Chemical modifications of histones and DNA, such as histone methylation, histone acetylation, and DNA methylation, play critical roles in epigenetic gene regulation. Many of the enzymes that add or ...remove such chemical modifications are known, or might be suspected, to be sensitive to changes in intracellular metabolism. This knowledge provides a conceptual foundation for understanding how mutations in the metabolic enzymes SDH, FH, and IDH can result in cancer and, more broadly, for how alterations in metabolism and nutrition might contribute to disease. Here, we review literature pertinent to hypothetical connections between metabolic and epigenetic states in eukaryotic cells.
In this review, we describe speculative ideas and early stage research concerning the flow of genetic information from the nuclear residence of genes to the disparate, cytoplasmic sites of protein ...synthesis. We propose that this process of information transfer is meticulously guided by transient structures formed from protein segments of low sequence complexity intrinsic disorder. These low complexity domains are ubiquitously associated with regulatory proteins that control gene expression and RNA biogenesis, but they are also found in the central channel of nuclear pores, the nexus points of intermediate filament assembly, and the locations of action of other well-studied cellular proteins and pathways. Upon being organized into localized cellular positions via mechanisms utilizing properly folded protein domains, thereby facilitating elevated local concentration, certain low complexity domains adopt cross-β interactions that are both structurally specific and labile to disassembly. These weakly tethered assemblies, we propose, are built to relay the passage of genetic information from one site to another within a cell, ensuring that the process is of extreme fidelity.
Cellular granules lacking boundary membranes harbor RNAs and their associated proteins and play diverse roles controlling the timing and location of protein synthesis. Formation of such granules was ...emulated by treatment of mouse brain extracts and human cell lysates with a biotinylated isoxazole (b-isox) chemical. Deep sequencing of the associated RNAs revealed an enrichment for mRNAs known to be recruited to neuronal granules used for dendritic transport and localized translation at synapses. Precipitated mRNAs contain extended 3′ UTR sequences and an enrichment in binding sites for known granule-associated proteins. Hydrogels composed of the low complexity (LC) sequence domain of FUS recruited and retained the same mRNAs as were selectively precipitated by the b-isox chemical. Phosphorylation of the LC domain of FUS prevented hydrogel retention, offering a conceptual means of dynamic, signal-dependent control of RNA granule assembly.
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► A crystallized small molecule recruits mRNAs into granule-like aggregates ► The granule-like aggregates contain known granule RNAs ► Aggregates from brain lysates are enriched in mRNAs encoding synaptic proteins ► 3′ UTR lengths of recruited RNAs are significantly longer than average
Aggregation of RNA-binding proteins, including FUS, from mammalian cells within hydrogels retains mRNAs previously associated with RNA granules. Phosphorylation of FUS influences mRNA retention suggesting a mechanism for regulating granule assembly and composition.
The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby ...assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD.
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•Low-complexity domains of FET proteins form reversible polymeric fibers•Polymeric fiber formation correlates with transcriptional activation capacity•The C-terminal domain (CTD) of RNA polymerase II binds to polymeric fibers•Prebound CTD is released from polymeric fibers by phosphorylation
Polymeric fibers formed by low-complexity polypeptide domains can fuse to DNA-binding domains and potentiate transcription; biochemical evidence indicates that this is due to the ability of the fibers to bind to the CTD of RNA polymerase and then release it upon CTD phosphorylation.
Many DNA and RNA regulatory proteins contain polypeptide domains that are unstructured when analyzed in cell lysates. These domains are typified by an over-representation of a limited number of amino ...acids and have been termed prion-like, intrinsically disordered or low-complexity (LC) domains. When incubated at high concentration, certain of these LC domains polymerize into labile, amyloid-like fibers. Here, we report methods allowing the generation of a molecular footprint of the polymeric state of the LC domain of hnRNPA2. By deploying this footprinting technique to probe the structure of the native hnRNPA2 protein present in isolated nuclei, we offer evidence that its LC domain exists in a similar conformation as that described for recombinant polymers of the protein. These observations favor biologic utility to the polymerization of LC domains in the pathway of information transfer from gene to message to protein.
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•A footprinting method was used to probe cross-β structure of LC domain polymers•Similar footprints were obtained from hydrogels, liquid-like droplets, and nuclei•Mutations impeding hydrogel binding map to the core of the LC domain footprint•Hydrogel and liquid-like droplet formation is driven by cross-β polymerization
A chemical footprinting method reveals that polymers of low-complexity domains exhibit similar cross-β structure in hydrogels, liquid-like droplets, and nuclei of mammalian cells, suggesting a common underlying structural basis.
Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral ...sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.
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•The toxic, C9orf72-encoded PRn poly-dipeptide binds many intracellular targets•The toxic PRn poly-dipeptide binds polymeric forms of low complexity sequences•Aliphatic alcohols melt both low complexity polymers and intracellular puncta•RNA granules may bind intermediate filaments for proper localization within cells
Toxic proline-arginine poly-dipeptides, which are encoded by C9orf72 hexanucleotide expansions frequently found in amyotrophic lateral sclerosis, bind to polymers of low complexity domain proteins.
Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of ...unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.
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► A biotinylated small molecule precipitates RNA granule proteins from cell lysates ► Low complexity sequences in these proteins form hydrogels ► Amyloid-like fibers within the gels can trap LCS domains from other proteins ► The cell-free in vitro reactions model RNA granule architecture and formation
RNA-binding proteins with regions of low complexity sequence can form hydrogels in vitro comprised of amyloid-like fibers either via nucleation by a small molecule or by self-organization. Unlike pathologic amyloids, the fibers are dynamic and can incorporate low complexity domains from different proteins, suggesting a basis for assembly of RNA granules within cells.
The P7C3 class of aminopropyl carbazole chemicals fosters the survival of neurons in a variety of rodent models of neurodegeneration or nerve cell injury. To uncover its mechanism of action, an ...active derivative of P7C3 was modified to contain both a benzophenone for photocrosslinking and an alkyne for CLICK chemistry. This derivative was found to bind nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme involved in the conversion of nicotinamide into nicotinamide adenine dinucleotide (NAD). Administration of active P7C3 chemicals to cells treated with doxorubicin, which induces NAD depletion, led to a rebound in intracellular levels of NAD and concomitant protection from doxorubicin-mediated toxicity. Active P7C3 variants likewise enhanced the activity of the purified NAMPT enzyme, providing further evidence that they act by increasing NAD levels through its NAMPT-mediated salvage.
Yeast ataxin-2, also known as Pbp1, senses the activity state of mitochondria in order to regulate TORC1. A domain of Pbp1 required to adapt cells to mitochondrial activity is of low sequence ...complexity. The low-complexity (LC) domain of Pbp1 forms labile, cross-β polymers that facilitate phase transition of the protein into liquid-like or gel-like states. Phase transition for other LC domains is reliant upon widely distributed aromatic amino acids. In place of tyrosine or phenylalanine residues prototypically used for phase separation, Pbp1 contains 24 similarly disposed methionine residues. Here, we show that the Pbp1 methionine residues are sensitive to hydrogen peroxide (H2O2)-mediated oxidation in vitro and in living cells. Methionine oxidation melts Pbp1 liquid-like droplets in a manner reversed by methionine sulfoxide reductase enzymes. These observations explain how reversible formation of labile polymers by the Pbp1 LC domain enables the protein to function as a sensor of cellular redox state.
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•Phase separation of the yeast ataxin-2 methionine-rich LC domain is sensitive to H2O2•The dissolved droplets are specifically revived by methionine sulfoxide reductases•Methionine to tyrosine variants confer resistance to H2O2in vitro and in cells•Yeast ataxin-2 senses cellular redox state to modulate TORC1 activity
Many proteins that are able to undergo phase transitions contain repeats rich in hydrophobic residues. By contrast, yeast ataxin-2 condensates rely on an abundance of methionines, providing a means for redox-sensitive regulation of its material properties.
Low complexity (LC) head domains 92 and 108 residues in length are, respectively, required for assembly of neurofilament light (NFL) and desmin intermediate filaments (IFs). As studied in isolation, ...these IF head domains interconvert between states of conformational disorder and labile, β-strand-enriched polymers. Solid-state NMR (ss-NMR) spectroscopic studies of NFL and desmin head domain polymers reveal spectral patterns consistent with structural order. A combination of intein chemistry and segmental isotope labeling allowed preparation of fully assembled NFL and desmin IFs that could also be studied by ss-NMR. Assembled IFs revealed spectra overlapping with those observed for β-strand-enriched polymers formed from the isolated NFL and desmin head domains. Phosphorylation and disease-causing mutations reciprocally alter NFL and desmin head domain self-association yet commonly impede IF assembly. These observations show how facultative structural assembly of LC domains via labile, β-strand-enriched self-interactions may broadly influence cell morphology.
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