Histones, ubiquitous in eukaryotes as DNA-packing proteins, find their evolutionary origins in archaea. Unlike the characterized histone proteins of a number of methanogenic and themophilic archaea, previous research indicated that HpyA, the sole histone encoded in the model halophile Halobacterium salinarum, is not involved in DNA packaging. Instead, it was found to have widespread but subtle effects on gene expression and to maintain wild type cell morphology. However, the precise function of halophilic histone-like proteins remain unclear. Here we use quantitative phenotyping, genetics, and functional genomics to investigate HpyA function. These experiments revealed that HpyA is important for growth and rod-shaped morphology in reduced salinity. HpyA preferentially binds DNA at discrete genomic sites under low salt to regulate expression of ion uptake, particularly iron. HpyA also globally but indirectly activates other ion uptake and nucleotide biosynthesis pathways in a salt-dependent manner. Taken together, these results demonstrate an alternative function for an archaeal histone-like protein as a transcriptional regulator, with its function tuned to the physiological stressors of the hypersaline environment.

The formation of G-quadruplex structures can regulate telomerase activity and the expression of oncogenes at the transcriptional and translational levels. Therefore, stabilization of G-quadruplex DNA structures by small molecules has been recognized as a promising strategy for anticancer drug therapy. One of the major challenges in this field is to impart stabilizing molecules with selectivity toward quadruplex structures over duplex DNAs, and to maintain specificity toward a particular quadruplex topology. Herein we report the synthesis and binding interactions of indenopyrimidine derivatives, endowed with drug-like properties, with oncogenic promoters of c-myc and c-kit, telomeric and duplex DNAs. The results show specific stabilization of promoter over telomeric quadruplexes and duplex DNAs. Molecular modeling studies support the experimental observations by unraveling the dual binding mode of ligands by exploiting the top and bottom quartets of a G-quadruplex structure. This study underscores the potential of the indenopyrimidine scaffold, which can be used to achieve specific G-quadruplex-mediated anticancer activity.

Stabilization of G-quadruplex DNA structures by small molecules has emerged as a promising strategy for the development of anticancer drugs. Since G-quadruplex structures can adopt various topologies, attaining specific stabilization of a G-quadruplex topology to halt a particular biological process is daunting. To achieve this, we have designed and synthesized simple structural scaffolds based on an indolylmethyleneindanone pharmacophore, which can specifically stabilize the parallel topology of promoter quadruplex DNAs (c-MYC, c-KIT1, and c-KIT2), when compared to various topologies of telomeric and duplex DNAs. The lead ligands (InEt2 and InPr2) are water-soluble and meet a number of desirable criteria for a small molecule drug. Highly specific induction and stabilization of the c-MYC and c-KIT quadruplex DNAs (ΔT1/2 up to 24 °C) over telomeric and duplex DNAs (ΔT1/2 ∼ 3.2 °C) by these ligands were further validated by isothermal titration calorimetry and electrospray ionization mass spectrometry experiments (Ka ∼ 10(5) to 10(6) M(-1)). Low IC50 (∼2 μM) values were emerged for these ligands from a Taq DNA polymerase stop assay with the c-MYC quadruplex forming template, whereas the telomeric DNA template showed IC50 values >120 μM. Molecular modeling and dynamics studies demonstrated the 5'- and 3'-end stacking modes for these ligands. Overall, these results demonstrate that among the >1000 quadruplex stabilizing ligands reported so far, the indolylmethyleneindanone scaffolds stand out in terms of target specificity and structural simplicity and therefore offer a new paradigm in topology specific G-quadruplex targeting for potential therapeutic and diagnostic applications.

Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high rRNA:mRNA ratio (80–90%: ~10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA, and therefore, requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here, we show that after the discontinuation of the previous version of RiboZero (Illumina, San Diego, CA, USA) that was useful in partially or completely depleting rRNA from archaea, archaeal transcriptomics studies have experienced a slowdown. To overcome this limitation, here, we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper, we provide insights into the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts.

DNA-binding proteins with roles in chromatin architecture and transcriptional regulation are present in all three domains of life. Histones that package DNA and regulate gene expression in eukaryotes find their evolutionary origin in the domain of life Archaea. Previously characterised archaeal histones have a somewhat conserved functional role in nucleosome formation and DNA packaging. However, previous research has indicated that the histone-like proteins of high salt-adapted archaea, or halophiles, appear to function differently. The sole histone protein encoded by the model halophilic species Halobacterium salinarum is non-essential, is involved in direct and indirect transcriptional regulation, and does not appear to package DNA. Here we use protein-DNA binding assays, computational analysis, and quantitative phenotyping to compare DNA binding patterns across halophilic histone proteins, bacterial and archaeal TFs, NAPs, and eukaryotic histones. Like TFs, halophilic histones bind the genome too sparsely to compact the genome. However, unlike TFs, binding occurs in both coding and intergenic regions. Unlike histones, halophilic histone occupancy is not depleted at the start sites of genes, and halophilic genomes lack the dinucleotide periodicity known to facilitate histone binding. We detect unique sequence preferences for histone binding in halophiles. Together these data suggest that the non-essentiality and genome-wide binding features of halophilic histone-like proteins are conserved across halophiles; they bind DNA in ways resembling both TFs and chromatin proteins, but do not appear to play a role in forming chromatin.

Most cells in eukaryotic species—from yeast to humans—possess histone proteins that pack and unpack DNA in response to environmental cues. These essential proteins regulate genes necessary for important cellular processes, including development and stress protection.

Despite intense recent research interest in archaea, the scientific community has experienced a bottleneck in the study of genome-scale gene expression experiments by RNA-seq due to the lack of commercial and specifically designed rRNA depletion kits. The high ratio rRNA:mRNA (80-90%: ∼10%) in prokaryotes hampers global transcriptomic analysis. Insufficient ribodepletion results in low sequence coverage of mRNA and therefore requires a substantially higher number of replicate samples and/or sequencing reads to achieve statistically reliable conclusions regarding the significance of differential gene expression between case and control samples. Here we show that after the discontinuation of the previous version of RiboZero (Illumina) that was useful to partially deplete rRNA from halophilic archaea, archaeal transcriptomics studies have experienced a standstill. To overcome this limitation, here we analyze the efficiency for four different hybridization-based kits from three different commercial suppliers, each with two sets of sequence-specific probes to remove rRNA from four different species of halophilic archaea. We conclude that the key for transcriptomic success with the currently available tools is the probe-specificity for the rRNA sequence hybridization. With this paper we provide insights to the archaeal community for selecting certain reagents and strategies over others depending on the archaeal species of interest. These methods yield improved RNA-seq sensitivity and enhanced detection of low abundance transcripts.

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