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Multimodal multiscale analysis reveals flexible DNA contacts of bacterial nucleoprotein filaments

ABSTRACT: Gene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the overall structures, arrangements of DNA-binding domains (DBDs), positions of DBD–DNA contacts, and determinants of genomic distribution for these linear and bridged filaments are uncertain. To connect H-NS structures that silence genes in vivo with features elucidated in vitro, we developed a multimodal multiscale analysis that combines a new method for chromatin reconstitution and mapping (Nitro-seq), ChIP-seq, tethered-nuclease mapping of DBD–DNA contacts (TEN-map), ·OH footprinting, molecular dynamics, and bioinformatics. We find that DNA sequence principally governs H-NS-filament location with indistinguishable sequence specificity in bridged or linear forms with or without the H-NS modifiers StpA and Hha and that DBD–DNA contacts vary in orientation and position with ~10-bp average spacing. Our results support a hemi-sequestration model of linear-to-bridged filament switching.

ORGANISM(S): Escherichia coli

PROVIDER: GSE157512 | GEO | 2023/09/01

REPOSITORIES: GEO

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Histidine-dependent protein methylation is required for compartmentalization of CTP synthase

Project description:CTP synthase (CTPS) forms compartmentalized filaments in response to substrate availability and environmental nutrient status. However, the physiological role of filaments and mechanisms for filament assembly are not well understood. Here, we provide evidence that CTPS forms filaments in response to histidine influx during glutamine starvation. CTPS protein levels remained stable in the presence of histidine during nutrient deprivation, followed by rapid cell growth following nutrient replenishment. Tetramer conformation-based filament formation restricted CTPS enzyme activity duringnutrient deprivation. Furthermore, we demonstrated that filament formation controlled at two levels. Starvation stress/glutamine deficiency activates the GCN2/ATF4/MTHFD2 axis to accelerate the folate cycle in mitochondria for energy homeostasis. In addition, histidine promotes re-methylation of homocysteine by donating one-carbon to the cytosolic folate cycle. CTPS filament formation induced by histidine-mediated methylation maybe a strategy usedby cancer cells to maintain homeostasis and ensure a growth advantage in adverse environments.

2018-08-29 | GSE105251 | GEO

RfaH counter-silences inhibition of transcript elongation by H-NS–StpA H-NS nucleoprotein filaments in pathogenic E. coli

Project description:Expression of virulence genes in pathogenic E. coli is controlled in part by the transcription silencer H-NS and its paralogs (e.g., StpA), which sequester DNA in multi-kb nucleoprotein filaments to inhibit transcription initiation, elongation, or both. Some activators counter-silence initiation by displacing H-NS from promoters. How H-NS inhibition of elongation is overcome is not understood. In uropathogenic E. coli (UPEC), elongation regulator RfaH aids expression of some H-NS-silenced pathogenicity operons (e.g., hlyCABD encoding hemolysin). RfaH associates with elongation complexes (ECs) via direct contacts to a transiently exposed, nontemplate DNA-strand sequence called ops (operon polarity suppressor). RfaH–ops interactions establish long-lived RfaH–EC contacts that allow RfaH to recruit ribosomes to the nascent mRNA and to suppress transcriptional pausing and termination. Using ChIP-seq, we mapped the genome-scale distributions of RfaH, H-NS, StpA, RNA polymerase (RNAP), and σ70 in the UPEC strain CFT073. We identify 8 RfaH-activated operons, all of which were bound by H-NS and StpA. Four are new additions to the RfaH regulon. Deletion of RfaH caused premature termination whereas deletion of H-NS and StpA allowed elongation without RfaH. Thus, RfaH is an elongation counter-silencer of H-NS. Consistent with elongation counter-silencing, deletion of StpA alone decreased the effect of RfaH. StpA increases DNA bridging, which inhibits transcript elongation via topological constraints on RNAP. Residual RfaH effect when both H-NS and StpA were deleted was attributable to targeting of RfaH-regulated operons by a minor H-NS paralog, Hfp. These operons have evolved higher levels of H-NS–binding features, explaining minor-paralog targeting.

2022-10-06 | GSE212064 | GEO

TAF15 amyloid filaments in neurodegenerative disease

Project description:Frontotemporal dementia (FTD) is the most common form of dementia after Alzheimer's disease and results from frontotemporal lobar degeneration (FTLD). The pathological hallmarks of FTLD are neuronal inclusions of specific, abnormally assembled proteins1. In the majority of cases, the inclusions contain amyloid filament assemblies of TAR DNA-binding protein 43 (TDP-43) or tau, with distinct filament structures characterising different FTLD subtypes2,3. The presence, identities and structures of amyloid filaments in the remaining ~10% of FTLD cases are unknown, but are widely believed to be composed of the protein fused in sarcoma (FUS). As such, these cases are commonly referred to as FTLD-FUS. Here, we used cryogenic electron microscopy (cryo-EM) to determine the structures of amyloid filaments extracted from the prefrontal and temporal cortices of four individuals with FTLD-FUS. Unexpectedly, we found abundant amyloid filaments of the FUS homologue TATA-binding protein-associated factor 15 (TAF15), rather than of FUS itself. The filament fold is formed from residues 7 to 99 in the low-complexity domain (LCD) of TAF15 and was identical between individuals. The formation of TAF15 amyloid filaments with a characteristic fold in FTLD establishes TAF15 proteinopathy in neurodegenerative disease. Furthermore, we found TAF15 filaments with the same fold in the motor cortex and brainstem of two of the individuals, who also showed changes associated with amyotrophic lateral sclerosis (ALS), suggesting that TAF15 proteinopathy may underlie a disease spectrum of FTLD and ALS. The structure of TAF15 amyloid filaments provides a basis for the development of model systems of neurodegenerative disease, as well as for the design of diagnostic and therapeutic tools targeting TAF15 proteinopathy.

2023-12-07 | PXD044821 | Pride

The Trichomonas PACK proteome - The Cytoskeleton of Parabasalian Parasites Comprises Proteins that Share Properties Common to Intermediate Filament Proteins

Project description:Certain protist lineages bear cytoskeletal structures that are germane to them and define their individual group. Trichomonadida are excavate parasites united by a unique cytoskeletal framework, which includes tubulin-based structures such as the pelta and axostyle, but also other filaments such as the striated costa whose protein composition remains unknown. We determined the proteome of the detergent-resistant cytoskeleton of Tetratrichomonas gallinarum. 203 proteins with homology to Trichomonas vaginalis were identified, which contain significantly more long coiled-coil regions than control protein sets. Five candidates were shown to associate with previously described cytoskeletal structures including the costa and the expression of a single T. vaginalis protein in T. gallinarum induced the formation of accumulated, striated filaments. Our data suggests that filament-forming proteins of protists other than actin and tubulin share common structural properties with metazoan intermediate filament proteins, while not being homologous. These filament-forming proteins might have evolved many times independently in eukaryotes, or simultaneously in a common ancestor but with different evolutionary trajectories downstream in different phyla. The broad variety of filament-forming proteins uncovered, and with no homologs outside of the Trichomonadida, once more highlights the diverse nature of eukaryotic proteins with the ability to form unique cytoskeletal filaments.

2016-10-10 | PXD003212 | Pride

A prophage encoded actin-like protein mediates viral DNA segregation/replication in bacteria [Set2]

Project description:Active segregation of DNA in bacteria is catalyzed by cytomotive structures. Mediators of subcellular plasmid positioning are Walker-type ATPases (ParA), actin-like proteins, or tubulin homologs. These motor proteins are coupled to the DNA via adaptor proteins that recognize specific DNA motifs. Here, we describe that a temperate phage, CGP3, integrated into the genome of Corynebacterium glutamicum ATCC 13032 encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms dynamic filamentous structures upon phage induction. The co-transcribed AlpA protein binds to a specific region of the phage DNA, possibly functioning as an adaptor protein that connects circular phage DNA to the tips of the AlpC filaments. The AlpC filaments transport phage DNA to the cell membrane of the host cell. Furthermore, both AlpA and AlpC are required for efficient phage replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites.

2015-05-01 | GSE45906 | GEO

A prophage encoded actin-like protein mediates viral DNA segregation/replication in bacteria [Set1]

2015-05-01 | GSE45905 | GEO

Transcriptomes of the developing larval ovaries of Drosophila melanogaster during terminal filament formation

Project description:Insect ovaries are made up of tubular egg producing subunits called ovarioles, whose number largely determines the number of eggs that can be potentially laid. Ovariole number is directly determined by the number of cellular structures called terminal filaments, which are stacks of cells that assemble in the larval ovary. Elucidating the developmental and regulatory mechanisms of terminal filament formation is thus key to understanding the regulation of insect reproduction through ovariole number regulation. We systematically measured mRNA expression of all cells in the Drosophila larval ovary at the beginning, middle and end of terminal filament formation using RNA-seq. We also separated somatic and germ line cells during these stages and assessed their tissue-specific gene expression during larval ovary development.

2021-10-18 | GSE172015 | GEO

Posttranslational modifications mediate the structural diversity of tauopathy strains

Project description:Tau aggregation is the defining pathological hallmark of tauopathies, but structural insights are revealing key differences between filaments across this class of disorders. Using cryo-electron microscopy, we identify a 107-residue fragment, K274-E380, which forms the insoluble core of tau filaments in the tauopathy corticobasal degeneration (CBD). This tau conformer is distinct from those identified in Alzheimer’s Disease (AD), Pick’s Disease, and Chronic Traumatic Encephalopathy, pointing towards a “one strain per disease” paradigm. Each disease-specific conformer can pack into multiple fibril subtypes and, despite similarities in secondary structure elements between tau filaments from CBD and AD, mass spectrometry revealed key differences in posttranslational modifications (PTMs). Given the abundance of lysine residues in the insoluble tau filament core in both CBD and AD, ubiquitination and acetylation are identified as key PTMs that compete to modify specific residues, which may ultimately determine filament morphology.

2020-02-14 | PXD016862 | Pride

Global H-NS counter-silencing by LuxR activates quorum sensing gene expression [RNA-seq]

Project description:Bacteria coordinate cellular behaviors using a cell-cell communication system termed quorum sensing. In Vibrio harveyi, the master quorum sensing transcriptional factor LuxR directly regulates >100 genes in response to changes in population density. Here, we show that LuxR derepresses quorum sensing loci by competing with H-NS, a global transcriptional repressor that oligomerizes on DNA to form filaments and bridges. We first identified H-NS as a repressor of bioluminescence gene expression, for which LuxR is a required activator. In an hns deletion strain, LuxR is no longer necessary for transcription activation of the bioluminescence genes, suggesting that the primary role of LuxR is to displace H-NS to derepress gene expression. Using RNA-seq and ChIP-seq, we determined that H-NS and LuxR co-regulate and co-occupy 28 promoters driving expression of 63 genes across the genome. ChIP-PCR assays show that as autoinducer concentration increases, LuxR protein accumulates at co-occupied promoters while H-NS protein disperses. LuxR is sufficient to evict H-NS from promoter DNA in vitro, which is dependent on LuxR DNA binding activity. From these findings, we propose a model in which LuxR serves as a counter-silencer at H-NS-repressed quorum sensing loci by disrupting H-NS nucleoprotein complexes that block transcription.

2019-11-01 | GSE136191 | GEO

Global H-NS counter-silencing by LuxR activates quorum sensing gene expression

2019-11-01 | GSE136050 | GEO

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