Deirdre Fahy

Rad4–Rad23 interaction with SWI/SNF links ATP-dependent chromatin remodeling with nucleotide excision repair

Chromatin rearrangement occurs during nucleotide excision repair (NER). Here we show that Snf6 and Snf5, two subunits of the SWI/SNF chromatin-remodeling complex in Saccharomyces cerevisiae, copurify with the NER damage-recognition heterodimer Rad4–Rad23. This interaction between SWI/SNF and Rad4–Rad23 is stimulated by UV irradiation. We demonstrate that NER in the transcriptionally silent, nucleosome-loaded HML locus is reduced in yeast cells lacking functional SWI/SNF. In addition, using a restriction enzyme accessibility assay, we observed UV-induced nucleosome rearrangement at the silent HML locus. Notably, this rearrangement is markedly attenuated when SWI/SNF is inactivated. These results indicate that the SWI/SNF chromatin-remodeling complex is recruited to DNA lesions by damage-recognition proteins to increase DNA accessibility for NER in chromatin.

Role of the mammalian SWI/SNF chromatin remodeling complex in the cellular response to UV damage.

Mammalian cells exhibit complex cellular responses to DNA damage, including cell cycle arrest, DNA repair and apoptosis. Defects in any one of these responses can result in carcinogenesis. Absence of the chromatin remodeling complex Swi/Snf is found in many instances of cancer, and we have investigated its role in the UV damage response. The human carcinoma cell line SW13 is deficient in Swi/Snf and is very sensitive to UV radiation. In contrast, SW13 cells with ectopic Brg1 expression regain active Swi/Snf and become significantly more resistant to UV radiation. Sensitivity to UV light correlates well with dramatic UV induced apoptosis in SW13 cells, but not in SW13 cells expressing Brg1. We show that SW13 cells synchronized at the G1/S border progress into S phase after UV irradiation, and this checkpoint deficiency is corrected after Brg1 expression is restored. Interestingly, Brg1 expression in SW13 cells restores expression of two DNA damage responsive genes, Gadd45a and p21. Furthermore, Gadd45a induction and p21 degradation were observed in the Brg1-expressing SW13 cells after UV irradiation. Our findings demonstrate that Swi/Snf protects cells against deleterious consequences of UV induced DNA damage. These results also indicate that Swi/Snf may play a role in replication checkpoint activation after UV damage via regulation of the two PCNA-binding proteins Gadd45a and p21.

Deciphering the Roles of the Histone H2B N-Terminal Domain in Genome-Wide Transcription†

ABSTRACT Histone N-terminal domains are frequent targets of posttranslational modifications. Multiple acetylated lysine residues have been identified in the N-terminal domain of H2B (K6, K11, K16, K17, K21, and K22), but little is known about how these modifications regulate transcription. We systematically mutated the N-terminal domain of histone H2B, both at known sites of lysine acetylation and elsewhere, and characterized the resulting changes in genome-wide expression in each mutant strain. Our results indicate that known sites of lysine acetylation in this domain are required for gene-specific transcriptional activation. However, the entire H2B N-terminal domain is principally required for the transcriptional repression of a large subset of the yeast genome. We find that the histone H2B repression (HBR) domain, comprised of residues 30 to 37, is necessary and sufficient for this repression. Many of the genes repressed by the HBR domain are located adjacent to telomeres or function in vitamin and carbohydrate metabolism. Deletion of the HBR domain also confers an increased sensitivity to DNA damage by UV irradiation. We mapped the critical residues in the HBR domain required for its repression function. Finally, comparisons of these data with previous studies reveal that a surprising number of genes are coregulated by the N-terminal domains of histone H2B, H3, and H4.

A single amino acid change in histone H4 enhances UV survival and DNA repair in yeast

Single amino acid changes at specific DNA contacts of histones H3 and H4 generate SWI/SNF-independent (Sin) mutants in yeast. We have analyzed the effect of the Sin mutation at R45 of histone H4 on cell survival following UV irradiation, nucleotide excision repair (NER) and chromatin structure. We find that this mutation renders yeast cells more resistant to UV damage and enhances NER at specific chromatin loci. In the transcriptionally silent HML, repressed GAL10 and the constitutively active RPB2 loci, H4 R45 mutants exhibit enhanced repair of UV-induced cyclobutane pyrimidine dimers (CPDs) compared to wild-type (wt). However, the H4 R45 mutation does not increase the transcription of NER genes, disrupt transcriptional silencing of the HML locus or alter repression in the GAL10 locus. We have further shown that the H4 R45C mutation increases the accessibility of nucleosome DNA in chromatin to exogenous nucleases and may expedite nucleosome rearrangements during NER. Taken together, our results indicate that the increased repair observed in Sin mutants is a direct effect of the altered chromatin landscape caused by the mutation, suggesting that such subtle changes in the conserved histone residues can influence the accessibility of DNA repair factors in chromatin.

Repair-Independent Chromatin Assembly onto Active Ribosomal Genes in Yeast after UV Irradiation

ABSTRACT Chromatin rearrangements occur during repair of cyclobutane pyrimidine dimers (CPDs) by nucleotide excision repair (NER). Thereafter, the original structure must be restored to retain normal genomic functions. How NER proceeds through nonnucleosomal chromatin and how open chromatin is reestablished after repair are unknown. We analyzed NER in ribosomal genes (rDNA), which are present in multiple copies but only a fraction are actively transcribed and nonnucleosomal. We show that removal of CPDs is fast in the active rDNA and that chromatin reorganization occurs during NER. Furthermore, chromatin assembles on nonnucleosomal rDNA during the early events of NER but in the absence of DNA repair. The resumption of transcription after removal of CPDs correlates with the reappearance of nonnucleosomal chromatin. To date, only the passage of replication machinery was thought to package ribosomal genes in nucleosomes. In this report, we show that early events after formation of UV photoproducts in DNA also promote chromatin assembly.

Type 1 diacylglycerol acyltransferases of Brassica napus preferentially incorporate oleic acid into triacylglycerol

Highlight Fatty acid composition determines oil qualities. Not only the selectivity of BnDGAT1 enzymes, but also the concentration of the fatty acid substrates, determines the oil composition in Brassica napus seeds.

Impact of salt stress, cell death, and autophagy on peroxisomes: quantitative and morphological analyses using small fluorescent probe N-BODIPY

Plant peroxisomes maintain a plethora of key life processes including fatty acid β-oxidation, photorespiration, synthesis of hormones, and homeostasis of reactive oxygen species (ROS). Abundance of peroxisomes in cells is dynamic; however mechanisms controlling peroxisome proliferation remain poorly understood because measuring peroxisome abundance is technically challenging. Counting peroxisomes in individual cells of complex organs by electron or fluorescence microscopy is expensive and time consuming. Here we present a simple technique for quantifying peroxisome abundance using the small probe Nitro-BODIPY, which in vivo fluoresces selectively inside peroxisomes. The physiological relevance of our technique was demonstrated using salinity as a known inducer of peroxisome proliferation. While significant peroxisome proliferation was observed in wild-type Arabidopsis leaves following 5-hour exposure to NaCl, no proliferation was detected in the salt-susceptible mutants fry1-6, sos1-14, and sos1-15. We also found that N-BODIPY detects aggregation of peroxisomes during final stages of programmed cell death and can be used as a marker of this stage. Furthermore, accumulation of peroxisomes in an autophagy-deficient Arabidopsis mutant atg5 correlated with N-BODIPY labeling. In conclusion, the technique reported here enables quantification of peroxisomes in plant material at various physiological settings. Its potential applications encompass identification of genes controlling peroxisome homeostasis and capturing stress-tolerant genotypes.

Cytochrome b5 Coexpression Increases Tetrahymena thermophila Δ6 Fatty Acid Desaturase Activity in Saccharomyces cerevisiae

ABSTRACT Very-long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, are important to the physiology of many microorganisms and metazoans and are vital to human development and health. The production of these and related fatty acids depends on Δ6 desaturases, the final components of an electron transfer chain that introduces double bonds into 18-carbon fatty acid chains. When a Δ6 desaturase identified from the ciliated protist Tetrahymena thermophila was expressed in Saccharomyces cerevisiae cultures supplemented with the 18:2Δ9,12 substrate, only 4% of the incorporated substrate was desaturated. Cytochrome b5 protein sequences identified from the genome of T. thermophila included one sequence with two conserved cytochrome b5 domains. Desaturation by the Δ6 enzyme increased as much as 10-fold when T. thermophila cytochrome b5s were coexpressed with the desaturase. Coexpression of a cytochrome b5 from Arabidopsis thaliana with the Δ6 enzyme also increased desaturation. A split ubiquitin growth assay indicated that the strength of interaction between cytochrome b5 proteins and the desaturase plays a vital role in fatty acid desaturase activity, illustrating the importance of protein-protein interactions in this enzyme activity.

Reducing saturated fatty acids in Arabidopsis seeds by expression of a Caenorhabditis elegans 16:0–specific desaturase

Plant oilseeds are a major source of nutritional oils. Their fatty acid composition, especially the proportion of saturated and unsaturated fatty acids, has important effects on human health. Because intake of saturated fats is correlated with the incidence of cardiovascular disease and diabetes, a goal of metabolic engineering is to develop oils low in saturated fatty acids. Palmitic acid (16:0) is the most abundant saturated fatty acid in the seeds of many oilseed crops and in Arabidopsis thaliana. We expressed FAT-5, a membrane-bound desaturase cloned from Caenorhabditis elegans, in Arabidopsis using a strong seed-specific promoter. The FAT-5 enzyme is highly specific to 16:0 as substrate, converting it to 16:1∆9; expression of fat-5 reduced the 16:0 content of the seed by two-thirds. Decreased 16:0 and elevated 16:1 levels were evident both in the storage and membrane lipids of seeds. Regiochemical analysis of phosphatidylcholine showed that 16:1 was distributed at both positions on the glycerolipid backbone, unlike 16:0, which is predominately found at the sn-1 position. Seeds from a plant line homozygous for FAT-5 expression were comparable to wild type with respect to seed set and germination, while oil content and weight were somewhat reduced. These experiments demonstrate that targeted heterologous expression of a desaturase in oilseeds can reduce the level of saturated fatty acids in the oil, significantly improving its nutritional value.

Characterization of Cytokinetic Mutants Using Small Fluorescent Probes.

Cytokinesis is a powerful paradigm for addressing fundamental questions of plant biology including molecular mechanisms of development, cell division, cell signaling, membrane trafficking, cell wall synthesis, and cytoskeletal dynamics. Genetics was instrumental in identification of proteins regulating cytokinesis. Characterization of mutant lines generated using forward or reverse genetics includes microscopic analysis for defects in cell division. Typically, failure of cytokinesis results in appearance of multinucleate cells, formation of cell wall stubs, and isotropic cell expansion in the root elongation zone. Small fluorescent probes served as a very effective tool for the detection of cytokinetic defects. Such probes stain living or formaldehyde-fixed specimens avoiding complex preparatory steps. Although resolution of the fluorescence probes is inferior to electron microscopy, the procedure is fast, easy, and does not require expensive materials or equipment. This chapter describes techniques for staining DNA with the probes DAPI and SYTO82, for staining membranes with FM4-64, and for staining cell wall with propidium iodide.