Dynamic landscape of protein occupancy across the Escherichia coli chromosome
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA596475 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10734200_1.fastq.gz | Fastqsanger.gz | |||
| SRR10734200_2.fastq.gz | Fastqsanger.gz | |||
| SRR10734201_1.fastq.gz | Fastqsanger.gz | |||
| SRR10734201_2.fastq.gz | Fastqsanger.gz | |||
| SRR10734202_1.fastq.gz | Fastqsanger.gz |
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Project description:In bacteria, adaptive regulation of gene expression occurs through the precise interactions of hundreds of DNA binding proteins across the chromosome. Decades of research has revealed the identity and functions of many of these regulators. However, a systems-level understanding of gene expression requires simultaneous, comprehensive monitoring of these interactions as a function of genetic and environmental perturbations. Here we present a high-resolution in vivo protein occupancy display (IPOD-HR) technology that enables quantitative and comprehensive monitoring of DNA-protein interactions across a bacterial chromosome. The global nature of IPOD-HR permits simultaneous activity profiling of all known sequence specific transcription factors and discovery of novel condition-dependent DNA-binding proteins. We show that global IPOD-HR profiles can be used for de novo discovery of sequence specificity motifs for active transcription factors. IPOD-HR also reveals many large domains of extended protein occupancy that define relatively stable, transcriptionally silent regions with unique sequence and gene functional features. IPOD-HR thus provides a unique form of systems-level access to transcriptional regulatory states that will allow rapid profiling and characterization of both known and novel regulatory logic in bacteria. We provide here IPOD-HR occupancy results across a range of genetic and physiological perturbations, as detailed in the accompanying manuscript.
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Project description:Protein occupancy of Mycoplasma pneumoniae chromosome
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Project description:Free-living bacteria adapt to environmental change by reprogramming gene expression through precise interactions of hundreds of DNA-binding proteins. A predictive understanding of bacterial physiology requires us to globally monitor all such protein-DNA interactions across a range of environmental and genetic perturbations. Here, we show that such global observations are possible using an optimized version of in vivo protein occupancy display technology (in vivo protein occupancy display-high resolution, IPOD-HR) and present a pilot application to Escherichia coli. We observe that the E. coli protein-DNA interactome organizes into 2 distinct prototypic features: (1) highly dynamic condition-dependent transcription factor (TF) occupancy; and (2) robust kilobase scale occupancy by nucleoid factors, forming silencing domains analogous to eukaryotic heterochromatin. We show that occupancy dynamics across a range of conditions can rapidly reveal the global transcriptional regulatory organization of a bacterium. Beyond discovery of previously hidden regulatory logic, we show that these observations can be utilized to computationally determine sequence specificity models for the majority of active TFs. Our study demonstrates that global observations of protein occupancy combined with statistical inference can rapidly and systematically reveal the transcriptional regulatory and structural features of a bacterial genome. This capacity is particularly crucial for non-model bacteria that are not amenable to routine genetic manipulation.
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Project description:Protein occupancy is a means of identifying all the regions in the genome that are bound to proteins. A simple protocol is used whereby cross-linked chromatin is digested with DNase to remove all non-crosslinked, naked, DNA. To distinguish the regions that are bound by RNA polymerase from the rest, a ChAPseq (chromatin affinity precipitation) with TAP-tagged alpha subunit of RNApol, was performed.
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Project description:Using a high-density tiling array containing 25-mer oligonucleotides at the resolution of one every four base pairs across the entire genome, we developed In vivo Protein Occupancy Display (IPOD), a technology that reveals protein occupancy across an entire bacterial chromosome at the resolution of individual binding sites. Application to Escherichia coli reveals thousands of protein occupancy peaks, highly enriched within and in close proximity to non-coding regulatory regions.
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Project description:Kinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e. all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded ~50% phospho-occupancy, and the cumulative phospho-occupancy changed by only ~20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained, in part, by ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. The low inherent phospho-occupancy promotes microtubule attachment to kinetochores while the high sensitivity of kinetochore-microtubule attachments to small changes in phospho-occupancy drives error correction and ensures high mitotic fidelity.
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Project description:Meiotic prophase I (MPI) is the most important event in mammalian meiosis. Even though MPI-related gene expression has been extensively investigated, the status of the chromosome binding proteins (CBPs) and the corresponding complexes in this process has been not scrutinized yet. A quantitative proteomics focused onto the MPI-related CBPs was accomplished in this study, in which the mouse primary spermatocytes in 4 different subphases of MPI were collected and the chromosome-enriched proteins (ChEPs) within the cells were extracted and identified in quantitative mode. According to a stringent criterion, in total of 1136 CBPs in the MPI subphases were identified and quantified. Looking at the dynamic patterns of CBP abundance in response to MPI progression, the patterns were broadly divided to two groups, high abundance in leptotene (L) and zygotene (Z) or that in pachytene (P) and diplotene (D). Most importantly, 26 CBP complexes were found, while some MPI-dependent features of these complexes were perceived for the first time. The functions of these complexes were categorized to DNA replication, pre-initial complex of transcription, transcriptional regulation and synapsis. In agreement with the current theory, the dynamic changes of the CBP complex abundance appeared declined DNA replication and activated transcription and synapsis activities in P and D, and Z and P phase; in converse to the traditional prediction, the abundance response of CBP complexes to MPI emerged condensin activity weakened in P and D phase. Profiling of protein complexes thus offered convincing evidence how important the CBP complexes partake in MPI regulation.
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Project description:Protein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation and translation. We have developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing (Baltz and Munschauer et al. 2012). Our current work focuses on streamlining and extending protein occupancy profiling on poly(A)-RNA. Our objectives are to identify previously unknown protein-bound transcripts and, more importantly, to assess global and local differences in protein occupancy across different biological conditions. To this end, we have implemented poppi, the first pipeline for differential analysis of protein occupancy profiles. We have applied our analysis pipeline to pinpoint changes in occupancy profiles of MCF7 cells against already published HEK293 cells. We generated protein occupancy cDNA libraries for two biological replicates. Briefly, we crosslinked 4SU-labeled MCF7 cells and purified protein-mRNA complexes using oligo(dT)-beads. The precipitate was treated with RNAse I to reduce the protein-crosslinked RNA fragments to a length of about 30-60 nt. To remove non-crosslinked RNA, protein-RNA complexes were precipitated with ammonium sulfate and blotted onto nitrocellulose. The RNA was recovered by Proteinase K treatment, ligated to cloning adapters, and reverse transcribed. The resulting cDNA libraries were PCR-amplified and next-generation sequenced.
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