Dataset Information

Higher-order structure of DNA determines its positioning in cell-size droplets under crowded conditions.

ABSTRACT:

Background

It is becoming clearer that living cells use water/water (w/w) phase separation to form membraneless organelles that exhibit various important biological functions. Currently, it is believed that the specific localization of biomacromolecules, including DNA, RNA and proteins in w/w microdroplets is closely related to their bio-activity. Despite the importance of this possible role of micro segregation, our understanding of the underlying physico-chemical mechanism is still unrefined. Further research to unveil the underlying mechanism of the localization of macromolecules in relation to their steric conformation in w/w microdroplets is needed.

Principal findings

Single-DNA observation of genome-size DNA (T4 GT7 bacteriophage DNA; 166kbp) by fluorescence microscopy revealed that DNAs are spontaneously incorporated into w/w microdroplets generated in a binary aqueous polymer solution with polyethylene glycol (PEG) and dextran (DEX). Interestingly, DNAs with elongated coil and shrunken conformations exhibit Brownian fluctuation inside the droplet. On the other hand, tightly packed compact globules, as well as assemblies of multiple condensed DNAs, tend to be located near the interface in the droplet.

Conclusion and significance

The specific localization of DNA molecules depending on their higher-order structure occurs in w/w microdroplet phase-separation solution under a binary aqueous polymer solution. Such an aqueous solution with polymers mimics the crowded conditions in living cells, where aqueous macromolecules exist at a level of 30-40 weight %. The specific positioning of DNA depending on its higher-order structure in w/w microdroplets is expected to provide novel insights into the mechanism and function of membraneless organelles and micro-segregated particles in living cells.

SUBMITTER: Nishio T

PROVIDER: S-EPMC8694483 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Our previous work have shown that certain subpopulations of Klebsiella pneumoniae exhibit significant phenotypic changes under simulated microgravity (SMG), including enhanced biofilm formation and cellulose synthesis, which may be evoked by changes in gene expression patterns. It is well known that prokaryotic cells genomic DNA can be hierarchically organized into different higher-order three-dimensional structures, which can highly influence gene expression. It is remain elusive whether phenotypic changes induced by SMG in the subpopulations of K. pneumoniae are driven by genome higher-order structural changes. Here, we investigated the above-mentioned issue using the wild-type (WT) K. pneumoniae (WT was used as a control strain and continuously cultivated for 2 weeks under standard culture conditions of normal gravity) and two previous identified subpopulations (M1 and M2) obtained after 2 weeks of continuous incubation in a SMG device. By the combination of genome-wide chromosome conformation capture (Hi-C), RNA-seq and whole-genome methylation (WGS) analyses, we found that the along with the global chromosome interactions change, the compacting extent of M1, M2 subpopulations were much looser under SMG and even with an increase in active, open chromosome regions. In addition, transcriptome data showed that most differentially expressed genes (DEGs) were upregulated, whereas a few DEGs were downregulated in M1 and M2. The functions of both types DEGs were mainly associated with membrane fractions. Additionally, WGS analysis revealed that methylation levels were lower in M1 and M2. Using combined analysis of multi-omics data, we discovered that most upregulated DEGs were significantly enriched in the boundary regions of the variable chromosomal interaction domains (CIDs), in which genes regulating biofilm formation were mainly located. These results suggest that K. pneumoniae may regulate gene expression patterns through DNA methylation and changes in genome structure, thus resulting in new phenotypes in response to altered gravity.