Project description:Significant insight about biological networks arises from the study of network motifs âoverly 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 Â°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 â??long-livedâ?? [1], and that is involved in memory during cell state inheritance in metazoans [2]. 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: [1] 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. [2] 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: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:We used complementary DNA microarray to analyze the gene expression profiles in different proglottids of Moniezia expansa. A total of 4056 sequences including full length and partial complementary DNAs representing novel, known, and control genes were analyzed. We utilized cDNA array for detection of gene expression profiles of different proglottids of M.expansa. The present study provides some interesting data to better understanding the mechanisms of procreation and may suggest some potential target molecules for a more effective treatment on verminosis. 1.Construction of microarray By clustering the 2,642 sequences from the cDNA library, 1,078 unigenes of M.expansa, including full-length and partial cDNAs representing novel or known genes, were obtained for array construction. The negative control spots of non-tapeworm origin in the chip were the rice genes (48 spots). In addition, the array included 9 known genes(54 spots) as positive control genes that were provided by our library and 24 empty spots. Unigenes and all control genes were cloned into plasmid vector. The DZH (TC) chips were constructed by United Gene Holdings Limited of China (Shanghai, China) according to the method described by Li et al. The cDNA inserts were amplified by use of the polymerase chain reaction (PCR) using universal primers to plasmid vector sequences and were then purified. All PCR products were examined by agarose gel electrophoresis to ensure the quality and the identity of the amplified clones as expected. Then the amplified PCR products were dissolved in a buffer containing 3ÃSSC solution. The solution with amplified PCR products were spotted onto glass slides (model DZH-TC) using a Cartesian PixSys 7500 motion control robot (Cartesian Technologies, Irvine, CA., USA) fitted with ChipMaker Micro-Spotting Technology (TeleChem International, Sunnyvale, CA., USA). The glass slides were then hydrated for 2 h in 70% humidity, dried for 0.5 h at room temperature, and UV crosslinked (65 mj/cm). They were further processed at room temperature by soaking in 0.2% sodium dodecyl sulfate (SDS) for 10 min, distilled H2O for 10 min, and 0.2% sodium borohydride (NaBH4) for 10 min. The slides were dried again and ready for use. 2.RNA preparation and probe labeling Total RNAs were isolated from 6 M.expansas with scolex-neck proglottids, immature proglottids, mature proglottids and gravid proglottids. Tissue samples were ground into a fine powder in a 10 cm ceramic mortar (RNase-free) and were then homogenized in TRIzol (Biostar, Shanghai, China). After centrifugation, the supernatant was separated from the organic phase and was extracted in an equal volume of chloroform. The aqueous phase was then precipitated by an equal volume of isopropanol at 4Â°C, centrifuged to pellet the RNA and dissolved in Milli-Q H2O. The fluorescent cDNA probes were prepared through reverse transcription with Cy3- or Cy5-deoxy UTP (Amersham Pharmacia Biotech, Piscataway, NJ, USA) as follows: 5 Î¼g of oligo(dT18) was added and annealed to 3Î¼g of mRNA by heating the mixture to 70ËC for 10 min, and then chilling it on ice. The final reaction buffer mixture contained dNTPs (200 Î¼M dATP, dCTP and dGTP; 60 Î¼M dTTP; and 60 Î¼M Cy3- or Cy5-dUTP), 2 Î¼l of Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and 1Ãreaction buffer 10Î¼l. The reactions were carried out at 42 ÂºC for 2 h. RNA was hydrolyzed by adding 4 Î¼l of 2.5 M NaOH, incubating the mixture at 65 ÂºC for 10 min and then neutralizing it with 4 Î¼l of 2.5 M HCl. The RNA samples from the scolex-neck proglottids were labeled with Cy3-dUTP and those from immature proglottids, mature proglottids and gravid proglottids, respectively, were labeled with Cy5-dUTP. The two color probes were then mixed and diluted to 500 Î¼l with TE, and concentrated using a Microcon YM-30 filter (Millipore, Bedford, MA, USA) to 10 Î¼l. The sample was then vacuum dried. 3.Hybridization The probe was dissolved in 20 ml of hybridization solution (5ÃSSC (0.75M NaCl and 0.075M sodium citrate), 0.4% SDS, 50% formamide). Microarrays were pre-hybridized with a hybridization solution containing 0.5 mg/ml of denatured salmon sperm DNA at 42Â°C for 6 h. Fluorescent probe mixtures were denatured at 95Â°C for 5 min and then applied onto the pre-hybridized chip under a cover glass. Chips were hybridized at 42Â°C for 15-17 h. Next, the hybridized chips were each washed at 60Â°C for 10 min in solutions of 2ÃSSC and 0.2% SDS, 0.1ÃSSC and 0.2% SDS, 0.1ÃSSC and then dried at room temperature. 4.Detection and analysis The chips were scanned with a ScanArray 4000 (GSI Lumonics, Bellerica, MA, USA) at two wavelengths, 635 and 532 nm, to detect emission from both Cy5 and Cy3, respectively. The acquired images were analyzed using GenePix Pro 3.0 software. The intensities of each spot at the two wavelengths represent the quantity of Cy3-dUTP and Cy5-dUTP. Ratios of Cy5 to Cy3 were computed using the GenePix Pro 3.0 median of ratio method. Overall intensities were normalized using the corresponding GenePix default normalization factor. All spots flagged âBadâ or âNot Foundâ by GenePix software were removed from the final data. Only genes with raw intensity values for both Cy3 and Cy5 of >200 were chosen for differential analysis. Genes were identified as differentially expressed if the ratio was >2 or <0.5, or the absolute value of base 2 logarithm of the ratio was >1 or < -1. mature proglottids, immature proglottids, gravid proglottids vs common reference scolex-neck proglottids tissue. 2 biological replicates for each experiments.

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