scholarly journals Microscale Thermophoresis Analysis of Chromatin Interactions

2018 ◽  
pp. 177-197 ◽  
Author(s):  
Ivan Corbeski ◽  
Velten Horn ◽  
Ramon A. van der Valk ◽  
Ulric B. le Paige ◽  
Remus T. Dame ◽  
...  
Keyword(s):  
Microscale Thermophoresis ◽  
Chromatin Interactions

scholarly journals A novel ligand of the translationally controlled tumor protein (TCTP) identified by virtual drug screening for cancer differentiation therapy

2021 ◽  
Author(s):  
Nicolas Fischer ◽  
Ean-Jeong Seo ◽  
Sara Abdelfatah ◽  
Edmond Fleischer ◽  
Anette Klinger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Virtual Screening ◽  
Small Molecules ◽  
Large Scale ◽  
Differentiation Therapy ◽  
P53 Expression ◽  
Microscale Thermophoresis ◽  
Cycle Arrest ◽  
G1 Cell Cycle Arrest ◽  
Mcf 7

SummaryIntroduction Differentiation therapy is a promising strategy for cancer treatment. The translationally controlled tumor protein (TCTP) is an encouraging target in this context. By now, this field of research is still at its infancy, which motivated us to perform a large-scale screening for the identification of novel ligands of TCTP. We studied the binding mode and the effect of TCTP blockade on the cell cycle in different cancer cell lines. Methods Based on the ZINC-database, we performed virtual screening of 2,556,750 compounds to analyze the binding of small molecules to TCTP. The in silico results were confirmed by microscale thermophoresis. The effect of the new ligand molecules was investigated on cancer cell survival, flow cytometric cell cycle analysis and protein expression by Western blotting and co-immunoprecipitation in MOLT-4, MDA-MB-231, SK-OV-3 and MCF-7 cells. Results Large-scale virtual screening by PyRx combined with molecular docking by AutoDock4 revealed five candidate compounds. By microscale thermophoresis, ZINC10157406 (6-(4-fluorophenyl)-2-[(8-methoxy-4-methyl-2-quinazolinyl)amino]-4(3H)-pyrimidinone) was identified as TCTP ligand with a KD of 0.87 ± 0.38. ZINC10157406 revealed growth inhibitory effects and caused G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. ZINC10157406 (2 × IC50) downregulated TCTP expression by 86.70 ± 0.44% and upregulated p53 expression by 177.60 ± 12.46%. We validated ZINC10157406 binding to the p53 interaction site of TCTP and replacing p53 by co-immunoprecipitation. Discussion ZINC10157406 was identified as potent ligand of TCTP by in silico and in vitro methods. The compound bound to TCTP with a considerably higher affinity compared to artesunate as known TCTP inhibitor. We were able to demonstrate the effect of TCTP blockade at the p53 binding site, i.e. expression of TCTP decreased, whereas p53 expression increased. This effect was accompanied by a dose-dependent decrease of CDK2, CDK4, CDK, cyclin D1 and cyclin D3 causing a G0/G1 cell cycle arrest in MOLT-4, SK-OV-3 and MCF-7 cells. Our findings are supposed to stimulate further research on TCTP-specific small molecules for differentiation therapy in oncology.

scholarly journals Novel Method for Quantifying AhR-Ligand Binding Affinities Using Microscale Thermophoresis

Biosensors ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 60
Author(s):  
Anne Stinn ◽  
Jens Furkert ◽  
Stefan H. E. Kaufmann ◽  
Pedro Moura-Alves ◽  
Michael Kolbe
Keyword(s):  
Ligand Binding ◽  
Environmental Pollutants ◽  
Screening Strategy ◽  
Pharmacological Intervention ◽  
Binding Affinities ◽  
Free System ◽  
Microscale Thermophoresis ◽  
Aryl Hydrocarbon ◽  
Promising Target ◽  
A Cell

The aryl hydrocarbon receptor (AhR) is a highly conserved cellular sensor of a variety of environmental pollutants and dietary-, cell- and microbiota-derived metabolites with important roles in fundamental biological processes. Deregulation of the AhR pathway is implicated in several diseases, including autoimmune diseases and cancer, rendering AhR a promising target for drug development and host-directed therapy. The pharmacological intervention of AhR processes requires detailed information about the ligand binding properties to allow specific targeting of a particular signaling process without affecting the remaining. Here, we present a novel microscale thermophoresis-based approach to monitoring the binding of purified recombinant human AhR to its natural ligands in a cell-free system. This approach facilitates a precise identification and characterization of unknown AhR ligands and represents a screening strategy for the discovery of potential selective AhR modulators.

scholarly journals Glucocorticoid receptor wields chromatin interactions to tune transcription for cytoskeleton stabilization in podocytes

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Hong Wang ◽  
Aiping Duan ◽  
Jing Zhang ◽  
Qi Wang ◽  
Yuexian Xing ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Glucocorticoid Receptor ◽  
Protective Effect ◽  
Histone Modification ◽  
Regulatory Networks ◽  
Target Gene ◽  
Regulatory Elements ◽  
Chromatin Interactions ◽  
Super Enhancer

AbstractElucidating transcription mediated by the glucocorticoid receptor (GR) is crucial for understanding the role of glucocorticoids (GCs) in the treatment of diseases. Podocyte is a useful model for studying GR regulation because GCs are the primary medication for podocytopathy. In this study, we integrated data from transcriptome, transcription factor binding, histone modification, and genome topology. Our data reveals that the GR binds and activates selective regulatory elements in podocyte. The 3D interactome captured by HiChIP facilitates the identification of remote targets of GR. We found that GR in podocyte is enriched at transcriptional interaction hubs and super-enhancers. We further demonstrate that the target gene of the top GR-associated super-enhancer is indispensable to the effective functioning of GC in podocyte. Our findings provided insights into the mechanisms underlying the protective effect of GCs on podocyte, and demonstrate the importance of considering transcriptional interactions in order to fine-map regulatory networks of GR.

scholarly journals Reproducibility and accuracy of microscale thermophoresis in the NanoTemper Monolith: a multi laboratory benchmark study

2021 ◽  
Author(s):  
Blanca López-Méndez ◽  
Bruno Baron ◽  
Chad A. Brautigam ◽  
Thomas A. Jowitt ◽  
Stefan H. Knauer ◽  
...  
Keyword(s):  
Microscale Thermophoresis ◽  
Benchmark Study

scholarly journals Large organized chromatin lysine domains help distinguish primitive from differentiated cell populations

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Seyed Ali Madani Tonekaboni ◽  
Benjamin Haibe-Kains ◽  
Mathieu Lupien
Keyword(s):  
Transposable Elements ◽  
Histone Modifications ◽  
Cell Types ◽  
Regulatory Elements ◽  
Cell Populations ◽  
Genomic Features ◽  
Differentiated Cells ◽  
Chromatin Interactions ◽  
Topologically Associating Domains ◽  
Highly Expressed Genes

AbstractThe human genome is partitioned into a collection of genomic features, inclusive of genes, transposable elements, lamina interacting regions, early replicating control elements and cis-regulatory elements, such as promoters, enhancers, and anchors of chromatin interactions. Uneven distribution of these features within chromosomes gives rise to clusters, such as topologically associating domains (TADs), lamina-associated domains, clusters of cis-regulatory elements or large organized chromatin lysine (K) domains (LOCKs). Here we show that LOCKs from diverse histone modifications discriminate primitive from differentiated cell types. Active LOCKs (H3K4me1, H3K4me3 and H3K27ac) cover a higher fraction of the genome in primitive compared to differentiated cell types while repressive LOCKs (H3K9me3, H3K27me3 and H3K36me3) do not. Active LOCKs in differentiated cells lie proximal to highly expressed genes while active LOCKs in primitive cells tend to be bivalent. Genes proximal to bivalent LOCKs are minimally expressed in primitive cells. Furthermore, bivalent LOCKs populate TAD boundaries and are preferentially bound by regulators of chromatin interactions, including CTCF, RAD21 and ZNF143. Together, our results argue that LOCKs discriminate primitive from differentiated cell populations.

scholarly journals HiChIP and Hi-C Protocol Optimized for Primary Murine T Cells

2021 ◽  
Vol 4 (3) ◽  
pp. 49
Author(s):  
Tomas Zelenka ◽  
Charalampos Spilianakis
Keyword(s):  
T Cells ◽  
Long Range ◽  
Three Dimensional ◽  
Cell Types ◽  
Range Interaction ◽  
Chromatin Interactions ◽  
Optimized Protocol ◽  
Long Range Interaction ◽  
Chromatin Compactness ◽  
Detailed Protocol

The functional implications of the three-dimensional genome organization are becoming increasingly recognized. The Hi-C and HiChIP research approaches belong among the most popular choices for probing long-range chromatin interactions. A few methodical protocols have been published so far, yet their reproducibility and efficiency may vary. Most importantly, the high frequency of the dangling ends may dramatically affect the number of usable reads mapped to valid interaction pairs. Additionally, more obstacles arise from the chromatin compactness of certain investigated cell types, such as primary T cells, which due to their small and compact nuclei, impede limitations for their use in various genomic approaches. Here we systematically optimized all the major steps of the HiChIP protocol in T cells. As a result, we reduced the number of dangling ends to nearly zero and increased the proportion of long-range interaction pairs. Moreover, using three different mouse genotypes and multiple biological replicates, we demonstrated the high reproducibility of the optimized protocol. Although our primary goal was to optimize HiChIP, we also successfully applied the optimized steps to Hi-C, given their significant protocol overlap. Overall, we describe the rationale behind every optimization step, followed by a detailed protocol for both HiChIP and Hi-C experiments.

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