Project description:The Drosophila MSL complex mediates dosage compensation by increasing transcription of the single X chromosome in males approximately two-fold. This is accomplished through recognition of the X chromosome and subsequent acetylation of histone H4K16 on X-linked genes. Initial binding to the X is thought to occur at a subset of sites. However, the consensus sequence motif of entry sites (M-bM-^@M-^\MSL recognition elementM-bM-^@M-^] or MRE) is only slightly enriched on the X (~2 fold), and only a fraction of them is utilized by the MSL complex. Here we ask whether chromatin context could distinguish between utilized and non-utilized copies of the motif, by comparing their relative enrichment for histone modifications and chromosomal proteins mapped in the NHGRI modENCODE project. Through a comparative analysis of the chromatin features in male S2 cells, which contain MSL complex, and female Kc cells, which lack the complex, we find that the presence of active chromatin modifications, together with an elevated local GC content in surrounding sequence, has strong predictive value for functional MSL entry sites, independent of MSL binding. We tested these sites for function in Kc cells by RNAi knockdown of Sxl, resulting in induction of MSL complex. We show that ectopic MSL expression in Kc cells leads to H4K16 acetylation around these sites, and a relative increase in X chromosome transcription. Collectively, our results support a model in which a pre-existing active chromatin environment, coincident with H3K36me3, contributes to MSL entry site selection. The consequences of MSL targeting of the male X chromosome include increase in nucleosome lability, enrichment for H4K16 acetylation and JIL-1 kinase, and depletion of linker histone H1 on active X-linked genes. Our finding serves as a model to understand how chromatin and local sequence features are involved in the selection of functional protein binding sites in the genome. The key Drosophila female sex determinant protein, SXL, represses dosage compensation by inhibiting MSL2 translation. Loss of SXL results in the expression, stabilization, and targetting of the MSL complex in female cells. Therefore, depletion of SXL by RNA interference (RNAi) in female Kc cells will lead to a MSL2-dependent increase in transcription from the female X chromosomes, consistent with the induction of dosage compensation. In this experiment, we generated ChIP-chip profiles of H4K16 acetylation (H4K16ac) in Kc cells of control (GFP) and Sxl RNAi. For ChIP, we used Upstate (now Millipore) anti-H4K16ac antibody, catalog # 07-329, lot #JBC1355376.

Project description:Active genes on the X chromosome of Drosophila males are upregulated by the Male-specific lethal (MSL) complex containing five MSL proteins and two non-coding roX RNAs. To probe the targeting mechanism, we have solved the structure of the MSL3 chromodomain, designed point mutations in key residues that disrupt putative methyl-lysine recognition, and tested their effect on full length MSL3 function. Transgenic males expressing these site-directed point mutants or MSL3short, a naturally occurring MSL3 form lacking the chromodomain, are unhealthy and developmentally delayed. Genomewide analyses of the binding patterns of these mutants support a two-step model: the first step is chromodomain-independent association with “chromatin entry sites” carrying GA-rich MSL recognition elements (MREs). The second step involves spreading from entry sites to the majority of active genes on the X. Either deleting or introducing point mutations in the MSL3 chromodomain disrupts this second step. In vitro studies demonstrate that chromodomain mutants have diminished interaction with recombinant nucleosomes methylated at H3K36. We propose that MSL spreading depends, to a large extent, on the integrity of the MSL3 chromodomain to interact with lysine-methylated nucleosomes. Chromatin immunoprecipitation using TAP-tag was performed as described previously (Alekseyenko et al., 2006). The immunoprecipitated material was eluted from the beads by adding 10 ?L (100 U) of AcTEV protease into 450 ?L of TEV buffer followed by incubation at 25°C for 1 h. To reverse the cross-links, samples were brought to 0.3M NaCl and 1% SDS and incubated at 65°C for 12 h. The samples were then extracted with phenol/chloroform/isoamyl alcohol followed by chloroform, and precipitated by ethanol in the presence of glycogen. The resulting precipitated DNA and the input DNA were amplified using a DNA linker as described (Strutt et al., 1997). LM-PCR (25 cycles) was performed using Platinum Pfu (Invitrogen). The resulting DNA was labeled and hybridized to arrays by NimbleGen Systems, Inc. Two-channel tiling arrays containing 388,000 probes were designed based on FlyBase 3.2. Chromosomes X and 19.6Mb of 2L were covered. The ChIP sample of interest was hybridized on one channel and genomic DNA was used as the reference on the other channel. Each ChIP-chip experiment was performed in duplicate.

Project description:Thermomacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, a M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supra-normal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to up-regulation of 55 genes. Genome re-sequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism or transport. These mutations included 7 non-synonymous substitutions, 4 insertions and 1 deletion. One of the insertion mutations mapped to pseudogene, Msed_1517, and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that include the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula was naturally lacking this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low affinity high velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated spontaneous arsenate resistant mutants derived from CuR1 all underwent mutation in pitA and non-selectively became copper resistant. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching. The study comprises 5 samples, described in detail below. WT_CuR1: Differential transcriptional response of Metallosphaera sedula DSM 5348, WT, to the supra-normal copper resistant spontaneous Metallosphaera sedula mutant, CuR1 under normal growth conditions. This experiment was done to analyze the differential transcription of WT cells compared with CuR1 cells at mid log phase. WT-15_CuR1-15: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT and CuR1 15 minutes post copper challenge. The copper cultures were harvested 15 minutes after the shock. WT-60_CuR1-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT and CuR1 60 minutes post copper challenge. The copper cultures were harvested 60 minutes after the shock. WT-15_WT-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula WT 15 and 60 minutes post copper challenge. The copper cultures were harvested 15 and 60 minutes after the shock, respectively. CuR1-15_CuR1-60: Differential transcription of Metallosphaera cells under sub-inhibitory copper challenge (2.0 mM). This experiment was done to analyze the differential transcription of Metallosphaera sedula CuR1 15 and 60 minutes post copper challenge. The copper cultures were harvested 15 and 60 minutes after the shock, respectively.

Project description:Escherichia coli strain MG1655 was grown in a Zn-depleted custom-built chemostat. Culture volume (120 ml), temperature (37 oC) and stirring speed (440 rpm) were maintained. Steady state values for pH and OD600 were 6.9 and 0.6, respectively. Chemostats were grown for 50 h to allow five culture volumes to pass through the vessel and allow an apparent (pseudo-)steady state to be reached. At this point, ZnSO4.7H2O in water was added to a final concentration of 0.2 M in the chemostat. A 10 ml sample of culture was taken using a polypropylene pipette tip immediately prior to Zn addition and 2.5, 7, 10 and 30 min after addition. Samples were harvested into RNAprotect and total RNA was purified using QiagenM-bM-^@M-^Ys RNeasy Mini kit (using the supplierM-bM-^@M-^Ys protocol) prior to use in microarray analysis. A control experiment was also carried out in which water was added. Biological experiments were carried out twice (i.e. control (water added) and experiment (ZnSO4 added) chemostats were grown separately twice), and a dye swap performed for each experiment, providing at least two technical repeats for each of the two biological repeats at all five time points. Slides were analysed so that time points M-bM-^@M-^\with ZnM-bM-^@M-^] were compared to the same time point M-bM-^@M-^\without ZnM-bM-^@M-^] (e.g. 30 min after Zn was added was compared to 30 min after water being added). Cells were grown in glycerol-glycerophosphate medium (GGM) lacking Zn. Final concentrations in GGM are: MES (40.0 mM), NH4Cl (18.7 mM), KCl (13.4 mM), beta-glycerophosphate (7.64 mM), glycerol (5.00 mM), K2SO4 (4.99 mM), MgCl2 (1.00 mM), EDTA (134 microM), CaCl2.2H2O (68.0 microM), FeCl3.6H2O (18.5 microM), H3BO3 (1.62 microM), CuCl2.2H2O (587 nM), CoNO3.6H2O (344 nM), and (NH4)6Mo7O24.4H2O (80.9 nM) in MilliQ water (Millipore). Bulk elements (MES, NH4Cl, KCl, K2SO4 and glycerol in MilliQ water at pH 7.4 (batch growth) or 7.6 (continuous culture)) were passed through a column containing Chelex-100 ion exchange resin (Bio-Rad) to remove contaminating cations. Trace elements (with no added Zn) and a CaCl2 solution were then added to give the final concentrations shown above prior to autoclaving. After autoclaving, MgCl2 and beta-glycerophosphate were added at the final concentrations shown above. All chemicals were of AnalaR grade of purity or higher. Chelex-100 was packed into a Bio-Rad Glass Econo-column (approximately 120 mm M-CM-^W 25 mm) that had previously been soaked in 3.5% nitric acid for 5 d. E. coli strain MG1655 was grown a custom-built chemostat made entirely of non-metal parts. Glass growth vessels and flow-back traps were soaked extensively (approximately two months) in 10% nitric acid before being rinsed thoroughly in MilliQ water. Vent filters (Vent Acro 50 from VWR) were connected to the vessel using PTFE tubing. Metal-free pipette tips were used (MAXYMum Recovery Filter Tips from Axygen). Culture volume was maintained at 120 ml using an overflow weir in the chemostat vessel. The dilution rate (and hence the specific growth rate) was 0.1 h-1. The vessel was inoculated using one of the side-arms. Flasks were placed on KMO 2 Basic IKA-Werke stirrers (arbitrary setting: 400). Stirring speed was calibrated (440 rpm) and matched between vessels using a handheld laser tachometer (Compact Instruments Ltd). Temperature was kept constant at 37 oC. Twice daily, a 2-ml sample was taken and used to test the pH (which stayed constant at pH 6.9), OD600 (0.6 at steady state), glycerol limitation, and contamination on nutrient agar plates. E. coli strain MG1655 was grown in a M-bM-^@M-^\Zn-freeM-bM-^@M-^] chemostat. Chemostats were grown for 50 h to allow five culture volumes to pass through the vessel and allow an apparent (pseudo-)steady state to be reached. At this point, ZnSO4.7H2O in water was added to a final concentration of 0.2 M in the chemostat. A 10 ml sample of culture was taken using a polypropylene pipette tip immediately prior to Zn addition and 2.5, 7, 10 and 30 min after addition. Total RNA was stabilised using RNAprotect (Qiagen). Total RNA was purified using QiagenM-bM-^@M-^Ys RNeasy mini kit. A control experiment was carried out in which water was added. Microarray slides were set up so that time points M-bM-^@M-^\with ZnM-bM-^@M-^] were compared to the same time point M-bM-^@M-^\without ZnM-bM-^@M-^] (e.g. 30 min after Zn was added was compared to 30 min after water being added). For each time point, equal quantities of RNA obtained after addition of zinc and addition of water were labelled by using nucleotide analogues of dCTP containing either Cy3 or Cy5 fluorescent dyes (Perkin Elmer). For each microarray slide, one sample was labelled with Cy3-dCTP, while the other sample was labelled with Cy5-dCTP. RNA (15-20 micrograms) was annealed to 9 micrograms pd(N)6 random hexamers (Amersham Biosciences) by heating at 65 oC for 10 min, followed by 10 min at 22 oC. This was supplemented with 4 microlitres of 5M-CM-^W First-strand buffer (Invitrogen), 2 microlitres dNTP mixture (5 mM each dATP, dTTP, dGTP and 2 mM dCTP), 2 microlitres 0.1 M DTT, 2 microlitres 1 mM Cy3 or Cy5 (Perkin Elmer) and 1 microlitre Superscript II reverse transcriptase (200 U) (Invitrogen). This was incubated at 42 oC for 2 hours. The reaction was terminated by the addition of NaOH (5 microlitres of 1M) and HCl (5 microlitres of 1M) before the addition of TE buffer (200 microlitres). Labelled cDNA was purified using a Qiaquick PCR purification kit (Qiagen). The Cy3-dCTP-labelled sample was mixed with the Cy5-dCTP-labelled sample and re-suspended in 120 microlitres salt-based hybridisation buffer (supplied with the microarray slides). This was heated at 95 oC for 3 min then cooled on ice for 3 min. The mixture was pipetted onto a microarray slide, sealed with a GeneFrame and coverslip in a hybridization chamber and incubated for 18 h at 42 M-BM-0C. Following hybridization, microarray slides (minus GeneFrame and coverslip) were washed in a series of pre-warmed (37 M-BM-0C) SSC buffers for 5 min each at 37oC: 1M-CM-^W SSC/0.1 % SDS, 1M-CM-^W SSC, 0.2M-CM-^W SSC and 0.01M-CM-^W SSC. Microarray slides were dried by centrifugation at 500 M-CM-^W g for 2 min before scanning. Slides were scanned on an Affymetrix 428 scanner. The average signal intensity and local background correction were obtained using a commercially available software package from Biodiscovery, Inc (Imagene, version 4.0 and GeneSight, version 3.5). Spots automatically flagged as bad, negative or poor in the Imagene software were removed before the statistical analysis was carried out in GeneSight. The mean values from each channel were log2 transformed and normalised using the Lowess method to remove intensity-dependent effects in the log2(ratios) values. The Cy3/Cy5 fluorescent ratios were calculated from the normalized values. Biological experiments were carried out twice times, and a dye swap performed for each experiment, providing at least two technical repeats for each of the two biological repeats at all five time points. Data from the independent experiments were combined. Genes that were differentially expressed M-bM-^IM-% twofold and displayed and P value of < 0.05 (as determined by a t test) were defined as being statistically significantly differentially transcribed.

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Sequence-specific targeting of dosage compensation in Drosophila favors an active chromatin context

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