Unknown,Transcriptomics,Genomics,Proteomics

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High-resolution mapping of the spatial organization of Caulobacter crescentus chromosome by chromosome conformation capture in conjunction with next-generation sequencing (Hi-C)

ABSTRACT: Chromosomes must be highly compacted and organized within cells, but how this is achieved in vivo remains poorly understood. We report the first use of Hi-C to map the structure of bacterial chromosomes at a high, unprecedented resolution. Analysis of Hi-C data and polymer modeling indicates that the Caulobacter crescentus chromosome consists of multiple, largely independent spatial domains likely comprised of supercoiled plectonemes arrayed into a bottlebrush-like fiber. These domains are stable throughout the cell cycle and re-established concomitantly with DNA replication. We provide strong evidence that domain boundaries are established by highly-expressed genes and the formation of plectoneme-free regions. Additionally, we use Hi-C to demonstrate that supercoiling, the histone-like protein HU, and SMC act at different length-scales and in complementary ways to organize and structure chromosomes within cells. Hi-C experiments were performed on untreated wild type swarmer cells and drug-treated swarmer cells (Novobiocin or Rifampicin) of Caulobacter crescentus CB15N. Hi-C were also performed on swarmer cells of smc knock-out and hup1hup2 knock-out mutants of Caulobacter crescentus. Time-course Hi-C were performed on a synchronous population of Caulobacter crescentus at 5, 10, 30, 45, 60 and 75 minutes after synchronization. The progression of DNA replication during the cell cycle was followed by total genomic DNA sequencing.

ORGANISM(S): Caulobacter crescentus CB15

SUBMITTER: TUNG LE

PROVIDER: E-GEOD-45966 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Double-strand breaks (DSBs) can lead to the loss of genetic information and cell death. Consequently, cells in all domains of life have evolved mechanisms to repair DSBs, including through homologous recombination. Although recombination has been well characterized, the spatial organization of this process in living cells remains poorly understood. Here, we introduced site-specific DSBs in Caulobacter crescentus, and then used time-lapse microscopy to visualize the homology search, DSB repair, and the resegregation of chromosomal DNA. Even loci tethered to opposite cell poles can efficiently release, pair to enable recombination-based repair, and then resegregate to their original locations. Resegregation occurs independent of DNA replication and without disrupting global chromosome organization. Origin-proximal regions are resegregated by the same machinery, ParABS, used to segregate undamaged chromosomes following DNA replication. In contrast, origin-distal regions efficiently resegregate after a DSB independent of ParABS, and likely without dedicated segregation proteins. Instead, we propose that a physical, spring-like force drives the resegregation of origin-distal loci after DSB repair. Caulobacter cells were depleted of DnaA for 1.5 h before synchronization. Swarmer cells were then released into DnaA depleting conditions (without IPTG) and double-strand breaks were induced for 1 h by the addition of 500 ?M vanillate. For control sample, no vanillate was added. Formadehyde (Sigma) was then added to the final concentration of 1%. Formadehyde crosslinks protein-DNA and DNA-DNA together, thereby capturing the structure of the chromosome at the time of fixation. Fixation was performed at the cell density of OD600 = 0.2. The crosslinking reactions were allowed to proceed for 30 minutes at 25 °C before quenching with 2.5 M glycine at a final concentration of 0.125 M. Fixed cells were then pelleted by centrifugation and subsequently washed twice with 1x M2 buffer (6.1 mM Na2HPO4, 3.9 mM KH2PO4, 9.3 mM NH4Cl, 0.5 mM MgSO4, 10 ?M FeSO4, 0.5 mM CaCl2) before resuspending in 1x TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA) to a final concentration of 107 cells per µl. Resuspended cells were then divided into 25 µl aliquots and stored at -80 °C for no more than 2 weeks. Each Hi-C experiment was performed using two of the 25 µL aliquots. Chromosome conformation capture with next-generation seqeuncing (Hi-C) was carried out exactly as described previously (Le et al., 2013 PMID: 24158908)

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High-resolution mapping of the spatial organization of Caulobacter crescentus chromosome by chromosome conformation capture in conjunction with next-generation sequencing (Hi-C)

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