Project Description
The binding of H-NS to the bacterial DNA is initiated by electrostatic interactions between the positive residues on the H-NS linker and the negatively charged DNA. The initial contacts established by the linker then promotes engagement by the C-terminal DNA-binding domain of H-NS proteins.
These findings were published in PNAS in 2017.
Gao Y et al. Charged residues in the H-NS linker drive DNA binding and gene silencing in single cells. PNAS. 2017. 114(47). 12560-12565. doi: 10.1073/pnas.1716721114.
[More information on the Kenney Lab and the Yan Lab]
Figure: Binding of H-NS to DNA via electrostatic interactions between the positive residues on the H-NS linker and the DNA. H-NS binding to DNA leads to gene silencing. When the H-NS linker is removed or mutated, DNA binding is reduced and its gene silencing function is lost.
The study identifies a role for the H-NS linker region in initiating H-NS binding to bacterial DNA. The findings in this study highlight the significance of five positively-charged residues on the linker (2 arginines and 3 lysines) in promoting electrostatic interactions with the negatively-charged DNA. The initial contacts made by the H-NS linker promotes further engagement by its C-terminal DNA-binding domain. H-NS binding leads to the stiffening of the DNA filament and make it inaccessible to the DNA transcription machineries, thereby silencing the expression of genes located within this stretch of DNA.
Understanding the basics
The bacterial genome is organized into complex structures called nucleoids, wherein the millimeters-long, circular DNA is condensed into micron-sized dense assemblies of DNA, RNA, and nucleoid-associated proteins (NAPs). Despite being densely compacted, the DNA in the nucleoid remains accessible to DNA transcription and protein synthesis machinery, suggesting that the nucleoid is a structurally-organized entity. Several factors drive the compaction of DNA in the nucleoid, including DNA supercoiling, macromolecular crowding, association of NAPs to the DNA, and the presence of specific DNA sequences that affect the flexibility of the DNA. Several studies have combined high-throughput techniques such as chromatin immunoprecipitation with either microarrays (ChIP-chip) or next-generation sequencing (ChIP-Seq) to characterize DNA binding by nucleoid-associated proteins such as H-NS, Fis, HU, IHF, etc. Such studies described how NAPs are important for establishing and maintaining the nucleoid structure.
Alterations to the chromatin structure occur as a consequence of the limitations acquired through its condensation. As chromatin is condensed into the primary nucleosome structure, DNA becomes less accessible for transcription factors. With the loosening of this chromatin structure, however, transcription machinery is better able to access the genomic DNA, and transcription is thus promoted. Hence, nucleosome organization and dynamics are regularly modified by the combined influence of covalent post-translational modifications (PTMs), histone chaperones, ATP-dependent nucleosome remodelers and histone variants.
During an infection, an external virulent agent like bacteria, virus or fungi, invades into body tissues and proliferates, causing disease. The ability of a bacteria to invade a cell or tissue, to establish an infection within the body and to avoid or even exploit the immune response is often dependent on the bacteria’s ability to manipulate the host cytoskeleton, and exploit various biochemical pathways that respond to changes in mechanical stimuli. While some molecular mechanisms may be unique to a particular pathogen, some may be conserved across species.
The Study in Detail
Key Findings
- When the H-NS linker was deleted or replaced with a dummy linker, H-NS was unable to silence the hdfr gene, which, in turn, is a repressor of flhDC, the flagella regulating gene in bacteria. Therefore, H-NS linker mutations reduced motility in bacterial cells.
- H-NS linker mutations also failed to repress another target, csgD (fused to GFP), leading to increase in fluorescence.
- When the five charged residues of the H-NS linker region were reintroduced into the dummy linker Q15 (contains 15 glutamine residues), H-NS was once again able to repress its target genes, hdfr and csgD.
- Mutant H-NS without a linker or having a dummy linker do not polymerize along the DNA. Instead, they formed small patches of filaments on the DNA.
- The H-NS linker mutants formed oligomers similar to the wild-type, suggesting that the mutations in the linker primarily affect H-NS protein’s DNA-binding and gene silencing abilities.
- H-NS proteins colocalized with the nucleoids, where they clustered as dense foci. On the other hand, H-NS linker mutants were not able to form such foci.
- An absence of DNA binding in the H-NS linker mutant did not affect nucleoid compaction, as evidenced by slightly smaller nucleoids in the presence of H-NS linker mutants compared to H-NS wild-type.
- The H-NS proteins bound to DNA by electrostatic interactions between the positively charged (2 arginines and 3 lysines) and the negatively-charged DNA via the formation of approximately 5 hydrogen bonds.
Methods and Controls used in the study
- Atomic Force Microscopy was employed to assess the binding of wild-type H-NS and H-NS linker mutants to DNA.
- Single-Molecule Localization Microscopy (SMLM) was used to study the colocalization of H-NS with the nucleosomes.
- Structured Illumination Microscopy (SIM) was employed to assess the extent of nucleoid compaction upon binding by the H-NS proteins.
- Multiple Molecular Dynamics (MD) simulations were used to postulate that the binding of H-NS to DNA was initiated through electrostatic interactions between the positively charged residues on the H-NS linker and the negatively-charged DNA.
Applications and Future Directions
- By bringing to light the role of H-NS proteins in silencing bacterial genes, such as those involved in their virulence in hosts, the study provide new possibilities for anti-bacterial treatment that mimic the H-NS gene silencing mechanism.
- The study also highlights other important roles that linker domains can play in protein function, in addition to their well-known functions in internally connecting their multiple protein domains and providing flexibility to the proteins.
