Dataset Information

DNA Affinity chromatografy on Mycoplasma pneumoniae extracts I

ABSTRACT: Determining the gene regulatory network of an organism is fundamental to achieving a global understanding of cell behavior. In general, studies of transcription regulation are limited to the annotated transcription factors, not considering other non-canonical regulators. Here we describe the first systematic analysis of the DNA-interactome of a bacterium with a minimal proteome (Mycoplasma pneumoniae). We first determined by DNA affinity chromatography and intact chromatin isolation all potential DNA binding proteins. We then mapped the DNA binding of these factors by ChIP-seq, as well as their functionality by gain- and loss-of-function experiments, by transcriptomics and proteomics. This identified new DNA binding proteins and novel regulators with moonlighting properties like proteases and metabolic enzymes and allowed to reconstruct the gene regulatory network.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Mycoplasma Pneumoniae (strain Atcc 29342 / M129)

SUBMITTER: Eva Yus

LAB HEAD: Luis Serrano

PROVIDER: PXD007674 | Pride | 2019-11-12

REPOSITORIES: Pride

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Publications

Determination of the Gene Regulatory Network of a Genome-Reduced Bacterium Highlights Alternative Regulation Independent of Transcription Factors.

Yus Eva E Lloréns-Rico Verónica V Martínez Sira S Gallo Carolina C Eilers Hinnerk H Blötz Cedric C Stülke Jörg J Lluch-Senar Maria M Serrano Luis L

Cell systems 20190821 2

Here, we determined the relative importance of different transcriptional mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae, by employing an array of experimental techniques under multiple genetic and environmental perturbations. Of the 143 genes tested (21% of the bacterium's annotated proteins), only 55% showed an altered phenotype, highlighting the robustness of biological systems. We identified nine transcription factors (TFs) and their targets, representing 43% of the genome, ...[more]

PMID: 31445891

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Project description:We previously demonstrated that inactivation of the replication checkpoint via a mec1 mutation led to chromosome breakage at replication forks initiated from virtually all origins of replication, after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Furthermore, we have shown that chromosomes break at replication forks that have suffered single-stranded DNA (ssDNA) formation. Here we sought to determine whether all replication forks containing ssDNA gaps have equal probability of producing double strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-Seq, that combines our previously described DSB labeling with NextGen sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed greater distance in MEC1 cells than in mec1 cells during the recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of the said transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. Our model provides an explanation for a long-standing problem in chromosome biology: why different replication inhibitors produce different spectra of chromosome breakage? We propose that different inhibitors elicit different transcription responses as well as destabilize replication forks, and, when the two processes collide, ssDNA at the replication fork suffers further strand breakage, causing DSBs. We queried the yeast genome for DSBs after cells were treated with 200 mM hydroxyurea during S phase. Samples were collected from 1) cells synchronized in G1 phase by alpha factor; 2) cells released from G1 into medium containing 200 mM hydroxyurea for 1 h; 3) cells recovering in fresh medium without hydroxyurea for 1 h after the 1 h exposure to HU. These samples are referred to as G1, HU 1h, and R 1h, respectively. The strains from which the samples were collected are indicated following the time point, e.g. G1_MEC1 or R 1h_mec1. The experiment with mec1 was done twice (Experiments A and B) and that with MEC1 was done once (Experiment C). In addition, a control experiment of in vitro digestion with BamHI using the G1_mec1 sample (G1_BamHI) was performed.

Dataset Information

DNA Affinity chromatografy on Mycoplasma pneumoniae extracts I

Publications

Determination of the Gene Regulatory Network of a Genome-Reduced Bacterium Highlights Alternative Regulation Independent of Transcription Factors.

Similar Datasets

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