Temporal and Spatial Transcriptional Profiles of Aging in Drosophila
ABSTRACT: Genetic analyses suggest that alterations in gene expression at the molecular and tissue levels can have profound effects on aging for multi-cellular organisms. However, much remains unknown about the normal pattern of genetic changes in different tissues and how these tissues interact during aging. To investigate tissue-specific aging systematically, we measured expression profiles of aging in Drosophila melanogaster in seven tissues representing nervous, muscular, digestive, renal, reproductive, and storage systems. In each tissue, we identified hundreds of age-related genes mostly showing gradual changes of transcript levels with age. Age-relatedgenes showed clear tissue-specific transcriptional patterns; less than 10% of age-related genes in each tissue shared expression patterns with any other tissue; less than 20% of age-related biological processes were shared between tissues. A significant portion of tissue-specific age-related genes are those involved in physiological functions regulated by the corresponding tissue. However, limited overlaps of age-related function groups among tissues particularly those involved in proteasome function suggest some common mechanisms of transcription regulation in aging across tissues. This study defined global, temporal and spatial changes associated withaging at the molecular and tissue levels. Analyses indicated that different tissues might age in different patterns or at different rates. This study addressed comprehensively the relationship of age-related changes among different tissues in one organism, providing a foundation to address tissue-specific regulation in aging. RNA was then amplified by a one-step linear amplification protocol to generate amplified RNA (aRNA). Experiment aRNA refers to amplified RNA from flies of 15, 20, 30, 45 and 60 days old, and reference aRNA refers to amplified RNA from flies of 3 days old, and experiment and reference aRNAs were labeled with fluorescent dye Cy3 and Cy5, respectively. For each tissue, RNA from the corresponding tissue of 3-day old flies was used as the reference RNA and expression profiles at each of the five age-points was measured twice by using independently prepared duplicated samples. Seven types of tissues or organs of the male fly strain w1118 , accessory gland, testis, brain, gut, malpighian tubule, dorsal thoracic muscle and abdominal fat body were hand dissected out of flies at age of 3, 15, 20, 30, 45 and 60 days old. Tissues or organs from four males of the same age were pooled together and used for each RNA sample preparation.
Project description:Fish and Chips: Expression Profiling in Non-traditional Model Systems Using a Cichlid Fish cDNA Microarray This series represents the 26 arrays that went into Renn et al 2004 (submitted January 16th), and one additional hybridization (GSM15240) that was not included in the publication analysis for statistical reasons.
Project description:We have exploited a spontaneously isolated mutant IgaA(T191P) that is near-maximally activated for the Rcs system, to identify a vast set of genes that respond, and report new regulatory properties of this signaling system in Salmonella enterica serovar Typhimurium. Microarray data show that the Rcs system normally functions as a positive regulator of SPI-2 and other genes important for growth of Salmonella in macrophages, although when highly activated, the system completely represses the SPI-1/SPI-2 virulence, flagellar, and fimbrial biogenesis pathways. The auxilliary protein RcsA, which works with RcsB to positively regulate colanic acid and other target genes, not only stimulates but also antagonizes the positive regulation of many genes in the igaA mutant. We show that RcsB represses motility through the 'RcsB box' in the promoter region of the master operon flhDC, and that RcsA is not required for this regulation. Curiously, RcsB selectively stimulates expression of the flagellar Type 3 secretion genes fliPQR; an RcsAB box located downstream of fliR influences this regulation. We show that excess colanic acid impairs swimming and inhibits swarming motility, consistent with the inverse regulation of the two pathways by the Rcs system. The work was published in Journal of Bacteriology: Wang Q, Zhao Y, McClelland M, Harshey RM. The RcsCDB signaling system and swarming motility in Salmonella enterica serovar Typhimurium: dual regulation of flagellar and SPI-2 virulence genes. J Bacteriol. 2007 Sep 28; [Epub ahead of print] PMID: 17905992 [PubMed - as supplied by publisher] Keywords: Comparative genomic hybridization Microarray was used to reveal the gene expression profiles in igaA(T191P) and related strains at two growth stages (log and stationary phases). Quantitative RT-PCR was used for detail studies.
Project description:The temporal expression of the 23 CfMNPV genes representing all four temporal classes including its 7 unique genes were determined by a modified oligonucleotide-based two-channel DNA microarray. Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene was also detected by the array analysis. The expression of four host genes varied several fold throughout virus infection. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements. We first developed a novel normalization protocol using Cy5-labeled CfMNPV viral genomic DNA (vgDNA) as equimolar reference standards for each probe in order to overcome the inherent variability problem of the traditional microarray normalization procedures including use of internal standards. Cy3-labeled cDNA was from total RNA isolated at different times post infection of Cf203 insect cells infected with CfMNPV. Host genes were unsuitable for normalization between microarrays. The DNA microarray results were selectively validated by quantitative RT-PCR (qRT-PCR). The nature of the polyhedrin antisense transcription was further investigated using long range RT-PCR analysis. Keywords: Time course, detection of antisense transcripts, viral genomic DNA normalization Total RNA was isolated from Cf203 cells at 0, 3, 6, 12, 24 and 48 h post infection, as well as from mock-infected Cf203 cells. The Cy3-labeled cDNA derived from 20 μg total RNA was co-hybridized with 10 ng/μl vgDNA to each array at 42ºC. Seven hybridizations in each experiment, two independent hybridization experiments were performed, and therefore 14 samples were analyzed and included in the sample submission including the mock-infected sample.
Project description:Acetylation of histone H3 lysine 56 is a covalent modification best-known as a mark of newly-replicated chromatin, but has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase, and a further specific decrease in turnover among early origins of replication, which switch from rapidly-replaced in G1 phase to stable-bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly-replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle, and suggest that H3K56 acetylation provides a positive feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location. To characterize incorporation of H3K56ac in yeast, several sets of ChIP-chip experiments were performed, along with a set of gene expression microarrays. The following describes each individual set, the number of biological and array replicates performed, dye-flip replicates if performed, the total number of arrays, and the applicable figures in the manuscript. Except for Item D (gene expression), all arrays were ChIP-chip experiments. A. Mid-log H3K56ac measurement. 3 biological replicates, 1 array replicate each of K56Ac ChIP with Grunstein Lab antibody. 2 biological replicates, 2 array replicates each of K56Ac ChIP with Upstate antibody. 7 total arrays. Figure 1A-C, 3, S1, S2A, S6, S7. B. Cell cycle - H3K56ac measurements. 1 biological replicate, 3 array replicates each of K56Ac ChIP with Upstate antibody. 17 time points (10 min. unavailable in third array replicate). 50 total arrays. Figures 2, 3, 4, S4, S5, S7, S8. C. G1 phase turnover. 1 biological replicate (3 time points) with 1 dye-flip replicate each time point (45 minutes replaced with 60 minutes for dye-flip replicate). 6 total arrays. Tables S2, S3. D. Cell cycle - mRNA measurements. 2 biological replicates, 1 array replicate each. 34 total arrays. Figure S3. E. M phase turnover rates. 2 biological replicates, second biological replicate has one dye-flip replicate. 17 total arrays. Figure 5. F. Deletion mutants in H3K56ac pathway for G1-arrested cells. Strains: wild-type parental strain (PKY4212; 2 biological replicates), asf1D (strain 2 biological replicates), vps75D (3 biological replicates, third biological replicate having two array replicates), rtt109D (2 biological replicates, each with one dye-flip replicate). 12 total arrays. Figure 6.
Project description:The goal of this study is to identify P. vivax genes whose expression is dependent on the intact spleen in experimental infections in Aotus monkeys. These studies were carried-out at the facilities of the “Fundación Centro de Primates de la Universidad del Valle”, Cali, Colombia and in the “Barcelona Centre for International Health Resarch” - CRESIB, Barcelona, Spain. This protocol was approved from the Ethical Committees of both Centres. A total of 4 Aotus lemurinus griseimembra naive animals were used in these experiments. Three animals were splenectomized whereas another had an intact spleen. A donor monkey was infected with P. vivax Sal-I strain and after peak parasitemias appeared a time-series infections into Sp-1, Sp-2, Sp-3, and Sp+2 animals were performed. Parasites from each infection were obtained from peripheral blood, monkey leukocytes were removed by MidMacs and only purified schizont stages were used for RNA extractions. Dual-hybridizations Cy3/Cy5 comparing the global expression of parasites obtained from different infections (Cy5) with a reference pool PvSp-1 obtained from splenectomized monkeys from CDC (PvSp-1) were perfomed using an Agilent's 60-mer platform representing the complete coding genome of P. vivax (1 oligonucleotide/2 kb coding sequences) GPL6667
Project description:The complete genome sequence of the P. vivax Sal-1 strain allowed the design of a first version array representing 1 oligo/2 kb of coding sequences (http://zblab.sbs.ntu.edu.sg/vivax/index.html). Here, proof-of-principle experiments using total RNA of parasites obtained from the Sal-1 strain, from P. falciparum and from parasites obtained directly from two human patients are presented. To determine the extent of cross-hybridization of P. falciparum with P. vivax, and to determine overlaps in expression profiles of the P. vivax Sal1 monkey-adapted strain vs wild isolates, single dual hybridization analyses were performed.
Project description:Significant insight about biological networks arises from the study of network motifs –overly abundant network subgraphs, but such wiring patterns do not specify when and how potential routes within a cellular network are used. To address this limitation, we introduce activity motifs, which capture patterns in the dynamic use of a network. Using this framework to analyze transcription in Saccharomyces cerevisiae metabolism, we find that cells use different timing activity motifs to optimize transcription timing in response to changing conditions: forward activation to produce metabolic compounds efficiently, backward shutoff to rapidly stop production of a detrimental product and synchronized activation for co-production of metabolites required for the same reaction Measuring protein abundance over a time course reveals that mRNA timing motifs also occur at the protein level. Timing motifs significantly overlap with binding activity motifs, where genes in a linear chain have ordered binding affinity to a transcription factor, suggesting a mechanism for ordered transcription. Finely timed transcriptional regulation is therefore abundant in yeast metabolism, optimizing the organism's adaptation to new environmental conditions. We generated a set of 13 time courses by measuring gene expression after a metabolic change. Yeast strain KCN118 (MATalpha ade2) was grown at 28 °C in 400 ml of synthetic complete media with 2% dextrose (SCD) to an OD600 of 0.6. Synthetic complete was prepared using the standard recipe, except 75 uM inositol was included. At OD600 of 0.6, 100 ml of cells were collected by centrifugation and frozen as a reference sample, and the remaining cells were rapidly collected by filtration, washed with distilled water and resuspended in 300 ml of one of the following media: SCE (SC + 2% ethanol), SCG (SC + 2% galactose), SM1 (SCD lacking amino acids A, R, N, C, Q, G, K, P, S, F and T), SM2 (SCD lacking amino acids L, I, V, W, H and M), S0 (SCD lacking all amino acids), S0G (no amino acids, 2% galactose) or S0E (no amino acids, 2% ethanol). The data appears in Figures 2 and 4 of the manuscript, as it relates to the global analysis of all the arrays used in the dataset. All time courses consist of the following time points (in min): 15, 30, 60, 120, 240, and were hybridized against the t = 0 time point of cells grown in SCD. Each time course was performed as one single biological replicate and one technological replicate, except where noted below. Specifically, the 13 time courses break down into the following groups: Media key: SCD (synthetic complete, not including inositol) SCE (SC + 2% ethanol) SCG (SC + 2% galactose) SM1 (SCD lacking amino acids A, R, N, C, Q, G, K, P, S, F and T) SM2 (SCD lacking amino acids L, I, V, W, H and M) S0 (SCD lacking all amino acids) S0G (no amino acids, 2% galactose) S0E (no amino acids, 2% ethanol). ino = inositol aa = all amino acids supplemented The following group descriptions are the media as described above, followed by the description as indicated in the long title of the individual arrays: 1. S0 (5 arrays) = SD 2. SCD (6 arrays, t = 240 min has 2 tech replicates) = SD+aa 3. SM2 (5 arrays) = SD+aa:ARNCQGKPSDEFTY+ino 4. SM1 + ino (5 arrays) = SD+aa:LIVWHM+ino 5. S0 + ino (6 arrays, t = 240 min has 2 tech replicates) = SD+ino 6. S0E (5 arrays) = SEtOH 7. S0E + aa (7 arrays, t = 240 min and 60 min each have 2 tech replicates) = SEtOH+aa 8. S0E + aa + ino (5 arrays) = SEtOH+aa+ino 9. S0E + ino (8 arrays, t = 240 min, 15 min and 30 min each have 2 tech replicates) = SEtOH+ino 10. S0G (5 arrays) = Sgal 11. S0G + aa (5 arrays) = Sgal+aa 12. S0G + aa + ino (5 arrays) = Sgal+aa+ino 13. S0G + ino (6 arrays, t = 60 min has 2 tech replicates) = Sgal+ino
Project description:Histone modifications affect DNA-templated processes ranging from transcription to genomic replication. In this study, we examine the cell cycle dynamics of the trimethylated form of histone H3 lysine 4 (H3K4me3), a mark of active chromatin that is viewed as “long-lived” , and that is involved in memory during cell state inheritance in metazoans . We synchronized yeast using two different protocols, then followed H3K4me3 patterns as yeast passed through subsequent cell cycles. While most H3K4me3 patterns were conserved from one generation to the next, we found that methylation patterns induced by alpha factor or high temperature were erased within one cell cycle, during S phase. Early-replicating regions were erased before late-replicating regions, implicating replication in H3K4me3 loss. However, incomplete H3K4me3 erasure occurred at the majority of loci even when replication was prevented, suggesting that most erasure results from an active process. Indeed, deletion of the demethylase Jhd2 slowed erasure at most loci. Together, these results indicate overlapping roles for passive dilution and active enzymatic demethylation in erasing ancestral histone methylation states in yeast. References:  Ng HH, Robert F, Young RA, Struhl K (2003) Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Mol Cell 11: 709-719.  Ringrose L, Paro R (2004) Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu Rev Genet 38: 413-443. The overall design of the experiment consists of two cell cycle experiments, each consisting of the following subsets: gene expression, ChIP with anti-H3K4Me3 antibody, and ChIP input. The experiments are as follows: CCA - BY4741 bar1- cells synchronized by alpha factor arrest; CCTS - BY4741 cdc28(ts) cells synchronized by arrest at non-permissive temperature. The individual time courses are enumerated as follows: CCA gene expression (array title "Synchronized cells, xxx min, cell cycle CCA"), 18 arrays; CCA ChIP for anti-H3K4Me3 (array title "H3K4Me3 ChIP cell cycle CCA, xxx min"), 17 arrays, 1 replicate; CCA ChIP input (array title "ChIP Input cell cycle CCA, xxx min"), 18 arrays, 1 replicate; CCTS gene expression (appears in a different dataset as biological replicate "I"; array title "Synchronized cells, xxx min, biological replicate I"), 18 arrays; CCTS ChIP for anti-H3K4Me3 (array title "H3K4Me3 cell cycle CCTS, xxx min"), 2 technical replicates, 18 arrays per replicate; CCTS ChIP input (array title "ChIP Input cell cycle CCTS, xxx min"), 2 technical replicates, 18 arrays per replicate. Gene expression arrays were run against a reference of unsynchronized cells, ChIP-chip anti-H3K4Me3 samples were run against an IP (of the same epitope) of unsynchronized cells, and ChIP input samples were run against either a pool of all the time points in the time course (CCTS) or against sonicated DNA isolated from unsynchronized cells (CCA). Four additional experiments have been performed. They are referred to as series X in the title. Series 1: anti H3K4me3 CHIP time course of BY4741 bar1 delete cells (wt) synchronized with alpha factor and released in the cell cycle, 9 arrays, 1 replicate. Series 2: anti H3K4me3 CHIP time course of BY4741 bar1 jhd2 delete cells (jhd2 delete) synchronized with alpha factor and released in the cell cycle, 7 arrays, 1 replicate. Series 3: anti H3K4me3 CHIP time course of CYM36 cells (cdc7ts) synchronized with alpha factor and released in the cell cycle at the permissive 24C or at the restrictive 37C temperature, 22 arrays, 2 replicates. Series 4: anti H3K4me3 CHIP time course of BY4741 bar1 delete cells (wt) grown in galactose and synchronized with alpha factor and either released in the cell cycle in glucose media or alpha factor arrested and switched to glucose media, 6 arrays, 1 replicate.
Project description:The use of genome-wide RNA abundance profiling by microarrays and deep sequencing has spurred a revolution in our understanding of transcriptional control. However, changes in mRNA abundance reflect the combined effect of changes in RNA production, processing, and degradation, and thus, mRNA levels provide an occluded view of transcriptional regulation. To partially disentangle these issues, we carry out genome-wide RNA Polymerase II (“Pol2”) localization profiling in budding yeast in two different stress response time courses. While mRNA changes largely reflect changes in transcription, there remains a great deal of variation in mRNA levels that is not accounted for by changes in Pol2 abundance. We find that genes exhibiting “excess” mRNA produced per Pol2 are enriched for those with overlapping cryptic transcripts, indicating a pervasive role for nonproductive or regulatory transcription in control of gene expression. Finally, we characterize changes in Pol2 localization when Pol2 is genetically inactivated using the rpb1-1 temperature-sensitive mutation. We find that Pol2 is lost from chromatin after roughly an hour at the restrictive temperature, and that there is a great deal of variability in the rate of Pol2 loss at different loci. Together, these results provide a global perspective on the relationship between Pol2 and mRNA production in budding yeast. The array studies consisted of 4 time courses in which the genome-wide location of RNA Polymerase II was mapped via chromatin immunoprecipitation (ChIP) in BY4741 S. cerevisiae cells or in such cells bearing the temperature sensitive mutation rpb1, in response to heat shock at 37 degrees C and/or 1.5 mM diamide. All samples were hybridized as the ChIP sample (Cy3 labeled, channel 1) versus its cognate ChIP input sample (Cy5 labeled, channel 2). The first time course was a heat shock in wild-type BY4741 cells. The second time course was a heat shock in BY4741 rpd1 cells. The third time course was a diamide treatment of BY4741 rpd1 cells. The fourth time course was a 10 minute heat shock in BY4741 rpd1 cells followed by treatment from 15-60 minutes with diamide (3 samples). In the first three time courses the time range is 0-120 minutes (5 samples). A total of 18 arrays were used in this study. No additional replicates or dye-flip experiments were performed.