HI-NESS: a family of genetically encoded DNA labels based on a bacterial nucleoid-associated protein.

The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic dyes are often used for chromosome labelling in vivo. The mode of binding of these dyes to DNA cause its distortion, elongation, and partial unwinding. The structural changes induce DNA damage and interfere with the binding dynamics of chromatin-associated proteins, consequently perturbing gene expression, genome replication, and cell cycle progression. We have developed a minimally-perturbing, genetically encoded fluorescent DNA label consisting of a (photo-switchable) fluorescent protein fused to the DNA-binding domain of H-NS - a bacterial nucleoid-associated protein. We show that this DNA label, abbreviated as HI-NESS (H-NS-based indicator for nucleic acid stainings), is minimally-perturbing to genomic processes and labels chromosomes in eukaryotic cells in culture, and in zebrafish embryos with preferential binding to AT-rich chromatin.

Shedding Light on Bacterial Chromosome Structure: Exploring the Significance of 3C-Based Approaches.

The complex architecture of DNA within living organisms is essential for maintaining the genetic information that dictates their functions and characteristics. Among the many complexities of genetic material organization, the folding and arrangement of DNA into chromosomes play a critical role in regulating gene expression, replication, and other essential cellular processes. Bacteria, despite their apparently simple cellular structure, exhibit a remarkable level of chromosomal organization that influences their adaptability and survival in diverse environments. Understanding the three-dimensional arrangement of bacterial chromosomes has long been a challenge due to technical limitations, but the development of Chromosome Conformation Capture (3C) methods revolutionized our ability to explore the hierarchical structure and the dynamics of bacterial genomes. Here, we review the major advances in the field of bacterial chromosome structure using 3C technology over the past decade.

ChIP-qPCR of FLAG-Tagged Proteins in Bacteria.

DNA-protein interactions occur in biological processes such as genome replication, gene transcription, DNA repair, and chromatin compaction and organization. Mapping the distribution of the DNA-bound proteins on the chromosome is essential for understanding their associated biological process. Chromatin immunoprecipitation (ChIP) involves the antibody-mediated enrichment of DNA fragments bound by a target protein and has become one of the most powerful techniques for exploring the distribution of proteins on the chromosome. By incorporating quantitative polymerase chain reaction (qPCR) downstream of the ChIP assay, ChIP-qPCR was developed to describe binding profiles of DNA-associated proteins at a candidate locus. In this chapter, we describe ChIP-qPCR. We provide a step-by-step protocol for the preparation of a ChIP library of a 3× FLAG-tagged protein in bacteria, describe how downstream qPCR experiments can be performed with the appropriate controls, and explain how the data is analyzed. This chapter provides reliable technical guidance for ChIP-qPCR studies in bacteria.

The environmentally-regulated interplay between local three-dimensional chromatin organisation and transcription of proVWX in E. coli.

Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS (histone-like nucleoid structuring protein)-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. By consolidating our in vivo investigations with earlier in vitro and in silico studies that provide mechanistic details of how H-NS re-models DNA in response to osmolarity, we report that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. Our results also reveal additional structural features associated with changes in proVWX transcript levels such as the decompaction of local chromatin upstream of the operon, highlighting that further complexity underlies the regulation of this model operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria.

Determination of the 3D Genome Organization of Bacteria Using Hi-C.

The spatial organization of genomes is based on their hierarchical compartmentalization in topological domains. There is growing evidence that bacterial genomes are organized into insulated domains similar to the Topologically Associating Domains (TADs) detected in eukaryotic cells. Chromosome conformation capture (3C) technologies are used to analyze in vivo DNA proximity based on ligation of distal DNA segments crossed-linked by bridging proteins. By combining 3C and high-throughput sequencing, the Hi-C method reveals genome-wide interactions within topological domains and global genome structure as a whole. This chapter provides detailed guidelines for the preparation of Hi-C sequencing libraries for bacteria.

Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM.

Although the distribution of DNA-binding proteins inside the cell nucleus can be analyzed by immunolabeling or by tagging proteins with GFP, we cannot establish whether the protein is bound to DNA or not. Here, we describe a novel approach that allows imaging of the in situ interaction between a GFP-fusion protein and DNA in the cell nucleus, using fluorescence resonance energy transfer (FRET). We used fluorescence lifetime imaging microscopy (FLIM) as a reliable tool to detect protein in contact with DNA. The method was successfully applied to the DNA-binding proteins histone H2B and the glucocorticoid receptor and to the heterochromatin-associated proteins HP1alpha and HP1beta.