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At least 19 recordsLinked to original sources

A First-in-Class Chemical-Induced Proximity System Achieves Dose-Dependent Control of Tumor Protein P53 Gene Activation in Preclinical Models of Gastric Cancer.

The tumor protein P53 (TP53) gene has long been studied in cancer research with genomic and epigenetic aberrations playing a driving role in cancer pathology, yet even after decades of work, only a few methods have been developed to specifically target TP53 therapeutically. Some cancers are driven by loss-of-function TP53 mutations, while others have wild-type TP53 in a transcriptionally repressed state; the latter is exploitable by advances in epigenome editing. In our previous work, we demonstrated that deactivated CRISPR/Cas9 systems (dCas9), combined with an FK-506-binding protein (FKBP) recruitment protein tag and chemical epigenetic modifier (CEM) small molecules, can elicit gene-specific changes in expression in a dose-dependent manner. Here, we describe the development, application, and characterization of the dCas9-FKBP-CEM technology to increase TP53 expression. We demonstrate that catalyzing increased TP53 expression via dCas9-FKBP-CEM87 induced apoptosis, cell cycle arrest, and tumor growth inhibition in a dose-dependent manner in preclinical models of gastric cancer.

CRISPR

A bivalent molecular glue linking lysine acetyltransferases to oncogene-induced cell death.

Developing cancer therapies that induce specific death of malignant cells is critical for preventing relapse. Highly effective strategies, such as immunotherapy, exemplify this principle. Here, we provide the mechanistic basis for a small-molecule approach that leverages chemically induced proximity (CIP) to kill diffuse large B cell lymphoma, the most common non-Hodgkin lymphoma. We developed lysine acetyltransferase (KAT)-based TCIPs (transcriptional/epigenetic chemical inducers of proximity), or KAT-TCIPs, which redirect p300/CREB-binding protein (CBP) to activate cell-death networks repressed by the oncogenic driver BCL6. Our lead KAT-TCIP reprograms the epigenome to initiate apoptosis. The crystal structure of the chemically induced p300-BCL6 complex reveals how chance protein-protein interactions may be exploited to confer the potency and selectivity of KAT-TCIPs. Thus, oncogenic drivers can be co-opted to activate robust cell death. Consistent with their gain-of-function mechanism, TCIPs recruiting different transcriptional activators-p300, BRD4, or CDK9-produce distinct genomic responses, suggesting specialized therapeutic uses.

Humans

Rapid and reversible epigenome editing by endogenous chromatin regulators.

Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.

CRISPR-Cas Systems

Spatial Mapping and Interactome Profiling of m6A-Modified R-Loops via Chemically Inducible Split-APEX2 Proximity Labeling.

m6A-Modified R-loops (m6A-R-loops) play crucial roles in epigenetic regulation and genome stability, yet resolving their spatial distribution and protein interactomes in live cells remains challenging. To address this, we developed m6A-R-loop proximity labeling (m6A-RLPL), a chemically inducible split-APEX2 proximity labeling technology integrating dual-target recognition using the RNA-DNA hybrid binding domain of RNase H1 for R-loop targeting and m6A reader protein's YTH domain for m6A recognition, coupled with an abscisic acid (ABA)-inducible dimerization system for signal amplification. This technology revealed host m6A-R-loops enriched with nucleoli under normal conditions. When applied to herpes simplex virus (HSV) infection, it further demonstrated viral m6A-R-loops undergoing dramatic accumulation within phase-separated granules in replication compartments during late-stage infection. Proximity proteomics identified ZC3H4 and CCDC124 as essential regulators maintaining these structures, which serve as transcription sites for HSV late genes, with disruption selectively impairing viral transcription. m6A-RLPL establishes a generalizable approach for spatially resolved profiling of m6A-R-loop interactomes and organizational dynamics in living systems.

Humans

The occurrence of lipid droplets in the proximal and distal tubules of the rat kidney after folic acid treatment.

Folic acid in high doses gives rise to an accumulation of lipid droplets in the kidney in addition to other changes in the epithelial of both proximal and distal tubules. With the administration of methionine a decrease of lipid droplets and an improvement of the structures of most of the membranes and mitochrondria are observed. These findings have been discussed in regard to the theory of the "chemically induced hyperplasia of the kidneys" related to folic acid.

Animals

Effect of the co-carcinogen benzo[e]pyrene on microsome-mediated chemical mutagenesis in Salmonella typhimurium.

Chemical agents that possess the ability to alter tumorigenicity of carcinogens (administered at subthreshold dose) constitute a major health hazard. We have employed the Ames Salmonella assay to examine the effect of co-carcinogenic benzo[e]pyrene (B[e]P) on microsome-mediated chemical mutagenesis. B[e]P enhanced the mutagenic activity induced by 2-acetylaminofluorene (2-AAF) and benzo[a]pyrene (B[a]P) in strains TA1538 and TA98. Enhancement was also noted with N-hydroxy-2-AAF (the proximal metabolite of 2-AAF) but not with an ultimate carcinogenic form (N-acetoxy-2-AAF). These results suggest the use of this approach to detect chemical agents that possess the ability to alter the activity of mutagenic or carcinogenic chemicals.

2-Acetylaminofluorene

Biological effects of alkali-treated protein and lysinoalanine: an overview.

The renal alterations induced by alkali-treated protein and lysinoalanine are reviewed and their biological implications discussed. Alkali-treated proteins and lysinoalanine, an unusual amino acid formed in proteins during alkali treatment, have been shown to produce a renal alteration characterized by nuclear and cytoplasmic enlargement, with alterations in DNA synthesis, mitotsis and nucleoprotein. These changes are localized in the straight portion of the proximal renal tubule and have been observed in rats but not in several other species. The nephrotoxic effect of synthetic lysinoalanine has been consistently demonstrated, but the ability of alkali-treated protein to induce renal alterations is apparently modified by factors other than lysinoalanine content. Factors which may influence the development of the kidney lesions in animals fed alkali-treatment protein are discussed, including nutritional factors, the chemical form of lysinoalanine in the protein, species differences, and metabolic fate. Other clinical and experimental conditions that result in similar renal alterations are presented for comparison with the lysinoalanine induced lesion, and possible functional consequences are considred.

Alanine

Metal binding sites of a gamma-carboxyglutamic acid-rich fragment of bovine prothrombin.

The metal binding sites of a gamma-carboxyglutamic acid-rich fragment derived from bovine prothrombin were examined using paramagnetic lanthanide ions to evaluate the role of gamma-carboxyglutamic acid resideus in metal binding. A gamma-carboxyglutamic acid-rich peptide, fragment 12-44, was isolated from a tryptic digest of prothrombin. Using 153Gd(III), fragment 12-44 was found to contain one high affinity metal binding site (KD = 0.55 microM) and four to six lower affinity metal binding sites (KD approximately 4 to 8 microM). The S-carboxymethyl derivative of fragment 12-44, in which the disulfide bond in fragment 12-44 was reduced and alkylated, contained no high affinity metal binding site and four or five lower affinity sites (KD = 8 microM). The effects of paramagnetic lanthanide ions on fragment 12-44 and its S-carboxymethyl derivative were studied by natural abundance 13C NMR spectroscopy. The 13C NMR spectrum of fragment 12-44 was recorded at 67.88 MHz and the resonances were assigned by comparison to the chemical shift of carbon resonances of amino acids and peptides previously studied. The proximity between bound metal ions and carbon atoms in fragment 12-44 was estimated using Gd(III), based upon the strategy that the magnitude of the change in the transverse relaxation rate of resonances of carbon nuclei induced by bound metal ions is related in part to the interatomic distances between bound metal and carbon nuclei. Titration of fragment 12-44 with Gd(III) resulted in the selective broadening of the gamma-carboxyl carbon, C gamma, C beta, and C alpha resonances of gamma-carboxyglutamic acid, and the C epsilon of the arginines. S-Carboxymethyl fragment 12-44, which lacked the high affinity metal binding site, showed markedly decreased perturbation of the C epsilon of the arginine residues upon titration with Gd(III). These studies indicate that gamma-carboxyglutamic acid residues in prothrombin fragment 12-44 participate in metal liganding. A high affinity metal binding site in fragment 12-44 is in close proximity of Arg 16 and Arg 25 and is stabilized by the disulfide bond. On the basis of these data, a model of the metal binding sites is proposed in which the high affinity site is composed of two gamma-carboxyglutamic acid residues which participate in intramolecular metal-dependent bridging of two regions of the polypeptide chain. The lower affinity metal binding sites, formed by single or paired adjacent gamma-carboxyglutamic acid residues, then may participate in intermolecular metal-dependent protein . protein or protein . membrane complex formation.

1-Carboxyglutamic Acid

Sodium- and potassium-activated ATPase. A possible target of aldosterone.

Na-K-ATPase activity was measured with an ultramicromethod in single portions of the proximal and distal convolution and of the thick ascending limb of Henle from adrenalectomized rats and after treatment with 5 mug aldosterone per 100 g body wt. The activity in all tubular structures returned to normal within 1 h after injection. This rapid activation of Na-K-ATPase induced by hormone was completely prevented by actinomycin D and cycloheximide. It appears that this aldosterone effect on Na-K-ATPase requires an intact protein synthetic process.

Adenosine Triphosphatases

Hi-C calibration by chemically induced chromosomal interactions.

The genome-wide chromosome conformation capture method, Hi-C, has greatly advanced our understanding of genome organization. However, its quantitative properties, including sensitivity, bias, and linearity, remain challenging to assess. Measuring these properties in vivo is difficult due to the heterogenous and dynamic nature of chromosomal interactions. Here, using Chemically Induced Chromosomal Interaction (CICI) method, we create stable intra- and inter-chromosomal interactions in G1-phase budding yeast across a broad range of contact frequencies. Hi-C analysis of these engineered cell populations demonstrates that static intra-chromosomal loops do not generate Topologically Associated Domains (TADs) and only promote 3D proximity within 10-60 kb flanking regions. At moderate sequencing depth, Hi-C is sensitive enough to detect interactions occurring in 5-10% of cells. It also shows no inherent bias toward intra- versus inter-chromosomal interactions. Furthermore, we observe a linear relationship between Hi-C signal intensity and contact frequency. These findings illuminate the intrinsic properties of the Hi-C assay and provide a robust framework for its calibration.

Chromosomes, Fungal

How Epitranscriptomic Machinery Senses Environmental Cues.

Environmental fluctuations remodel RNA modification landscapes, yet the routes that connect cue detection to writer-eraser-reader control remain dispersed across disciplines. Here, we consolidate upstream mechanisms capable of driving epitranscriptomic change and organize them by response speed. At the fastest proximal level, catalytic output can be modulated through shifts in substrate and cofactor availability, redox and ionic state, temperature, and direct chemical or metal interference with enzyme active sites, although transcriptome-wide RNA readouts may appear later. Over minutes to hours, cue-responsive signaling can reach the machinery through post-translational modification, partner switching, subcellular trafficking, and stress-induced condensates that may gate access to modified transcripts. Across hours to days, regulator abundance and specificity are reshaped by transcriptional programs, translational control, and protein quality-control pathways, enabling adaptation and, in some contexts, persistence. We propose a kinetics-to-sensors approach for interpreting time-resolved epitranscriptomic datasets and prioritizing perturbations that discriminate among candidate upstream inputs. We also outline conceptual gaps and experimental practices needed to establish causal cue-to-mark chains.

Epitranscriptome

Barrier effects on the kinetics of cohesin-mediated loop extrusion.

Chromosome organization mediated by structural maintenance of chromosome complexes is crucial in many organisms. Cohesin extrudes chromatin into loops that are thought to lengthen until it is obstructed by CTCF proteins. In complex cellular environments, the loop extrusion machinery may encounter other chromatin-binding proteins. How these proteins interfere with the cohesin-meditated extrusion process is largely unexplored, but recent experiments have shown that some proteins serve as physical barriers that block cohesin translocation. Other proteins containing a cohesin-interaction motif serve as chemical barriers to induce cohesin pausing through interactions with it. Here, we develop an analytically solvable approach for the loop extrusion model incorporating barriers to investigate the effect of the barrier on the passive extrusion process. To further quantify the impact of barriers, we calculate the mean looping time it takes for cohesin to translocate to form a stable loop before dissociation. Our finding reveals that the physical barrier can accelerate the loop formation, and the degree of acceleration is closely related to the impedance strength of the physical barrier. In particular, the synergy of the cohesin loading site and the physical barrier site accelerates loop formation more significantly. The proximity of the cohesin loading site to the barrier site facilitates the rapid formation of stable loops in long genomes, which implies loop extrusion and chromatin-binding proteins might shape functional genomic organization. Conversely, chemical barriers consistently impede loop formation, with increasing impedance strength of the chemical barrier leading to longer loop formation time. Our study contributes to a more comprehensive understanding of the complexity of the loop extrusion process, providing a new perspective on the potential mechanisms of gene regulation.

Cohesins

Studies on the kinetics of glycosidases from chemically-induced rat colonic tumours and normal rat colon.

K-m values of beta-N-acetylglucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase EC 3.2.1.30), beta-N-acetylgalactosaminidase (EC 3.2.1.53), beta-galactosidase (beta-D-galactoside galactohydrolase EC 3.2.1.23) and alpha-L-fucosidase (alpha-L-fucoside fucohydrolase EC 3.2.1.51) of distal colonic tumours, induced in rats by 1,2-dimethylhydrazine, were found to be significantly different compared with the values for the enzymes of the colonic mucosa of the control and tumour-bearing animals and of the proximal colonic tumours. The inhibition kinetics data also showed a significant difference between the enzymes of the distal colon tumours and of other experimental tissues. The data on the effect of pH on enzyme kinetics (pK values) showed no significant difference in the catalytic groups of the active centres of enzymes from tumours and from the control colonic mucosa. Tumour beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase compared with the enzymes from other experimental tissues were found to be different in their thermal inactivation kinetics. K-m values of 14 days old foetal intestinal beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase were significantly different from the values obtained for the adult mucosal enzymes but were similar to those of the distal colonic tumour enzymes.

Acetylgalactosamine

Liver and kidney damage induced by N-hydroxyparacetamol.

1. Liver and kidney glutathione are depleted in rats and mice following administration of N-hydroxyparacetamol. 2. Centrilobular hepatic necrosis and necrosis of renal proximal convoluted tubules were also found, the liver lesion predominantly in mice and the renal lesion predominantly in rats. Glutathione depletion was not responsible for this species difference. 3. These results indicate that N-hydroxyparacetamol is the metabolic precursor of the reactive toxic intermediate of paracetamol. They are also relevant to the pathogenesis of the renal damage associated with long term abuse of phenacetin containing compound analgesics.

Acetaminophen

A double-blind study on the effect of purified cellulose dietary fiber on 1,2-dimethylhydrazine-induced rat colonic neoplasia.

The incidence, distribution, size, and histopathology of grossly visible colonic tumors induced by parenteral administration of 1,2-dimethylhydrazine were examined in rats fed either a chemically defined fiber-free diet or a nutritionally and calorically equivalent diet containing a purified fiber component, microcrystalline cellulose. This double-blind study indicates that cellulose ingestion was associated with reduced numbers of animals involved with colonic neoplasia as well as a reduction in the total numbers of colonic tumors. Furthermore, this protective effect of cellulose appears to be time dependent and associated with a shift in tumor distribution from the proximal colon to a more distal site. Cellulose fiber had no apparent effect on colonic tumor size, histopathology, or the incidence of other tumors known to occur in this experimental animal model. This study strongly supports the hypothesis that fiber is an important protective agent against colonic neoplasia development. While the mechanism for this protective effect remains obscure, it appears to be temporally related to the duration of fiber ingestion as well as to a differential fiber effect on either the luminal content or the mucosa of the proximal and distal colon.

Adenocarcinoma

Adrenergic contribution to the motor transmission in the dog vas deferens.

Isolated vas deferens preparations from 9 dogs were subjected to electrical stimulation and chemical excitation under physiological conditions. The experimental smooth muscle cylinders were confined to the terminal 3 cm portions of either the distal 'urethral' segment or the proximal 'epididymal' segment. Intermittent field stimulation, at 60 sec intervals, was provided by a stimulator of low output impedance under constant parameters of frequency and voltage and an occasionally varied pulse width. Results from this examination completely confirmed the following: (i) a high degree of contractile sensitivity to minute doses of noradrenaline (0.03--0.03 micron; 10(-8)-10(-7) g/ml) and tyramine 0.58 micron (10(-7) g/ml; (ii) an apparent ease and rapidity of extinguishing the electrically-induced twitches by either small doses of phentolamine 0.25 micron (10(-7) g/ml) or phenoxybenzamine 5.8 micron (2 x 10(-6) g/ml); (iii) a complete absence of any inhibitory action by tyramine or noradrenaline on the electrically-induced twitches. The behavior of this motor transmission of the longitudinal muscle of the vas deferens to classical alpha-adrenoceptor blocking agents and the intense susceptibility to the motor actions of the putative neurotransmitter clearly fit in with a picture of an adrenergic implication in this mode of transmission.

Adrenergic alpha-Antagonists

Effects of locally applied chemicals on transretinal potential and horizontal cells in the isolated carp retina I. Amino acids.

Experiments were performed on isolated retinas of teh carp (Cyprinus carpio) to investigate the effects of locally applied amino acids on the transretinal potential and the membrane potential of horizontal cells. A minute quantity of each solution of an amino acid was applied locally at different layers by means of pressure-microinjection. The responses were induced by alternating central and annular stimuli of white light. Amino acids applied locally at the distal layer (receptor surface or outer plexiform layer) in the centrally stimulated area, caused, at concentrations of 25--50mM, rapid changes in the light-induced potential, whereas those microinjected into the inner plexiform layer did not. When applied locally at the distal layer, amino acids abolished the proximal PIII response and reduced the amplitude of S-potentials, particularly in response to a central stimulus. However, the resting potential of horizontal cells was depolarized only slightly by acidic amino acids. The results suggest that the passive membrane properties of horizontal cells are not altered significantly by the amino acids applied. Differential susceptibilities of the PII component to acidic and neutral amino acids were found; the former did not change or, in some cases, enlarged, whereas the latter abolished it. Possibly, neutral amino acids interfere with a neural mechanism generating the PII component.

Amino Acids