The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic... Show moreThe 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. Show less
Rashid, F.Z.M.; Mahlandt, E.; Vaart M, van der; Boer, D.E.C.; Varela Alvarez, M.; Henneman, B.; ... ; Dame, R.T. 2021
The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic... Show moreThe 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. Show less
Nucleoid associated proteins maintain the architecture of the bacterial chromosome and regulate gene expression, hinting that their role as transcription factors may involve local three-dimensional... Show moreNucleoid associated proteins maintain the architecture of the bacterial chromosome and regulate gene expression, hinting that their role as transcription factors may involve local three-dimensional chromosome re-modelling. This dissertation provides the first evidence to support this hypothesis. Using ensemble RT-qPCR and 3C-qPCR, in addition to in vivo and in vitro single molecule fluorescence microscopy techniques, the interplay between the expression of the H-NS-regulated, osmosensitive proVWX operon of Escherichia coli and structural re-modelling of the operon is shown. The formation of a loop anchored between the P2 promoter of proVWX and the terminus of the operon represses its expression. Destabilization of the loop activates transcription. The model presented in this thesis provides clues for how H-NS and H-NS-like proteins may regulate the expression of other operons and genes within their regulons. Show less
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... Show moreThe 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. Show less
