A modified CUT&amp;RUN-seq technique for qPCR analysis of chromatin-protein interactions

Panday, Arvind; Elango, Rajula; Willis, Nicholas A.; Scully, Ralph

doi:10.1016/j.xpro.2022.101529

Cited by 10 publications

(8 citation statements)

References 13 publications

(24 reference statements)

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“…We targeted this new reporter as a single copy to Rosa26 and confirmed the function of the Tus/ Ter RFB in two ways. First, to confirm that Tus binds the Ter array, we used anti-HA CUT&RUN-qPCR for HA-tagged Tus 52,53 , detecting ∼60-fold enrichment of Tus at the Ter array in Tus-transfected cells in comparison to empty-vector-transfected controls ( Supplemental Figure S6A ). Second, we made use of the fact that RAD51 is an early responder to the Tus/ Ter -stalled fork 50,54 .…”

Section: Resultsmentioning

confidence: 99%

Two-ended recombination at a Flp-nickase-broken replication fork

Elango,

Nilavar,

et al. 2024

Preprint

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SummaryCollision of a replication fork with a DNA nick is thought to generate a one-ended break, fostering genomic instability. Collision of the opposing converging fork with the nick could, in principle, form a second DNA end, enabling conservative repair by homologous recombination (HR). To study mechanisms of nickase-induced HR, we developed the Flp recombinase “step arrest” nickase in mammalian cells. Flp-nickase-induced HR entails two-ended, BRCA2/RAD51-dependent short tract gene conversion (STGC), BRCA2/RAD51-independent long tract gene conversion, and discoordinated two-ended invasions. HR induced by a replication-independent break and by the Flp-nickase differ in their dependence onBRCA1. To determine the origin of the second DNA end during Flp-nickase-induced STGC, we blocked the opposing fork using a site-specific Tus/Terreplication fork barrier. Flp-nickase-induced STGC remained robust and two-ended. Thus, collision of a single replication fork with a Flp-nick can trigger two-ended HR, possibly reflecting replicative bypass of lagging strand nicks. This response may limit genomic instability during replication of a nicked DNA template.

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Section: Resultsmentioning

confidence: 99%

Two-ended recombination at a Flp-nickase-broken replication fork

Elango,

Nilavar,

et al. 2024

Preprint

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“…A single clone for each condition was selected to perform further experiments (Figure S4). To evaluate the disruption, CTCF binding ability to IE8 was tested in both cell lines through Cleavage Under Targets and Release Using Nuclease (CUT&RUN) followed by qPCR [33]. Basal levels of CTCF occupancy were higher in MDA-MB-231 than in MDA-MB-436.…”

Section: Resultsmentioning

confidence: 99%

Chromatin insulation orchestrates matrix metalloproteinase gene cluster expression reprogramming in aggressive breast cancer tumors

Llinàs‐Arias

Orozco

Ensenyat-Mendez

et al. 2023

Preprint

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Background Triple-negative breast cancer (TNBC) is an aggressive subtype that exhibits a high incidence of distant metastases and lacks targeted therapeutic options. Here we explored how the epigenome may contribute to matrix metalloprotease (MMP) dysregulation given their key role in invasion, which is the first step of the metastatic process.Methods We combined RNA expression and chromatin interaction data to identify insulator elements potentially associated with invasion. We stably disrupted the CCCTC-Binding Factor (CTCF) binding site of a single insulator element in two TNBC cellular models. We characterized these models by combining Hi-C, ATAC-seq, and RNA-seq with functional experiments to determine invasive ability. Our findings were then also tested in a ductal carcinoma in situ (DCIS) cohort.Results We explored the clinical relevance of an insulator element located within the Chr11q22.2 locus, downstream of the MMP8 gene (IE8). This regulatory element resulted in a topologically associating domain (TAD) boundary that isolated nine MMP genes into two anti-correlated expression clusters. This expression pattern was strongly associated with worse relapse-free (HR = 1.57 [1.06 − 2.33]; p = 0.023) and overall (HR = 2.65 [1.31 − 5.37], p = 0.005) survival of TNBC patients. After CRISPR/Cas9-mediated disruption of IE8, cancer cells showed a switch in the MMP expression signature, specifically downregulating the pro-invasive MMP1 gene and upregulating the antitumorigenic MMP8 gene, resulting in reduced invasive ability. Finally, we observed that the imbalance in the MMP expression predicts DCIS that eventually progresses into invasive ductal carcinomas (AUC = 0.77, p < 0.01).Conclusion Our study demonstrates how the activation of an IE near the MMP8 gene determines the regional transcriptional regulation of MMP genes with opposing functional activity, ultimately influencing the invasive properties of aggressive forms of breast cancer.

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“…And a corresponding volume of DMSO (< 1%) was added to the DMSO group as control and incubated at 37 °C for 96 h. Cells were collected by trypsin digestion and dispensed into 0.5 × 10 6 / tubes. Subsequent steps were performed according to the experimental protocol [ 33 ]. Specific antibodies are used to bind to transcription complexes and pull-down specific fragments of DNA sequences by enzymatic cleavage and purification: TAF1 (TAF1 Rabbit mAb catalogue no.#12781S, D6J8B, CST) and NELFB (COBRA1 Rabbit mAb catalogue no.#14894S, D6K9A, CST) antibodies.…”

Section: Methodsmentioning

confidence: 99%

A G-quadruplex-binding platinum complex induces cancer mitochondrial dysfunction through dual-targeting mitochondrial and nuclear G4 enriched genome

Kuang,

Li,

Maksut

et al. 2024

J Biomed Sci

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Background G-quadruplex DNA (G4) is a non-canonical structure forming in guanine-rich regions, which play a vital role in cancer biology and are now being acknowledged in both nuclear and mitochondrial (mt) genome. However, the impact of G4-based targeted therapy on both nuclear and mt genome, affecting mt function and its underlying mechanisms remain largely unexplored. Methods The mechanisms of action and therapeutic effects of a G4-binding platinum(II) complex, Pt-ttpy, on mitochondria were conducted through a comprehensive approaches with in vitro and in vivo models, including ICP-MS for platinum measurement, PCR-based genetic analysis, western blotting (WB), confocal microscope for mt morphology study, extracellular flux analyzer, JC1 and Annexin V apoptosis assay, flow cytometry and high content microscope screening with single-cell quantification of both ROS and mt specific ROS, as well as click-chemistry for IF study of mt translation. Decipher Pt-ttpy effects on nuclear-encoded mt related genes expression were undertaken via RNA-seq, Chip-seq and CUT-RUN assays. Results Pt-ttpy, shows a highest accumulation in the mitochondria of A2780 cancer cells as compared with two other platinum(II) complexes with no/weak G4-binding properties, Pt-tpy and cisplatin. Pt-ttpy induces mtDNA deletion, copy reduction and transcription inhibition, hindering mt protein translation. Functional analysis reveals potent mt dysfunction without reactive oxygen species (ROS) induction. Mechanistic study provided first evidence that most of mt ribosome genes are highly enriched in G4 structures in their promoter regions, notably, Pt-ttpy impairs most nuclear-encoded mt ribosome genes’ transcription through dampening the recruiting of transcription initiation and elongation factors of NELFB and TAF1 to their promoter with G4-enriched sequences. In vivo studies show Pt-ttpy’s efficient anti-tumor effects, disrupting mt genome function with fewer side effects than cisplatin. Conclusion This study underscores Pt-ttpy as a G4-binding platinum(II) complex, effectively targeting cancer mitochondria through dual action on mt and nuclear G4-enriched genomes without inducing ROS, offering promise for safer and effective platinum-based G4-targeted cancer therapy. Graphical Abstract

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A modified CUT&RUN-seq technique for qPCR analysis of chromatin-protein interactions

Cited by 10 publications

References 13 publications

Two-ended recombination at a Flp-nickase-broken replication fork

Two-ended recombination at a Flp-nickase-broken replication fork

Chromatin insulation orchestrates matrix metalloproteinase gene cluster expression reprogramming in aggressive breast cancer tumors

A G-quadruplex-binding platinum complex induces cancer mitochondrial dysfunction through dual-targeting mitochondrial and nuclear G4 enriched genome

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