IPOD-HR, ChIP-seq, RNA-seq of E. coli nucleoid associated protein deletions
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA691963 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13433300_1.fastq.gz | Fastqsanger.gz | |||
| SRR13433300_2.fastq.gz | Fastqsanger.gz | |||
| SRR13433301_1.fastq.gz | Fastqsanger.gz | |||
| SRR13433301_2.fastq.gz | Fastqsanger.gz | |||
| SRR13433302_1.fastq.gz | Fastqsanger.gz |
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Project description:There is increasing evidence that microbes maintain a structured chromosome, which in turn impacts gene expression. However tools to profile the genome landscape and binding of key architectural proteins have been limited. We recently discovered densely occupied, multi-kilobase regions in E. coli that are transcriptionally silent, similar to eukaryotic heterochromatin. These regions, termed EPODs, overlap metabolic pathways and parasitic elements such as prophages. Here, we investigate the contributions of nucleoid associated proteins (NAPs) to these domains by examining the impacts of deleting NAPs on EPODs genome-wide in E. coli and an evolutionarily distant species, Bacillus subtilis. We identify key NAPs contributing to the silencing of specific EPODs, where deletion of a particular NAPs opens a chromosomal region to RNA polymerase binding. In E. coli, we distinguish novel xenogeneic silencing NAPs, Fis and Hfq, that are essential for cell viability in the presence of domesticated prophages. Furthermore, we show that changes in EPODs facilitate an extra layer of transcriptional regulation to prepare cells for exposure to exotic carbon sources. Our findings demonstrate a new suite of mechanisms through which genomic architecture primes bacteria for changing metabolic environments and silences harmful genomic elements
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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:Nucleoid remodeling facilitated by DNA supercoiling results in changes in nucleoid configuration and involves nucleoid-associated proteins (NAPs) and structural maintenance of chromosomes (SMC) proteins among others. Changes in nucleoid configurations regulated by NAPs are synchronized with cellular adaptation and influence the simultaneous expression of several genes. HU, a ubiquitous bacterial histone-like protein, is among the most conserved and abundant NAPs in eubacteria. In Escherichia coli, HU forms dimers by HUα self-association (HUαα) or by HUα-HUβ interactions (HUαβ). HUα is mostly expressed during lag and early exponential growth phase and HUβ is expressed only during the later exponential and stationary phase, pointing to distinct HUαα/DNA and HUαβ/DNA packaging of the nucleoid in regulating expression patterns during growth and stasis. Mutations or the deletion of HU transform the E. coli nucleoid to a different form and alter overall transcription program; thus, HU interactions with DNA directly affect global gene regulation. Recently, analysis of high-resolution contact maps of the E. coli nucleoid revealed an important role of HU to promote long-range DNA-DNA contacts within the nucleoid. Yet, the molecular connections between HU-DNA interactions and changes in nucleoid architecture that regulate gene expression globally remain unknown. Here, we explored the higher-order E. coli nucleoid organization by soft x-ray tomography (SXT) and revealed an effect of HU surface charges in overall nucleoid organization and rearrangements. We also studied global transcription by next-generation RNA sequencing (RNA-Seq) and found a link between nucleoid rearrangement and changes in global transcription. To determine the functional relationships of observed nucleoid rearrangement and HU-DNA interactions, we also characterized the overall organization of HU nucleoprotein complexes in solution by small angle x-ray scattering (SAXS). We found that HUαα-mediated DNA networks are different at different ionic strengths and pHs. By means of macromolecular crystallography, we additionally elucidate HUαα dependent molecular switches that modulate DNA networking. This integrative structural study explains how HUαα regulates dynamic transformations of the nucleoid by DNA bridging to control nucleoid rearrangement and global gene regulation.
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Project description:Plastids contain multiple copies of the plastid chromosome, folded together with proteins and RNA into nucleoids. The degree to which components of the plastid gene expression and protein biogenesis machineries are nucleoid associated, and the factors involved in plastid DNA organization, repair, and replication, are poorly understood. To provide a conceptual framework for nucleoid function, we characterized the proteomes of highly enriched nucleoid fractions of proplastids and mature chloroplasts isolated from the maize (Zea mays) leaf base and tip, respectively, using mass spectrometry. Quantitative comparisons with proteomes of unfractionated proplastids and chloroplasts facilitated the determination of nucleoid-enriched proteins. This nucleoid-enriched proteome included proteins involved in DNA replication, organization, and repair as well as transcription, mRNA processing, splicing, and editing. Many proteins of unknown function, including pentatricopeptide repeat (PPR), tetratricopeptide repeat (TPR), DnaJ, and mitochondrial transcription factor (mTERF) domain proteins, were identified. Strikingly, 70S ribosome and ribosome assembly factors were strongly overrepresented in nucleoid fractions, but protein chaperones were not. Our analysis strongly suggests that mRNA processing, splicing, and editing, as well as ribosome assembly, take place in association with the nucleoid, suggesting that these processes occur cotranscriptionally. The plastid developmental state did not dramatically change the nucleoid-enriched proteome but did quantitatively shift the predominating function from RNA metabolism in undeveloped plastids to translation and homeostasis in chloroplasts. This study extends the known maize plastid proteome by hundreds of proteins, including more than 40 PPR and mTERF domain proteins, and provides a resource for targeted studies on plastid gene expression. Details of protein identification and annotation are provided in the Plant Proteome Database.
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