ABSTRACT: untargeted global Mass Spectrometry-based Proteomics in ymr1∆ inp53∆ mutants released from a G1 into HU in AAA-imbalanced (-Trp) or AAA-balanced (+Trp) YPD medium
Project description:Diatoms are single celled photosynthetic bloom-forming algae that are responsible for at least 20% of global primary production. Nevertheless, more than 30% of the oceans are considered “ocean deserts” due to iron limitation. We used the diatom Phaeodactylum tricornutum as a model system to explore diatom’s response to iron limitation and its interplay with susceptibility to oxidative stress. By analyzing physiological parameters and proteome profiling, we defined two distinct phases: short-term (< 3 days, phase I) and chronic (> 5 days, phase II) iron limitation. While at phase I no changes in physiological parameters were observed, molecular markers for iron starvation, such as ISIP and flavodoxin, were highly upregulated. At phase II, down regulation of numerous iron-containing proteins was detected in parallel to reduction in growth rate, chlorophyll content, photosynthetic activity, respiration rate and antioxidant capacity. Intriguingly, while application of oxidative stress to phase I and II iron limited cells similarly oxidized the GSH pool, phase II iron limitation exhibited transient resistance to oxidative stress, despite the down regulation of many antioxidant proteins. By comparing proteomic profiles of P. tricornutum under iron limitation and metatranscriptomic data of an iron enrichment experiment conducted in the Pacific Ocean, we propose that iron limited cells in the natural environment resemble the phase II metabolic state. These results provide insights into the trade-off between maximal growth rate and susceptibility to oxidative stress as a possible key determinant in the response of diatoms to iron quota in the marine environment.
Project description:A conserved hallmark of eukaryotic chromatin architecture is the distinctive array of well-positioned nucleosomes downstream of transcription start sites (TSS). Recent studies indicate that trans-acting factors establish this stereotypical array. Here, we present the first genome-wide in vitro and in vivo nucleosome maps for the ciliate Tetrahymena thermophila. In contrast with previous studies in yeast, we find that the stereotypical nucleosome array is preserved in the in vitro reconstituted map, which is governed only by the DNA sequence preferences of nucleosomes. Remarkably, this average in vitro pattern arises from subsets of nucleosomes, rather than the whole array, being present in individual Tetrahymena genes. Variation in GC content contributes to the positioning of these sequence-directed nucleosomes, and affects codon usage and amino acid composition in genes. We propose that these ‘seed’ nucleosomes may aid the AT-rich Tetrahymena genome – which is intrinsically unfavorable for nucleosome formation – in establishing nucleosome arrays in vivo in concert with trans-acting factors, while minimizing changes to the coding sequences they are embedded within. All data are from the macronuclear genome. Datasets: 1) Log-phase cells, fixed chromatin, light MNase digest; 2) Log-phase cells, native chromatin, heavy MNase digest; 3) Starved cells, fixed chromatin, light MNase digest; 4) Starved cells, native chromatin, heavy MNase digest; 5) in vitro reconstituted chromatin, 50ul reaction, 4:10 histone:DNA ratio, light MNase digest; 6) in vitro reconstituted chromatin, 50ul reaction, 7:10 histone:DNA ratio, light MNase digest; 7) in vitro reconstituted chromatin, 150ul reaction, 4:10 histone:DNA ratio, light MNase digest; 8) in vitro reconstituted chromatin, 150ul reaction, 4:10 histone:DNA ratio, heavy MNase digest; Control dataset: 9): MNase-digested naked DNA
Project description:Here we adapt native elongating transcript sequencing (NET-seq) to develop transcription elongation factor associated nascent elongating transcript sequencing (TEF-seq). In this the RNA polymerase II (Pol2) transcription elongation complex (TEC) is immunoprecipitated via associated transcription elongation factors (TEFs). Sequencing from the 3' end of the Pol2-associated nascent transcript shows the position of the final incorporated nucleotide giving strand-specific, single nucleotide resolution maps of the level of TEF association with Pol2 during transcription.
Project description:The aim of the experiments was to determine the regulon of the Bacillus subtilis alternative sigma factor SigI. Biological relevance: To expand our knowledge about Bacillus subtilis transcriptional network under unfavorable conditions. Experimental workflow overview: Bacillus subtilis 168 trp+ (BaSysBio) was used as the genetic background. (i) sigI-rsgI knock-out, (ii) rsgI knock-out, and (iii) wr strains were cultured in LB medium to mid-exponential phase at 37°C and 52°C. Total RNA was isolated from 3 ml of the culture. rRNA was depleted from the samples with RiboMinus; subsequently RNA-seq libraries were prepared (Illumina compatible NEXTflex Rapid Directional RNA-Seq Kit, Bioo Scientific) and sequenced at the EMBL GeneCore facility. The experiment was performed in three independent replicates.
Project description:genome-wide ChIP-on-chip against TCF4 in LS174T cells, performed on NimbleGen Systems, Inc. human genome-wide 38-chip, hg17 array set followed by verification using biological replicates on a dedicated design.
Project description:Transcriptional profiling of infrarenal aortic tissue from Male 10-week-old C57BL/6J mice after AAA-induction with porcine pancreatic elastase, compared with sham-operated saline-injected mice. One day after AAA-induction, the mice were injected intraperitoneally with either lentiviral packaged miR-24 antagomir (anti-miR-24) or miR-24 mimic (pre-miR-24), or a scrambled microRNA control (scr-miR). Aortic samples were obtained 7 days after operation. The goal was to examine gene expression in developing AAA in this model, and to compare the effects of scr-miR, anti-miR-24 and pre-miR-24. Four condition experiment, one infrarenal aorta per array. Sham vs. scr-miR-PPE vs. anti-miR-24-PPE vs. pre-miR-24-PPE, all harvested at Day 7 post-operatively. After QC, the final analysis group (uploaded here) consisted of 18 arrays: Sham-Saline-treated (4 arrays), scr-miR-PPE-treated (5 arrays); pre-miR-24-PPE-treated (3 arrays); and anti-miR-24-PPE-treated (6 arrays).
Project description:We aim to understand the role that Cdx2 plays in specifying the rostro-caudal identity of differentiating motor neurons. We find that expressing Cdx2 in combination with FGF signaling is sufficient to produce motor neurons with a more caudal identity. ChIP-seq analysis of Cdx2 finds that it binds extensively throughout the Hox regions in progenitor motor neurons. Analysis of polycomb-associated chromatin over Hox regions in the subsequently generated motor neurons finds that Cdx2 binding corresponds to chromatin domains encompassing de-repressed caudal Hox genes. These results suggest a direct role for Cdx2 in specifying caudal motor neuron identity. ChIP-seq studies: We characterize the binding of Cdx2 in progenitor motor neurons using a V5 tagged doxycycline inducible Cdx2 ESC line (iCdx2). Progenitor motor neurons were generated after 4 days of in vitro differentiation of mouse embryonic stem cells using retinoic acid (RA) and hedgehog (Hh) signaling exposure at day 2. On day 3, the cells are exposed to Dox with and without accompanying FGF signaling. The genome-wide binding of the induced Cdx2 transcription factor is profiled using ChIP-seq with an anti-V5 antibody. An appropriate whole-cell extract control experiment for these ChIP-seq experiments is also included. We also examine the effect of induced Cdx2 expression on polycomb-associated chromatin structure in the resulting cellular populations by profiling the H3K27me3 chromatin mark using ChIP-seq. H3K27me3 experiments were performed after 5 days of in vitro differentiation using cells exposed to either: 1) RA & Hh to derive progenitor motor neurons, followed by Dox & FGF; 2) Dox & FGF alone; or 3) RA and Hh alone. There are 6 Illumina sequencing datasets included in this submission: two biological replicates of iCdx2 ChIP-seq in the presence of FGF; one sample of iCdx2 ChIP-seq in the absence of FGF; one H3K27me3 ChIP-seq in the presence of RA, Hh, Dox, and FGF; one H3K27me3 ChIP-seq in the presence of Dox and FGF; and one H3K27me3 ChIP-seq in the presence of RA and Hh.
Project description:The study of 5-hydroxylmethylcytosines (5hmC), the sixth base of the mammalian genome, as an epigenetic mark has been hampered by a lack of method to map it at single-base resolution. Previous affinity purification-based methods could not precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach for mapping 5hmC at base resolution. Application of this new method to the embryonic stem cells not only confirms widespread distribution of 5hmC in mammalian genome, but also reveals a strong sequence bias and strand asymmetry at sites of 5hmC. Additionally, the relative abundance of 5hmC varies significantly depending on the types of functional sequences, suggesting different mechanisms for 5hmC deposition and maintenance. Furthermore, we observe high levels of 5hmC and reciprocally low levels of 5mC at transcription factor binding sites, revealing a dynamic DNA methylation process at cis-regulatory elements. Base resolution sequencing of 5 hydroxymethylcytosine in human and mouse embryonic stem cells
Project description:Naive CD4+ T cells are the common precursors of multiple effector and memory T cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4+ T cells and their changes during the early phase of T cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy.<br>Periodate oxidation and aniline-catalyzed oxime ligation (PAL) technology was applied with subsequent quantitative LC-MS/MS (PAL-qLC-MS/MS) to generate a dataset describing the surface proteome of human naive CD4+ T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins, of which 24 were previously not known to be expressed on human naive CD4+ T cells or have no defined role within T cell activation. To independently confirm the proteomic dataset and to analyse the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous dataset, resulting in 229 surface proteins which are expressed on naive unstimulated and activated CD4+ T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation and predicted subcellular localization, and correlated the proteomics result with this transcriptional dataset.<br>This extensive surface atlas provides an overall naive CD4+ T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments.
Project description:Co-transcriptional splicing of introns is a defining feature of eukaryotic gene expression. We show that the mammalian spliceosome specifically associates with the S5P CTD isoform of RNA polymerase II (Pol II) as it elongates across spliced exons of protein coding genes, both in human Hela and murine lymphoid cell lines. Immuno-precipitation of MNase digested chromatin with phospho CTD specific antibodies reveals that components of the active spliceosome (both snRNA and proteins) form a specific complex with S5P CTD Pol II. Furthermore a dominant splicing intermediate formed by cleavage at intron 5’ss results in the tethering of upstream exons to this complex at all spliced exons. These are invariably connected to upstream spliced constitutive and less frequently to alternative exons. Finally S5P CTD Pol II accumulates over spliced exons but not adjacent introns. We propose that mammalian splicing employs a rapid, co-transcriptional splicing mechanism based on CTD phosphorylation transitions.