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The handle http://hdl.handle.net/1887/44297 holds various files of this Leiden University dissertation
Author: Valk, Ramon van der
Title: Responding to environmental cues : the adaptive qualities of chromatin compaction proteins
Issue Date: 2016-11-22
Summary
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The effective volume occupied by the genomes of all forms of life far exceeds that of the cells in which they are contained. All organisms have therefore developed mechanisms for compactly folding and functionally organizing their genetic material. Through recent advances in fluorescent microscopy and 3C-based technologies we finally have a first glimpse into the complex mechanisms governing the three-dimensional folding of genomes. This thesis describes the investigation of three mechanisms of DNA compaction and organization:
DNA bridging (by the bacterial chromatin protein H-NS) and DNA bending and wrapping (by the archaeal chromatin proteins HMfA and HMfB). These studies suggest that the DNA binding properties of these proteins are altered by physico-chemical conditions corresponding with different cellular environments. Due to the alteration of their DNA binding properties these proteins’ cellular functions, such as the regulation of transcription, might be modulated.
Bacteria frequently need to adapt to altered environmental conditions. Adaptation requires changes in gene expression, often mediated by global regulators of transcription. The nucleoid-associated protein H-NS is a key global regulator in Gram-negative bacteria, and is believed to be a crucial player in bacterial chromatin organization, through DNA bridging.
H-NS activity in vivo is modulated by physico-chemical factors (osmolarity, pH, temperature) and interaction partners. Mechanistically it is unclear how functional modulation of H-NS by such factors is achieved. In Chapter 2, it is shown that a diverse spectrum of factors can alter the ability of H-NS to bridge DNA. By altering the concentrations of divalent or monovalent ions it is possible to promote DNA bridging by H-NS, while leaving DNA binding mostly unaffected. Similarly, synergistic and antagonistic co-regulators modulate the DNA bridging efficiency of H-NS. DNA bridging can be promoted by H-NS interacting proteins such as Hha and YdgT, while other proteins such as H-NST and gp 5.5 are expected to have adverse effects.
Simulations of the structure of H-NS under some of these conditions revealed switching between a bridging capable and incapable form of H-NS.
It is not always a trivial task to understand and quantify the effects of proteins that bind to DNA. In recent years many new biophysical techniques have been established which can be used to address this issue. One such technique, Atomic force microscopy (AFM) imaging, has proven to be a powerful tool for the study of DNA-protein interactions due to its ability to image single molecules at the nanoscale. However, the use of AFM in force spectroscopy to study DNA-protein interactions has been limited. In Chapter 3 we discuss a high throughput, AFM based, pulling assay to measure the strength and kinetics of protein bridging of DNA molecules. As a model system, we investigated the interactions between DNA and H-NS.
Using this assay we were able to extract information on the strength of DNA-DNA bridges mediated by H-NS. In future studies this tool will be used to further quantify the effect of physico-chemical conditions and partner proteins on H-NS, such as those described in Chapter 2.
As mentioned above, genome organization is essential for all domains of life. In Chapter 4 we investigate the third domain of life, the archaea. The Archaea phylum is the newest domain in the phylogenetic tree, having only been recognized four decades ago. As the name indicates (stemming from the Greek arkhaios, meaning ancient or primitive) it is in fact believed to be the most ancient. Some archaeal species have been suggested to be direct predecessors of Eukarya, sharing much of the cellular machinery with modern eukaryotes.
Archaea are thus very interesting from an evolutionary point of view. Some archaeal species are attractive as minimal eukaryotic-like model systems for fundamental and applied (e.g.
drug design and evaluation) studies. Archaea can be found in a variety of environmental locations ranging from extreme temperatures and pressures, to human intestines. Archaea organize and compact their genomes in a variety of ways. Many archaeal species express homologues of eukaryotic histone H3 and H4. The dominant model, derived from in vitro data, suggests the formation of nucleosome-like structures involving a histone protein tetramer. However, recent in vivo studies point at the formation of differently sized histone- DNA complexes consisting of a number of dimeric units. Here we use the Methanothermus fervidus HMfA and HMfB proteins as a model system to investigate the assembly and structure of archaeal histone-DNA complexes. Our studies indicate that these histone-like proteins cooperatively multimerize along DNA, inducing strong compaction and suggest that multimerization beyond a tetramer is promoted by specific high-affinity sequences, reconciling the two models.
Risk analysis and technology assessment (RATA)
The RATA of this thesis involves the assessment of the risks as well as the socioeconomic benefits of the biological samples and findings. All the experiments and findings in this thesis are considered to be low risk, since the genetically modified Escherichia coli strains are non- pathogenic, and are unlikely to cause diseases if released in nature. As a safety precaution however these strains were contained to a lab environment. This was done to prevent genes that are not naturally found in Escherichia coli, such as genes coding for archaeal chromatin proteins or anti-biotic resistance, to transfer to other strains through horizontal gene transfer.
The experiments in this thesis were performed according to standard laboratory safety regulations, and measures were taken to reduce the risks (such as wearing of lab coats and gloves). Bacteria related experiments were performed in Biosafety level 1 designated labs, and careful measures were taken to dispose of any waste products. Experiments containing radioactivity were performed in labs especially designated and qualified for this purpose.
Some experiments required the use of micro/nanoparticles, such as the beads used in several biochemical assays and in the TPM experiments. No harmful effects have been associated with the use of these particles. Nevertheless, precautions were taken to reduce the risk of exposure, such as wearing gloves and minimizing the assay concentrations of these particles.
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Although the findings in this study may not be directly applicable in a business environment.
It is believed that they may eventually open new opportunities in the fields of antibiotics and treatments for diseases originating from gene misregulation or misfolding of the genome.
