Browsing by Subject "Histones"

Background

Methods and results

Conclusion

Protein-based compaction of the genome is a feature found in species across the tree of life. In Archaea, the majority of species contain a histone fold domain-containing protein, and these have been shown to compact DNA through the formation of nucleosomes and extended structures called hypernucleosomes. However, the role of the histone-like proteins of halophilic archaea is unclear. Previous work in the model species Halobacterium salinarum indicated that its sole histone gene, hpyA, is dispensable for growth and is expressed at very low levels. I hypothesize that the unique high-salt environment of halophilic archaea has selected for an alternative histone function, and that they function instead as transcription factors.

This hypothesis was addressed with genetic approaches including the creation of knockout and complementation strains, traditional microbiology techniques including growth assays and microscopy, and high-throughput genomics approaches: ChIP-Seq to study genome-wide binding, and RNA-Seq to study differential expression in ΔhpyA strain. It was found that hpyA is required for optimal growth in hypo-osmotic conditions, and exhibits strong salt-dependent binding patterns and gene regulation. It directly regulates genes involved in iron uptake, and indirectly regulates genes in ion transport and nucleotide metabolism. These results validate the link between histone function and the high-salt environment of halophilic archaea.

Similar to hpyA, I found that the sole histone gene of another model halophile: hstA of Haloferax volcanii, could be deleted, and that knockout cells remained viable. The genome-wide binding of both halophilic histones was studied, and compared with publicly available data regarding the binding patterns from transcription factors (TFs), nucleoid-associated proteins (NAPs), and eukaryotic histones. Halophilic histones bind in narrow, discrete, and relatively rare peaks, just like TFs; however, this binding is not enriched at the promoter, and they instead bind evenly in both intergenic and coding regions (like some NAPs). Their occupancy profile across gene start sites do not resemble those of histones or TFs. In terms of sequence specificity, HpyA exhibits a histone-like preference for 10bp periodicity, while HstA exhibits a TF-like trait in preferentially binding a palindromic sequence motif. When considering all the data, I conclude that halophilic histones blur the line between TFs, NAPs, and histones.

A major technical challenge in generating this data was the removal of rRNA prior to carrying out RNA-Seq. Several approaches were tested across four model species of halophiles, and the reasons for differences in performance for these approaches were analyzed. Methods that deliver efficient rRNA removal targeted to a particular species, or to halophilic archaea in general, are highlighted.

Together these results shed light on the unusual function and binding modes of the histone-like proteins of halophilic archaea. In combination with other recent work, they suggest that histone function is linked with the physical environment of archaeal species.