Enhancer RNA (eRNA) is a type of noncoding RNA transcribed from the enhancer. Although critical roles of eRNA in gene transcription control have been increasingly realized, the systemic landscape and potential function of eRNAs in cancer remains largely unexplored. Here, we report the integration of multi-omics and pharmacogenomics data across large-scale patient samples and cancer cell lines. We observe a cancer-/lineage-specificity of eRNAs, which may be largely driven by tissue-specific TFs. eRNAs are involved in multiple cancer signaling pathways through putatively regulating their target genes, including clinically actionable genes and immune checkpoints. They may also affect drug response by within-pathway or cross-pathway means. We characterize the oncogenic potential and therapeutic liability of one eRNA, NET1e, supporting the clinical feasibility of eRNA-targeted therapy. We identify a panel of clinically relevant eRNAs and developed a user-friendly data portal. Our study reveals the transcriptional landscape and clinical utility of eRNAs in cancer.
Heat shock factor (Hsf1) regulates the expression of molecular chaperones to maintain protein homeostasis. Despite its central role in stress resistance, disease and aging, the mechanisms that control Hsf1 activity remain unresolved. Here we show that in budding yeast, Hsf1 basally associates with the chaperone Hsp70 and this association is transiently disrupted by heat shock, providing the first evidence that a chaperone repressor directly regulates Hsf1 activity. We develop and experimentally validate a mathematical model of Hsf1 activation by heat shock in which unfolded proteins compete with Hsf1 for binding to Hsp70. Surprisingly, we find that Hsf1 phosphorylation, previously thought to be required for activation, in fact only positively tunes Hsf1 and does so without affecting Hsp70 binding. Our work reveals two uncoupled forms of regulation - an ON/OFF chaperone switch and a tunable phosphorylation gain - that allow Hsf1 to flexibly integrate signals from the proteostasis network and cell signaling pathways.DOI: http://dx.doi.org/10.7554/eLife.18638.001
Models for regulation of the eukaryotic heat shock response typically invoke a negative feedback loop consisting of the transcriptional activator Hsf1 and a molecular chaperone. Previously we identified Hsp70 as the chaperone responsible for Hsf1 repression and constructed a mathematical model that recapitulated the yeast heat shock response (Zheng et al., 2016). The model was based on two assumptions: dissociation of Hsp70 activates Hsf1, and transcriptional induction of Hsp70 deactivates Hsf1. Here we validate these assumptions. First, we severed the feedback loop by uncoupling Hsp70 expression from Hsf1 regulation. As predicted by the model, Hsf1 was unable to efficiently deactivate in the absence of Hsp70 transcriptional induction. Next, we mapped a discrete Hsp70 binding site on Hsf1 to a C-terminal segment known as conserved element 2 (CE2). In vitro, CE2 binds to Hsp70 with low affinity (9 µM), in agreement with model requirements. In cells, removal of CE2 resulted in increased basal Hsf1 activity and delayed deactivation during heat shock, while tandem repeats of CE2 sped up Hsf1 deactivation. Finally, we uncovered a role for the N-terminal domain of Hsf1 in negatively regulating DNA binding. These results reveal the quantitative control mechanisms underlying the heat shock response.
The coordination chemistry of hydrated and solvated vanadium(III), oxovanadium(IV), and dioxovanadium(V) ions in the oxygen donor solvents water, dimethylsulfoxide (dmso) and N,N′-dimethylpropyleneurea (dmpu) has been studied in solution by EXAFS and large angle X-ray scattering (LAXS) and in solid state by single crystal X-ray diffraction and EXAFS. The hydrated vanadium(III) ion has a regular octahedral configuration with a mean V-O bond distance of 1.99 Å. In the hydrated and dimethylsulfoxide solvated oxovanadium(IV) ions vanadium binds strongly to an oxo group at ca. 1.6 Å. The solvent molecule trans to the oxo group is very weakly bound, at ca. 2.2 Å, while the remaining four solvent molecules, with a mean V-O bond distance of 2.0 Å, form a plane slightly below the vanadium atom; the mean O=V-Operp bond angle is ca. 98°. In the dmpu solvated oxovanadium(IV) ion, the space demanding properties of the dmpu molecule leaving no solvent molecule in the trans position to the oxo group which reduces the coordination number to 5. The O=V-O bond angle is consequently much larger, 106°, and the mean V=O and V-O bond distances decrease to 1.58 and 1.97 Å, respectively. The hydrated and dimethylsulfoxide solvated dioxovanadium(V) ions display a very distorted octahedral configuration with the oxo groups in cis position with mean V=O bond distances of 1.6 Å and a O=V=O bond angle of ca. 105°. The solvent molecules trans to the oxo groups are weakly bound, at ca. 2.2 Å, while the remaining two have bond distances of 2.02 Å. The experimental studies of the coordination chemistry of hydrated and solvated vanadium(III,IV,V) ions are complemented by summarizing previously reported crystal structures to yield a comprehensive description of the coordination chemistry of vanadium with oxygen donor ligands.
Protein AMPylation is a conserved posttranslational modification with emerging roles in endoplasmic reticulum homeostasis. However, the range of substrates and cell biological consequences of AMPylation remain poorly defined. We expressed human and Caenorhabditis elegans AMPylation enzymes-huntingtin yeast-interacting protein E (HYPE) and filamentation-induced by cyclic AMP (FIC)-1, respectively-in Saccharomyces cerevisiae, a eukaryote that lacks endogenous protein AMPylation. Expression of HYPE and FIC-1 in yeast induced a strong cytoplasmic Hsf1-mediated heat shock response, accompanied by attenuation of protein translation, massive protein aggregation, growth arrest, and lethality. Overexpression of Ssa2, a cytosolic heat shock protein (Hsp)70, was sufficient to partially rescue growth. In human cell lines, overexpression of active HYPE similarly induced protein aggregation and the HSF1-dependent heat shock response. Excessive AMPylation also abolished HSP70-dependent influenza virus replication. Our findings suggest a mode of Hsp70 inactivation by AMPylation and point toward a role for protein AMPylation in the regulation of cellular protein homeostasis beyond the endoplasmic reticulum.AMPylation | FIC protein | chaperones | proteostasis | HSP70
The hydration of formamide (F), N-methylformamide (NMF), N,N-dimethylformamide (DMF), acetamide (A), N-methylacetamide (NMA), and N,N-dimethylacetamide (DMA) has been studied in aqueous solutions by means of FTIR spectra of HDO isotopically diluted in H2O. The difference spectra procedure has been applied to remove the contribution of bulk water and thus to separate the spectra of solute-affected HDO. To facilitate the interpretation of obtained spectral results, DFT calculations of aqueous amide clusters were performed. Molecular dynamics (MD) simulation for the cis and trans forms of NMA was also carried out for the SPC model of water. Infrared spectra reveal that only two to three water molecules from the surrounding of the amides are statistically affected, from among ca. 30 molecules present in the first hydration sphere. The structural-energetic characteristic of these solute-affected water molecules differs only slightly from that in the bulk and corresponds to the clathrate-like hydrogen-bonded cage typical for hydrophobic hydration, with the possible exception of F. MD simulations confirm such organization of water molecules in the first hydration sphere of NMA and indicate a practical lack of orientation and energetic effects beyond this sphere. The geometry of hydrogen-bonded water molecules in the first hydration sphere is very similar to that in the bulk phase, but MD simulations have affirmed subtle differences recognized by the spectral method and enabled their understanding. The spectral data and simulations results are highly compatible. In the case of F, NMF, and A, there is a visible spectral effect of water interactions with N-H groups, which have destabilizing influence on the amides hydration shell. There is no spectral sign of such interaction for NMA as the solute. The energetic stability of water H-bonds in the amide hydration sphere and in the bulk fulfills the order: NMA > DMA > A > NMF > bulk > DMF > F. Microscopic parameters of water organization around the amides obtained from the spectra, which have been used in the hydration model based on volumetric data, confirm the more hydrophobic character of the first three amides in this sequence. The increased stability of the hydration sphere of NMA relative to DMA and of NMF relative to DMF seems to have its origin in different geometries, and so the stability, of water cages containing the amides.
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