high visible light (650-700 micromol m(-2) s(-1) max for 6 h in a 13 h photoperiod) and supplemented with (+ UV-B) or without (-UV-B) 12 kJ m(-2) UV-B(BE) for 6 h daily. UV-B screening capacity of the leaf phenolics was determined at 315 nm during leaf development and compared with thymine dimers (TD) accumulation, as an indicator of UV-B-induced DNA damage and potential subsequent repair. The degree of damage was related to the phenolic contents of the leaves. UV-B screening capacity was increased ca. 4-fold in the parent line (+ UV-B), mainly due to UV-induced flavonoid (saponarin, lutonarin) accumulation in epidermal and subepidermal mesophyll tissue, relative to the flavonoid-deficient mutant. Nevertheless, in the parent line an 8-fold increase in TD levels occurred over the growth period of 18 days, whereas the mutant accumulated additional DNA damage, with 6- to 9-fold higher TD amounts. Surprisingly, under the high UV-B irradiation, growth and development of the primary leaves in both lines were only slightly reduced.     

Response of the estuarine cyanobacterium <Emphasis Type="Italic">Lyngbya aestuarii</Emphasis> to UV-B radiation

Growth response and changes in the spectral properties of methanolic extract of an estuarine cyanobacterium, Lyngbya aestuarii Agardh, to UV-B radiation were studied. Increase in growth accompanied by increase in chlorophyll a, protein and carbohydrate content was observed up to 12 h of UV-B irradiation followed by a decline with further increase in the duration of UV exposure. Carotenoid content progressively increased with the UV-B dose. The organism synthesized, to a significant extent, mycosporine amino acid-like substances (MAAs) upon UV-B exposure. The cells in the trichome became coiled followed by formation of small bundles as a response to UV-B radiation. SDS protein profile of the UV irradiated cells showed repression of 20 and 22 kDa proteins. However, irradiation with UV-B for 6–24 h led to overproduction of 84, 73, 60, 46, 40, 37 KDa proteins, possibly conferring protection to the organism from UV-B. UV irradiated cells cultured in florescent light for up to 7 days showed revival from UV damage of the pigments and macromolecular contents, suggesting existence of a repair mechanism in the organism.     

联合应用RNA-seq和ATAC-seq寻找FOXQ1转录因子的下游靶基因

结直肠癌(Colorectal cancer, CRC)是一种全球高发的恶性肿瘤,发病原因复杂且预后较差。近年来发现叉头框Q1(Forkhead box Q1,FOXQ1)基因作为一类核转录因子在结直肠癌中高表达,可控制下游基因转录活性。本实验拟探究CRC细胞中FOXQ1的转录调控功能并寻找其下游基因。方法:(1)构建低表达FOXQ1基因的稳定转染CRC细胞株;(2)应用RNA-seq检测FOXQ1敲低前后表达量显著差异的基因;(3)应用转座酶可接近性核染色质区域测序分析(Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq)检测FOXQ1敲低前后细胞染色质易接近性的变化;(4)进一步对FOXQ1敲低前后的RNA-seq和ATAC-seq数据进行一系列生物信息学分析,寻找CRC中FOXQ1转录调控的潜在下游基因。结果:应用RNA-seq筛选出了敲低FOXQ1后表达显著差异的基因EI24、TLR2、SMAD3,通过联合分析两细胞系的测序结果,发现FOXQ1基因敲低后,在DLD1和SW480两个细胞系中染色质易接近性均增强且表达量均上调的基因有61个,染色质易接近性均减弱且表达量均下调的基因有70个,且EI24、TLR2、SMAD3基因均位于重叠分析结果中,其中TLR2、SMAD3基因的染色质区域有明显变化,而EI24基因的染色质区域变化不明显。通过代谢通路分析找到了EI24、TLR2、SMAD3基因所富集的代谢通路。其中SMAD3、TLR2基因在炎症性肠病(Inflammatory bowel disease, IBD)通路中显著富集。EI24基因在p53信号通路(p53 signaling pathway)通路中显著富集。结论:基于染色质易接近性的变化和转录水平的研究发现:敲低FOXQ1基因对CRC细胞系中染色质的开放情况有较大的影响,且影响FOXQ1转录调控的下游基因的表达。找到了FOXQ1敲低后在SW480、DLD1中均发生变化的基因,为丰富FOXQ1转录因子的下游调控网络提供了研究基础。     

UV-B Irradiated Cell Lines Execute Programmed Cell Death in Various Forms

We investigated changes typical for apoptosis in various cell lines after UV-B irradiation. Using established methods for detection of apoptosis we demonstrate changes of cellular morphology, phosphatidylserine (PS) exposure, ollgonucleosomal DNA fragmentation and generation of hypochrome nuclei. To isolated high-molecular-weight (hmwt) DNA fragments we engaged a new method avoiding pulse field gel electrophoresis. Most UV-B irradiated cell lines showed oligonucleosomal DNA fragmentation, hypochrome nuclei, morphological changes, annexin-V binding and positive TUNEL reaction. However, no oligonucleosomal DNA fragmentation could be detected in Raji and HaCaT cells. Whereas HaCaT cells displayed all other changes typical for apoptosis, Raji cells were TUNEL negative, formed low amounts of hmwt DNA and showed an 'atypically' low hypochrome shift. Nevertheless, UV-B irradiated Raji cells excluded propidium iodide (PI), bound annexin-V and stopped proliferation. This suggests that Raji cells underwent growth arrest with exposure of PS being the only feature of apoptosis. However, in the presence of phagocytes expressing the phosphatidylserine receptor these cells would share the removal pathway with apoptotic cells. Since UV-B induced programmed cell death differs in dependence of cells under investigation, the failure to detect oligonucleosomal DNA fragmentation or chromatin condensation is not suitable to exclude programmed (apoptotic?) cell death.     

Responses to ultraviolet-B radiation (280–315 nm) of pea (Pisum sativum) lines differing in leaf surface wax

To test the hypothesis that leaf surface wax influences plant responses to UV-B, 6 lines of cultivated pea (Pisum sativum L.), selected as having more or less wax, were grown at 0 or 6.5 kJ m^-2 day^-1 plant-weighted UV-B against a background of 850–950 μmol m^-2 s^-1 photosynthetically active radiation. In the 4 lines with least leaf surface wax the amount of wax on adaxial and abaxial leaf surfaces was increased following exposure to 6.5 kJ m^-2 day^-1 UV-B, but UV-B decreased surface wax in Scout, which had the greatest wax deposits. On the adaxial leaf surface, UV-B radiation caused a shift in wax composition from alcohols to esters and hydrocarbons and the ratio of short to long chain length alkyl ester homologues was increased. There was no evidence of a shortening in carbon chain length of hydrocarbons, primary alcohols or fatty acids due to UV-B and no significant correlation between wax amount and UV reflectance from leaves. UV-B induced significant increases in UV-absorbing compounds in the expanded leaves and buds of most lines. UV-B reduced the growth of all lines. Foliage area (leaves plus stipules) declined by 5–30%, plant dry weight by 12–30%, and plant height by 24–38%. Reductions in growth occurred in the absence of any changes in chlorophyll fluorescence or photosynthetic rate. UV-B also had no major effect on carbon allocation patterns. The effects of UV-B on growth appeared to be due to changes in tissue extension and expansion. Indeed, many of the responses to UV-B observed in this study of pea appear more consistent with indirect effects being expressed in developing tissues rather than through the direct action of UV-B on mature tissues. There was no evidence that wax amount or biochemistry was associated with the sensitivity of the lines to UV-B radiation. Furthermore, induction of pigments was not correlated with changes in growth. However, lines with the greatest constitutive amounts of pigments in unexpanded bud tissues were most tolerant of elevated UV-B.