<i>In vitro</i>
            analysis of DNA–protein interactions by proximity ligation

Gústafsdóttir, Sigrún M.; Schlingemann, Joerg; Rada-Iglesias, Álvaro; Schallmeiner, Edith; Kamali‐Moghaddam, Masood; Wadelius, Claes; Landegren, Ulf

doi:10.1073/pnas.0611229104

Cited by 67 publications

(56 citation statements)

References 57 publications

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“…However, the p300 enhancer effect was abrogated when either M.Msp-I or M.Hpa-II methylated versions of the reporter were used ( Figure 5b). In order to confirm the direct binding of p300 to this LRP1B sequence and its possible perturbation by DNA methylation, we performed proximity ligation assay (Gustafsdottir et al, 2007) using DNA probes in the unmethylated, methylated and mutated forms. We showed a 64-fold increase in interactions between p300 and a DNA probe composed of the LRP1B intron 1 unmethylated sequence as compared with probes with a mutated binding site or with methylated *CG and *CCG sites ( Figure 5c).…”

Section: Lrp1b Underexpression Follows Thyroid Cancer Progression Andmentioning

confidence: 99%

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Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells

et al. 2010

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The low-density lipoprotein receptor-related protein (LRP1B), encoding an endocytic LDL-family receptor, is among the 10 most significantly deleted genes across 3312 human cancer specimens. However, currently the apparently crucial role of this lipoprotein receptor in carcinogenesis is not clear. Here we show that LRP1B inactivation (by chromosomal, epigenetic and microRNA (miR)-mediated mechanisms) results in changes to the tumor environment that confer cancer cells an increased growth and invasive capacity. LRP1B displays frequent DNA copy number loss and CpG island methylation, resulting in mRNA underexpression. By using CpG island reporters methylated in vitro, we found that DNA methylation disrupts a functional binding site for the histone-acetyltransferase p300 located at intron 1. We identified and validated an miR targeting LRP1B (miR-548a-5p), which is overexpressed in cancer cell lines as a result of 8q22 DNA gains. Restoration of LRP1B impaired in vitro and in vivo tumor growth, inhibited cell invasion and led to a reduction of matrix metalloproteinase 2 in the extracellular medium. We emphasized the role of an endocytic receptor acting as a tumor suppressor by modulating the extracellular environment composition in a way that constrains the invasive behavior of the cancer cells.

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Section: Lrp1b Underexpression Follows Thyroid Cancer Progression Andmentioning

confidence: 99%

“…Analysis of the interaction between DNA and p300 was performed by a proximity ligation assay as described by Gustafsdottir et al (2007), with the following modifications:…”

Section: Constructs and Expression Vectorsmentioning

confidence: 99%

Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells

et al. 2010

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“…The available technologies for sensitive protein detection typically use pairs of affinity reagents in a sandwich configuration. This is true for the popular sandwich ELISA (40), and for more recent highly sensitive techniques such as the biobarcode assay (41), singlemolecule counting assays (42)(43)(44), and homogeneous PLA (36,37,45).…”

Section: Discussionmentioning

confidence: 98%

Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer

Tavoosidana

Ronquist

Darmanis

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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202

173

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Prostasomes are microvesicles (mean diameter, 150 nm) that are produced and secreted by normal and malignant prostate acinar cells. It has been hypothesized that invasive growth of malignant prostate cells may cause these microvesicles, normally released into seminal fluid, to appear in interstitial space and therewith into peripheral circulation. The suitability of prostasomes as blood biomarkers in patients with prostate cancer was tested by using an expanded variant of the proximity ligation assay (PLA). We developed an extremely sensitive and specific assay (4PLA) for detection of complex target structures such as microvesicles in which the target is first captured via an immobilized antibody and subsequently detected by using four other antibodies with attached DNA strands. The requirement for coincident binding by five antibodies to generate an amplifiable reporter results in both increased specificity and sensitivity. The assay successfully detected significantly elevated levels of prostasomes in blood samples from patients with prostate cancer before radical prostatectomy, compared with controls and men with benign biopsy results. The medians for prostasome levels in blood plasma of patients with prostate cancer were 2.5 to sevenfold higher compared with control samples in two independent studies, and the assay also distinguished patients with high and medium prostatectomy Gleason scores (8/9 and 7, respectively) from those with low score (≤6), thus reflecting disease aggressiveness. This approach that enables detection of prostasomes in peripheral blood may be useful for early diagnosis and assessment of prognosis in organ-confined prostate cancer.

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“…While this manuscript was under review, we learned that Landegren and colleagues (41) developed another in vitro method for specific and sensitive analysis of interactions between proteins and nucleic acids by using the proximity ligation assay (PLA) (ref. 41, which is published in this issue of PNAS). In this method, the detection of protein binding also is achieved by quantification of nucleic acids.…”

Section: Methodsmentioning

confidence: 90%

Quantifying DNA–protein binding specificities by using oligonucleotide mass tags and mass spectroscopy

Zhang¹,

Kasif

Cantor

2007

Proc. Natl. Acad. Sci. U.S.A.

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The ability to determine the relative binding affinity of different transcription-factors (TF) to their DNA binding sites is fundamentally important for a comprehensive understanding of gene regulation. Here we present a general approach for multiplex quantification of DNA-TF binding specificities in vitro using oligonucleotide mass tag (OMT) labeling and mass spectroscopic quantification. An OMT is a short nucleic acid sequence with a distinct mass that can be resolved by a mass spectrometer. Each putative binding sequence is labeled with a unique OMT, and PCR amplification of OMTs is performed after removing nonbound DNA. Subsequently, a primer extension reaction is carried out, and the extension products are quantified by MALDI-TOF mass spectroscopy. Using the TF NF-B P50, we have quantified the binding specificities of up to 15 binding sequences in a single assay. The results from the multiplex assay are consistent with data from the traditional gel shift assay. The approach allows the competitive binding of multiple DNA sequences to the given protein in a homogeneous reaction. By using the commercially available homogeneous MassEXTEND platform (SEQUENOM), it is scalable for highthroughput DNA-TF binding applications, including genome-wide TF binding site mapping and analyses of SNPs in promoter regions.polymerase chain reaction ͉ transcription factor

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In vitro analysis of DNA–protein interactions by proximity ligation

Cited by 67 publications

References 57 publications

Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells

Chromosomal, epigenetic and microRNA-mediated inactivation of LRP1B, a modulator of the extracellular environment of thyroid cancer cells

Multiple recognition assay reveals prostasomes as promising plasma biomarkers for prostate cancer

Quantifying DNA–protein binding specificities by using oligonucleotide mass tags and mass spectroscopy

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