Coordinated activation of a cluster of MMP genes in response to UVB radiation.

Ultraviolet (UV) B radiation is a dangerous environmental stressor, which can lead to photoaging, inflammation, immune suppression and tumour formation. A recent report has shown the transcriptional activation of several skin-specific genes including matrix metalloproteases (MMPs) in response to UV irradiation. Here, we use a novel human keratinocyte model, HKerE6SFM, to demonstrate that UVB activates the transcription of most members of the 11q22.3 MMP gene cluster including MMP13, MMP12, MMP3, MMP1 and MMP10. Curiously, the expression of the well-characterized UVB-inducible MMP9, which is located outside of the cluster, remains unchanged. In accordance with the increased expression of the MMP gene cluster upon UVB irradiation, RNA polymerase II showed increased occupancy at their promoters following UVB irradiation. The results also demonstrate increased acetylated histone H3K9 levels at the promoters of the MMP13, MMP12, MMP3, MMP1 and MMP10 genes. These findings suggest a coordinated transcriptional activation of genes in the MMP cluster at 11q22.3 and that acetylation of histone H3 at lysine 9 has an important role in the UVB-dependent enhancement of transcription of MMP genes in this region.

Radiation induced transcriptional and post-transcriptional regulation of the hsa-miR-23a~27a~24-2 cluster suppresses apoptosis by stabilizing XIAP.

The non-coding transcriptome, in particular microRNAs (miRNA), influences cellular survival after irradiation. However, the underlying mechanisms of radiation-induced miRNA expression changes and consequently target expression changes are poorly understood. In this study we show that a single dose of 5Gy ɣ-radiation decreases expression of the miR-23a~27a~24-2 cluster in the human endothelial cell-line EA.hy926 and the mammary epithelial cell-line MCF10A. In the endothelial cells this was facilitated through transcriptional regulation by promoter methylation and also at the post-transcriptional level by reduced miRNA processing through phosphorylation of Argonaute (AGO). Furthermore, we demonstrate that all three mature cluster miRNAs reduce apoptosis by increasing expression of the common target protein XIAP. These findings link a temporal succession of transcriptional and post-transcriptional regulatory mechanisms of the miR~23a~24-2~27a cluster, enabling a dynamic stress response and assuring cellular survival after radiation exposure.

Photocaged Arabinose: A Novel Optogenetic Switch for Rapid and Gradual Control of Microbial Gene Expression.

Controlling cellular functions by light allows simple triggering of biological processes in a non-invasive fashion with high spatiotemporal resolution. In this context, light-regulated gene expression has enormous potential for achieving optogenetic control over almost any cellular process. Here, we report on two novel one-step cleavable photocaged arabinose compounds, which were applied as light-sensitive inducers of transcription in bacteria. Exposure of caged arabinose to UV-A light resulted in rapid activation of protein production, as demonstrated for GFP and the complete violacein biosynthetic pathway. Moreover, single-cell analysis revealed that intrinsic heterogeneity of arabinose-mediated induction of gene expression was overcome when using photocaged arabinose. We have thus established a novel phototrigger for synthetic bio(techno)logy applications that enables precise and homogeneous control of bacterial target gene expression.

Deletion of the Synechocystis sp. PCC 6803 kaiAB1C1 gene cluster causes impaired cell growth under light-dark conditions.

In contrast to Synechococcus elongatus PCC 7942, few data exist on the timing mechanism of the widely used cyanobacterium Synechocystis sp. PCC 6803. The standard kaiAB1C1 operon present in this organism was shown to encode a functional KaiC protein that interacted with KaiA, similar to the S. elongatus PCC 7942 clock. Inactivation of this operon in Synechocystis sp. PCC 6803 resulted in a mutant with a strong growth defect when grown under light-dark cycles, which was even more pronounced when glucose was added to the growth medium. In addition, mutants showed a bleaching phenotype. No effects were detected in mutant cells grown under constant light. Microarray experiments performed with cells grown for 1 day under a light-dark cycle revealed many differentially regulated genes with known functions in the ΔkaiABC mutant in comparison with the WT. We identified the genes encoding the cyanobacterial phytochrome Cph1 and the light-repressed protein LrtA as well as several hypothetical ORFs with a complete inverse behaviour in the light cycle. These transcripts showed a stronger accumulation in the light but a weaker accumulation in the dark in ΔkaiABC cells in comparison with the WT. In general, we found a considerable overlap with microarray data obtained for hik31 and sigE mutants. These genes are known to be important regulators of cell metabolism in the dark. Strikingly, deletion of the ΔkaiABC operon led to a much stronger phenotype under light-dark cycles in Synechocystis sp. PCC 6803 than in Synechococcus sp. PCC 7942.

NF-E2-related factor 2 regulates the stress response to UVA-1-oxidized phospholipids in skin cells.

Long-wavelength ultraviolet (UVA-1) radiation causes oxidative stress that modifies cellular molecules. To defend themselves against noxious oxidation products, skin cells produce detoxifying enzymes and antioxidants. We have recently shown that UVA-1 oxidized the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the stress-response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1- and UV-oxidized phospholipids on global gene expression in human dermal fibroblasts and keratinocytes. We identified a cluster of genes that were coinduced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit, aldo-keto reductases-1-C1 and -C2, and IL-8. These genes are members of the cellular stress response system termed "antioxidant response." Accordingly, the regulatory regions of all of these genes contain binding sites for NF-E2-related factor 2 (NRF2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation and DNA binding of NRF2. Silencing and deficiency of NRF2 suppressed the antioxidant response. Taken together, our data show that UVA-1-mediated lipid oxidation induces expression of antioxidant response genes, which is dependent on the redox-regulated transcription factor NRF2. Our findings suggest a different view on UV-generated lipid mediators that were commonly regarded as detrimental

Compromised repair of clustered DNA damage in the human acute lymphoblastic leukemia MSH2-deficient NALM-6 cells.

Ionizing radiation (IR) induces two classes of complex DNA damage, double-strand breaks (DSBs) and non-DSB bi-stranded oxidative clustered DNA lesions (OCDLs). OCDLs may consist of single strand breaks (SSBs), oxidized purines/pyrimidines and abasic sites within 5-10bp. These significant biological lesions are hypothesized to challenge the repair machinery and carry a high mutagenic potential. MSH2, a classical DNA mismatch repair protein, has been also implicated in other repair pathways associated with DSB and base lesion processing. MSH2 mutations have been identified in acute lymphoblastic leukemia (ALL) patients as well as in other types of cancers. Our research model involves two precursors B (pre-B) ALL human cell lines, NALM-6 cells, homozygous null for MSH2, and wild type 697 cells. Using a modified version of neutral and alkaline single cell gel electrophoresis (SCGE) with Escherichia coli repair enzymes as damage probes, the processing capacity of single strand breaks (SSBs), DSBs and OCDLs was assessed in NALM-6 and 697 cells exposed to a radiotherapy relevant gamma-ray dose of 5Gy. Using reverse transcriptase PCR and Western blotting we verified the complete lack of expression of MSH2 in the NALM-6 cells at the transcriptional and translational level. No differences were measured between NALM-6 and 697 cells in the induction levels of SSBs, DSBs and OCDLs after exposure to gamma-rays. However, 697 cells repaired each lesion more efficiently with significant differences observed after 1-3h post-irradiation. Lastly, our results indicate a significantly higher population of apoptotic 697 cells compared to NALM-6 cells 6-24h post-irradiation. Our studies suggest that MSH2 is probably involved in the processing of the biologically significant clustered DNA damages as well as the execution of apoptosis induced by ionizing radiation.

Identification of a biosynthetic gene cluster in rice for momilactones.

Rice diterpenoid phytoalexins such as momilactones and phytocassanes are produced in suspension-cultured rice cells treated with a chitin oligosaccharide elicitor and in rice leaves irradiated with UV light. The common substrate geranylgeranyl diphosphate is converted into diterpene hydrocarbon precursors via a two-step sequential cyclization and then into the bioactive phytoalexins via several oxidation steps. It has been suggested that microsomal cytochrome P-450 monooxygenases (P-450s) are involved in the downstream oxidation of the diterpene hydrocarbons leading to the phytoalexins and that a dehydrogenase is involved in momilactone biosynthesis. However, none of the enzymes involved in the downstream oxidation of the diterpene hydrocarbons have been identified. In this study, we found that a putative dehydrogenase gene (AK103462) and two functionally unknown P-450 genes (CYP99A2 and CYP99A3) form a chitin oligosaccharide elicitor- and UV-inducible gene cluster, together with OsKS4 and OsCyc1, the diterpene cyclase genes involved in momilactone biosynthesis. Functional analysis by heterologous expression in Escherichia coli followed by enzyme assays demonstrated that the AK103462 protein catalyzes the conversion of 3beta-hydroxy-9betaH-pimara-7,15-dien-19,6beta-olide into momilactone A. The double knockdown of CYP99A2 and CYP99A3 specifically suppressed the elicitor-inducible production of momilactones, strongly suggesting that CYP99A2, CYP99A3, or both are involved in momilactone biosynthesis. These results provide strong evidence for the presence on chromosome 4 of a gene cluster involved in momilactone biosynthesis.

Knockout of glutelin genes which form a tandem array with a high level of homology in rice by gamma irradiation.

In the course of evolution, a gene is often duplicated in tandem, resulting in a functional redundancy. The analysis of function of these genes by raising double mutant might be difficult because they are very tightly linked. We described here a mutant of such a tandem duplicated gene. glu1 is a gamma-ray-induced rice mutant, which lacks an acidic subunit of glutelin, a major seed storage protein. We found that glu1 harbors a 129.7-kb deletion involving two highly similar and tandem repeated glutelin genes, GluB5 and GluB4. The deletion eliminated the entire GluB5 and GluB4 gene except half of the first exon of GluB5. GluB5 and GluB4 have the same amino acid sequence in the acidic subunit, suggesting that only the mutation involving both GluB5 and GluB4 results in the lack of the glutelin acidic subunit deleted in glu1. Our finding suggests that gamma-ray can be an effective mutagen to analyze tandem repeated and functionally redundant genes.

On the chemical yield of base lesions, strand breaks, and clustered damage generated in plasmid DNA by the direct effect of X rays.

The purpose of this study was to determine the yield of DNA base damages, deoxyribose damage, and clustered lesions due to the direct effects of ionizing radiation and to compare these with the yield of DNA trapped radicals measured previously in the same pUC18 plasmid. The plasmids were prepared as films hydrated in the range 2.5 < Gamma < 22.5 mol water/mol nucleotide. Single-strand breaks (SSBs) and double-strand breaks (DSBs) were detected by agarose gel electrophoresis. Specific types of base lesions were converted into SSBs and DSBs using the base-excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). The yield of base damage detected by this method displayed a strikingly different dependence on the level of hydration (Gamma) compared with that for the yield of DNA trapped radicals; the former decreased by 3.2 times as Gamma was varied from 2.5 to 22.5 and the later increased by 2.4 times over the same range. To explain this divergence, we propose that SSB yields produced in plasmid DNA by the direct effect cannot be analyzed properly with a Poisson process that assumes an average of one strand break per plasmid and neglects the possibility of a single track producing multiple SSBs within a plasmid. The yields of DSBs, on the other hand, are consistent with changes in free radical trapping as a function of hydration. Consequently, the composition of these clusters could be quantified. Deoxyribose damage on each of the two opposing strands occurs with a yield of 3.5 +/- 0.5 nmol/J for fully hydrated pUC18, comparable to the yield of 4.1 +/- 0.9 nmol/J for DSBs derived from opposed damages in which at least one of the sites is a damaged base.

Differential response of genes for ferredoxin and ferredoxin:NADP+ oxidoreductase to nitrate and light in maize leaves.

In higher plants, ferredoxin (Fd) and Fd:NADP+ oxidoreductase (FNR, EC 1.18.1.2) are encoded by small multigene families, and the individuals transfer electrons to the dependent enzymes in the photosynthetic and the non-photosynthetic plastids. In maize, a C4 plant, expression of genes for the non-photosynthetic isoproteins, Fd VI and R-FNR, is responsive to nitrate in roots whereas the expression and the spatial distribution in the leaves have not been analysed. Here, we studied the expression pattern of a series of Fd and FNR genes in maize leaves in response to nitrate and light. Upon addition of nitrate, the transcripts for Fd VI and R-FNR rapidly accumulated in the leaves, whereas light did not induce accumulation. Expression of genes for the other isoproteins was not changed significantly by the nitrogen source. In the leaf, the transcripts for Fd VI and R-FNR were predominantly detected in mesophyll cells as were those for nitrate-assimilatory enzymes. Since R-FNR is an isoprotein transferring electrons from NADPH to non-photosynthetic type Fd, the redox equivalent is supplied in nitrate reduction, at least partially, via an oxidative pentose phosphate pathway, even in photosynthetic organs.

Clustered DNA damages induced in human hematopoietic cells by low doses of ionizing radiation.

Ionizing radiation induces clusters of DNA damages--oxidized bases, abasic sites and strand breaks--on opposing strands within a few helical turns. Such damages have been postulated to be difficult to repair, as are double strand breaks (one type of cluster). We have shown that low doses of low and high linear energy transfer (LET) radiation induce such damage clusters in human cells. In human cells, DSB are about 30% of the total of complex damages, and the levels of DSBs and oxidized pyrimidine clusters are similar. The dose responses for cluster induction in cells can be described by a linear relationship, implying that even low doses of ionizing radiation can produce clustered damages. Studies are in progress to determine whether clusters can be produced by mechanisms other than ionizing radiation, as well as the levels of various cluster types formed by low and high LET radiation.

Developmental and light-regulated expression of individual members of the light-harvesting complex b gene family in Pinus palustris.

Angiosperms requires light for multiple aspects of chloroplast development, including chlorophyll synthesis and induction of expression of the mRNAs encoding the major polypeptides of the light-harvesting complex of photosystem II (Lhcb genes). In contrast, many conifers, including pines, firs, and spruces, can accumulate chlorophyll and the light-harvesting chlorophyll a/b-binding proteins of photosystem II in complete darkness. To understand the factors responsible for the regulation of expression of individual Lhcb mRNAs in the pine Pinus palustris, we have prepared sequence-specific cDNA probes for each of three family members, Lhcb1*Pp1, Lhcb2*Pp1, and Lhcb2*Pp2, and have studied the expression of two of these, Lhcb1*Pp1 and Lhcb2*Pp2, in detail. The levels of expression of each sequence were disparate, and Lhcb1*Pp1-encoded transcripts were the most abundant in the light. Both Lhcb1*Pp1 and Lhcb2*Pp2 mRNAs were expressed in stems and cotyledons, but Lhcb1*Pp1 mRNA was present at about 10-fold lower levels in stems than in cotyledons, in contrast to Lhcb2*Pp2 mRNA, which was expressed at higher levels in stems than in cotyledons. Both Lhcb1*Pp1 and Lhcb2*Pp2 mRNAs were absent in embryos but were expressed during seedling development. The levels increased with age in both the light and the dark and in both cases were about 2-fold higher in the light than in the dark. Despite the expression of Lhcb1*Pp1 and Lhcb2*Pp2 mRNAs during development in darkness, the levels of both mRNAs increased in dark-grown seedlings given red light in the low fluence range within 2 h of treatment.

The genetic and molecular organization of the Dopa decarboxylase gene cluster of Drosophila melanogaster.

We report the complete molecular organization of the Dopa decarboxylase gene cluster. Mutagenesis screens recovered 77 new Df(2L)TW130 recessive lethal mutations. These new alleles combined with 263 previously isolated mutations in the cluster to define 18 essential genes. In addition, seven new deficiencies were isolated and characterized. Deficiency mapping, restriction fragment length polymorphism (RFLP) analysis and P-element-mediated germline transformation experiments determined the gene order for all 18 loci. Genomic and cDNA restriction endonuclease mapping, Northern blot analysis and DNA sequencing provided information on exact gene location, mRNA size and transcriptional direction for most of these loci. In addition, this analysis identified two transcription units that had not previously been identified by extensive mutagenesis screening. Most of the loci are contained within two dense subclusters. We discuss the effectiveness of mutagens and strategies used in our screens, the variable mutability of loci within the genome of Drosophila melanogaster, the cytological and molecular organization of the Ddc gene cluster, the validity of the one band-one gene hypothesis and a possible purpose for the clustering of genes in the Ddc region.

Blue and red light reversibly control psbA expression in the cyanobacterium Synechococcus sp. strain PCC 7942.

The three psbA genes encoding the photosystem II D1 protein in Synechococcus sp. strain PCC 7942 respond differentially to an increase in intensity of white light through transcriptional induction of psbAII and psbAIII and accelerated degradation of psbAI and psbAIII messages. We report that the genes exhibit a novel photoreversible response involving blue and red light that is almost indistinguishable from the high-white light response. Transfer of cells from white to low-fluence blue light caused a decrease in the level of the psbAI message and increased levels of psbAII and psbAIII messages, whereas transfer to red or far-red light had little effect. Five min of blue light was sufficient to trigger psbAII and psbAIII induction; five min of subsequent red irradiation attenuated this response, whereas subsequent green or far-red light (or darkness) had no effect. Response to both high and blue light was insensitive to inhibitors of photosynthetic electron transport. We propose that Synechococcus modulates photosystem II biosynthesis in a variable light environment through a photoreception signal pathway, which is independent of photosystem II activity and which is distinct from red/green-reversible control of chromatically adapting cyanobacteria and the red/far-red-reversible phytochrome of plants.

A desiccation-related Elip-like gene from the resurrection plant Craterostigma plantagineum is regulated by light and ABA.

The resurrection plant Craterostigma plantagineum tolerates an extreme loss of cellular water. Therefore this plant is being studied as model system to analyse desiccation tolerance at the molecular level. Upon dehydration, new transcripts are abundantly expressed in different tissues of the plant. One such desiccation-related nuclear gene (dsp-22 for desiccation stress protein) encodes a mature 21 kDa protein which accumulates in the chloroplasts. Sequence analysis indicates that dsp-22 is closely related to early light inducible genes (Elip) of higher plants and to a carotene biosynthesis related gene (cbr) isolated from the green alga Dunaliella bardawil. In contrast to other desiccation-related genes, light is an essential positive factor regulating the expression of dsp-22: ABA-mediated gene activation leads to the accumulation of the transcript only in the presence of light. During the desiccation process, light acts at the transcriptional and post-transcriptional levels. The implications of these different controls and the possible role of the dsp-22 protein in the desiccation/rehydration process are discussed.

Control of cab gene expression in synchronized Chlamydomonas reinhardtii cells.

In light-dark synchronized Chlamydomonas reinhardtii cultures transcripts of at least two members of the cab gene family coding for chlorophyll a/b binding proteins are highly abundant in the light, but almost undetectable in the dark. "Run-on" transcription assays in isolated nuclei were used to show that the rapid increase in cab mRNA levels during the light phase is primarily due to regulation at the transcriptional level. Functionally unrelated inhibitors such as dipyridyl and cycloheximide as well as anaerobic conditions block chlorophyll synthesis, presumably by interfering with the conversion of magnesium protoporphyrin monomethyl ester to protochlorophyllide. Under these conditions, cab mRNA does not accumulate and nuclei isolated from inhibitor-treated cells do not support cab gene transcription. Inhibitors such as dioxoheptanoic acid and diphenyl ether herbicides block earlier steps within the chlorophyll synthesis pathway without substantial effects on cab mRNA accumulation and transcription. A possible control of transcription by intermediates of the chlorophyll biosynthesis pathway is discussed.

Characterization of the human spr2 promoter: induction after UV irradiation or TPA treatment and regulation during differentiation of cultured primary keratinocytes.

We have isolated genomic clones from several members of the UV and TPA inducible human spr2 gene-family in order to analyse the regulation of these genes at a molecular level. From one of these members, the spr2-1 gene, we have identified and sequenced the regulatory region. By using CAT fusion plasmids and a liposome mediated transfection procedure we show that the isolated promoter region contains all the cis-elements necessary for induced expression after UV irradiation or phorbolester treatment of cultured human keratinocytes. Additionally the spr2-1 promoter is shown to be regulated aswell during the normal process of keratinocyte differentiation. This makes the spr2-1 promoter sequence an ideal tool to study the molecular mechanisms by which environmental agents such as UV radiation and chemical tumor promoters interfere with normal gene expression during cell proliferation and differentiation.

DNA damage activates transcription and transposition of yeast Ty retrotransposons.

A set of genes isolated from Saccharomyces cerevisiae showed increased transcript levels after yeast had been exposed to ultraviolet (UV) light or 4-nitroquinoline-1-oxide (4NQO). Included among these DNA damage responsive (DDR) genes were members of the Ty retrotransposon family of yeast. Northern hybridization analysis indicated that maximal levels of a 5.6 kb transcript encoded by the Ty elements accumulated in cells after 4 to 6 h of exposure to 4NQO. The induced levels of transcripts varied from two- to tenfold for different Ty probes although similar kinetics and dose responses were observed for transcripts hybridizing to the different Ty family members. Pulse labeling experiments suggested that the accumulation of Ty transcripts was due, in part, to an increased rate of Ty message synthesis. Transposition of Ty elements to two target loci encoding distinct alcohol dehydrogenase enzymes, ADH2 and ADH4, was examined in cells exposed to increasing doses of UV light or 4NQO. The frequency of Ty insertion into these genetic regions following DNA damaging treatments increased by as much as 17-fold compared with untreated cells. These results provide direct evidence that transposable elements can be activated by physical and chemical mutagens/carcinogens and that transpositional mutagenesis is induced by these agents in S. cerevisiae.

Isolation, characterization, and UV-stimulated expression of two families of genes encoding polypeptides of related structure in human epidermal keratinocytes.

By screening of a cDNA library made on mRNA isolated from UV-irradiated human epidermal keratinocytes for sequences whose relative concentration increases in the cytoplasm after irradiation, we have isolated 40 cDNA clones (T. Kartasova, B. J. C. Cornelissen, P. Belt, and P. van de Putte, Nucleic Acids Res. 15:5945-5962, 1987). Here we describe two distinct groups of cDNA clones which do not cross-hybridize to each other but nevertheless encode proteins of very similar primary structure. These polypeptides are small (8 to 10 kilodaltons) and exceptionally rich in proline, cysteine, and glutamine and have similar repeating elements not found elsewhere. The new proteins were designated sprI and sprII (small, proline rich). The presence of prolines and cysteines suggests that they may be either structural proteins with a strong secondary structure or metal-binding proteins such as metallothioneins. Southern blot and sequence analyses of the cDNAs indicate that at least the sprII group of clones represents a family of related genes. The nucleotide sequence of both groups seems to be conserved upon evolution. The level of mRNAs corresponding to the two groups of cDNAs is increased in the cytoplasm of human epidermal keratinocytes after both UV irradiation and treatment with 4-nitroquinoline 1-oxide or 12-O-tetradecanoylphorbol 13-acetate.