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1. The Hypersaline Archaeal Histones HpyA and HstA Are DNA Binding Proteins That Defy Categorization According to Commonly Used Functional Criteria

   https://doi.org/10.1128/mbio.03449-22  

   Sakrikar, Saaz ; Hackley, Rylee K. ; Martinez-Pastor, Mar ; Darnell, Cynthia L. ; Vreugdenhil, Angie ; Schmid, Amy K. ( April 2023 , mBio)

   Komeili, Arash (Ed.)

   ABSTRACT Histone proteins are found across diverse lineages of Archaea , many of which package DNA and form chromatin. However, previous research has led to the hypothesis that the histone-like proteins of high-salt-adapted archaea, or halophiles, function differently. The sole histone protein encoded by the model halophilic species Halobacterium salinarum , HpyA, is nonessential and expressed at levels too low to enable genome-wide DNA packaging. Instead, HpyA mediates the transcriptional response to salt stress. Here we compare the features of genome-wide binding of HpyA to those of HstA, the sole histone of another model halophile, Haloferax volcanii . hstA , like hpyA , is a nonessential gene. To better understand HpyA and HstA functions, protein-DNA binding data (chromatin immunoprecipitation sequencing [ChIP-seq]) of these halophilic histones are compared to publicly available ChIP-seq data from DNA binding proteins across all domains of life, including transcription factors (TFs), nucleoid-associated proteins (NAPs), and histones. These analyses demonstrate that HpyA and HstA bind the genome infrequently in discrete regions, which is similar to TFs but unlike NAPs, which bind a much larger genomic fraction. However, unlike TFs that typically bind in intergenic regions, HpyA and HstA binding sites are located in both coding and intergenic regions. The genome-wide dinucleotide periodicity known to facilitate histone binding was undetectable in the genomes of both species. Instead, TF-like and histone-like binding sequence preferences were detected for HstA and HpyA, respectively. Taken together, these data suggest that halophilic archaeal histones are unlikely to facilitate genome-wide chromatin formation and that their function defies categorization as a TF, NAP, or histone. IMPORTANCE 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. Although the histone fold domain originated in the domain of life Archaea , the function of archaeal histone-like proteins is not well understood relative to those of eukaryotes. We recently discovered that, unlike histones of eukaryotes, histones in hypersaline-adapted archaeal species do not package DNA and can act as transcription factors (TFs) to regulate stress response gene expression. However, the function of histones across species of hypersaline-adapted archaea still remains unclear. Here, we compare hypersaline histone function to a variety of DNA binding proteins across the tree of life, revealing histone-like behavior in some respects and specific transcriptional regulatory function in others. 

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2. Systematic Discovery of Archaeal Transcription Factor Functions in Regulatory Networks through Quantitative Phenotyping Analysis

   https://doi.org/10.1128/mSystems.00032-17  

   Darnell, Cynthia L. ; Tonner, Peter D. ; Gulli, Jordan G. ; Schmidler, Scott C. ; Schmid, Amy K. ; Shank, Elizabeth A. ( September 2017 , mSystems)

   ABSTRACT Gene regulatory networks (GRNs) are critical for dynamic transcriptional responses to environmental stress. However, the mechanisms by which GRN regulation adjusts physiology to enable stress survival remain unclear. Here we investigate the functions of transcription factors (TFs) within the global GRN of the stress-tolerant archaeal microorganism Halobacterium salinarum . We measured growth phenotypes of a panel of TF deletion mutants in high temporal resolution under heat shock, oxidative stress, and low-salinity conditions. To quantitate the noncanonical functional forms of the growth trajectories observed for these mutants, we developed a novel modeling framework based on Gaussian process regression and functional analysis of variance (FANOVA). We employ unique statistical tests to determine the significance of differential growth relative to the growth of the control strain. This analysis recapitulated known TF functions, revealed novel functions, and identified surprising secondary functions for characterized TFs. Strikingly, we observed that the majority of the TFs studied were required for growth under multiple stress conditions, pinpointing regulatory connections between the conditions tested. Correlations between quantitative phenotype trajectories of mutants are predictive of TF-TF connections within the GRN. These phenotypes are strongly concordant with predictions from statistical GRN models inferred from gene expression data alone. With genome-wide and targeted data sets, we provide detailed functional validation of novel TFs required for extreme oxidative stress and heat shock survival. Together, results presented in this study suggest that many TFs function under multiple conditions, thereby revealing high interconnectivity within the GRN and identifying the specific TFs required for communication between networks responding to disparate stressors. IMPORTANCE To ensure survival in the face of stress, microorganisms employ inducible damage repair pathways regulated by extensive and complex gene networks. Many archaea, microorganisms of the third domain of life, persist under extremes of temperature, salinity, and pH and under other conditions. In order to understand the cause-effect relationships between the dynamic function of the stress network and ultimate physiological consequences, this study characterized the physiological role of nearly one-third of all regulatory proteins known as transcription factors (TFs) in an archaeal organism. Using a unique quantitative phenotyping approach, we discovered functions for many novel TFs and revealed important secondary functions for known TFs. Surprisingly, many TFs are required for resisting multiple stressors, suggesting cross-regulation of stress responses. Through extensive validation experiments, we map the physiological roles of these novel TFs in stress response back to their position in the regulatory network wiring. This study advances understanding of the mechanisms underlying how microorganisms resist extreme stress. Given the generality of the methods employed, we expect that this study will enable future studies on how regulatory networks adjust cellular physiology in a diversity of organisms. 

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3. TbsP and TrmB jointly regulate gapII to influence cell development phenotypes in the archaeon Haloferax volcanii

   https://doi.org/10.1111/mmi.15225  

   Hackley, Rylee K. ; Hwang, Sungmin ; Herb, Jake T. ; Bhanap, Preeti ; Lam, Katie ; Vreugdenhil, Angie ; Darnell, Cynthia L. ; Pastor, Mar Martinez ; Martin, Johnathan H. ; Maupin‐Furlow, Julie A. ; et al ( January 2024 , Molecular Microbiology)

   Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt‐loving organismHaloferax volcaniiexhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related speciesHalobacterium salinarumby activating the expression of enzyme‐coding genes in the gluconeogenesis metabolic pathway. InHbt. salinarum, TrmB‐dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions inHfx. volcanii. The ∆trmBstrain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we nametrmBsuppressor, or TbsP (a.k.a. “tablespoon”). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild‐type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose‐dependent transcription ofgapII, which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients inHfx. volcanii.

    

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4. The Landscape of RNA-Protein Interactions in Plants: Approaches and Current Status

   https://doi.org/10.3390/ijms22062845  

   Burjoski, Vesper ; Reddy, Anireddy S. ( March 2021 , International Journal of Molecular Sciences)

   null (Ed.)

   RNAs transmit information from DNA to encode proteins that perform all cellular processes and regulate gene expression in multiple ways. From the time of synthesis to degradation, RNA molecules are associated with proteins called RNA-binding proteins (RBPs). The RBPs play diverse roles in many aspects of gene expression including pre-mRNA processing and post-transcriptional and translational regulation. In the last decade, the application of modern techniques to identify RNA–protein interactions with individual proteins, RNAs, and the whole transcriptome has led to the discovery of a hidden landscape of these interactions in plants. Global approaches such as RNA interactome capture (RIC) to identify proteins that bind protein-coding transcripts have led to the identification of close to 2000 putative RBPs in plants. Interestingly, many of these were found to be metabolic enzymes with no known canonical RNA-binding domains. Here, we review the methods used to analyze RNA–protein interactions in plants thus far and highlight the understanding of plant RNA–protein interactions these techniques have provided us. We also review some recent protein-centric, RNA-centric, and global approaches developed with non-plant systems and discuss their potential application to plants. We also provide an overview of results from classical studies of RNA–protein interaction in plants and discuss the significance of the increasingly evident ubiquity of RNA–protein interactions for the study of gene regulation and RNA biology in plants. 

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5. miR778 mediates gene expression, histone modification, and DNA methylation during cyst nematode parasitism

   https://doi.org/10.1093/plphys/kiac228  

   Bennett, Morgan ; Piya, Sarbottam ; Baum, Thomas J. ; Hewezi, Tarek ( May 2022 , Plant Physiology)

   Despite the known critical regulatory functions of microRNAs, histone modifications, and DNA methylation in reprograming plant epigenomes in response to pathogen infection, the molecular mechanisms underlying the tight coordination of these components remain poorly understood. Here, we show how Arabidopsis (Arabidopsis thaliana) miR778 coordinately modulates the root transcriptome, histone methylation, and DNA methylation via post-transcriptional regulation of the H3K9 methyltransferases SU(var)3-9 homolog 5 (SUVH5) and SUVH6 upon infection by the beet cyst nematode Heterodera schachtii. miR778 post-transcriptionally silences SUVH5 and SUVH6 upon nematode infection. Manipulation of the expression of miR778 and its two target genes significantly altered plant susceptibility to H. schachtii. RNA-seq analysis revealed a key role of SUVH5 and SUVH6 in reprograming the transcriptome of Arabidopsis roots upon H. schachtii infection. In addition, chromatin immunoprecipitation (ChIP)-seq analysis established SUVH5 and SUVH6 as the main enzymes mediating H3K9me2 deposition in Arabidopsis roots in response to nematode infection. ChIP-seq analysis also showed that these methyltransferases possess distinct DNA binding preferences in that they are targeting transposable elements under noninfected conditions and protein-coding genes in infected plants. Further analyses indicated that H3K9me2 deposition directed by SUVH5 and SUVH6 contributes to gene expression changes both in roots and in nematode feeding sites and preferentially associates with CG DNA methylation. Together, our results uncovered multi-layered epigenetic regulatory mechanisms coordinated by miR778 during Arabidopsis–H. schachtii interactions.

    

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