Cytosine modification of the dinucleotide CpG in the DNA regulatory region is an important epigenetic marker during early embryo development, cellular differentiation, and cancer progression. In clinical settings, such as anti-cancer drug treatment, it is desirable to develop research tools to characterize DNA sequences affected by epigenetic perturbations. Here, we describe the construction and characterization of two fusion endonucleases consisting of the (5)mCpG-binding domain of human MeCP2 (hMeCP2) and the cleavage domains of BmrI and FokI restriction endonucleases (REases). The chimeric (CH) endonucleases cleave M.HpaII (C(5)mCGG)-and M.SssI ((5)mCpG)-modified DNA. Unmodified DNA and M.MspI-modified DNA ((5)mCCGG) are poor substrates for the CH-endonucleases. Sequencing cleavage products of modified lambda DNA indicates that cleavage takes place outside the (5)mCpG recognition sequence, predominantly 4-17 bp upstream of the modified base (/N(4-17)(5)mCpG, where / indicates the cleavage site). Such (5)mCpG-specific endonucleases will be useful to study CpG island modification of the regulatory regions of tumor suppressor genes, and for the construction of cell-specific and tumor-specific modified CpG island databases.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1016/j.ab.2008.06.035 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Current Non-Viral-Based Strategies to Manufacture CAR-T Cells.
Int J Mol Sci
December 2024
Medizinische Klinik und Poliklinik II und Lehrstuhl für Zelluläre Immuntherapie, Universitätsklinikum Würzburg, 97080 Würzburg, Germany.
The successful application of CAR-T cells in the treatment of hematologic malignancies has fundamentally changed cancer therapy. With increasing numbers of registered CAR-T cell clinical trials, efforts are being made to streamline and reduce the costs of CAR-T cell manufacturing while improving their safety. To date, all approved CAR-T cell products have relied on viral-based gene delivery and genomic integration methods.
View Article and Find Full Text PDFFunctional anatomy of zinc finger antiviral protein complexes.
Nat Commun
December 2024
Laboratory of Retrovirology, The Rockefeller University, New York, NY, 10065, USA.
ZAP is an antiviral protein that binds to and depletes viral RNA, which is often distinguished from vertebrate host RNA by its elevated CpG content. Two ZAP cofactors, TRIM25 and KHNYN, have activities that are poorly understood. Here, we show that functional interactions between ZAP, TRIM25 and KHNYN involve multiple domains of each protein, and that the ability of TRIM25 to multimerize via its RING domain augments ZAP activity and specificity.
View Article and Find Full Text PDFMolecular protocol for genome-wide and cell-type-selective profiling of in vivo small noncoding RNA:target RNA interactions by CIMERA-seq.
STAR Protoc
December 2024
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address:
Small noncoding RNAs (sncRNAs) can regulate gene expression by guiding the RNA-induced silencing complex (RISC) to targeted transcripts for translational repression and/or target destabilization. Here, we present a robust benchtop protocol, termed CIMERA-seq, for the unambiguous profiling of sncRNA:target RNA interactions in a genome-wide and cell-type-selective manner. We describe steps for in vivo crosslinking and harvesting tissue, immunoprecipitation and covalent ligation of sncRNAs to target RNAs within the RISC, and sequencing of the resulting chimeric sncRNA:target RNA interactions.
View Article and Find Full Text PDFWidespread Impact of Natural Genetic Variations in CRISPR-Cas9 Outcomes.
CRISPR J
October 2024
Division of Artificial Intelligence in Medicine, Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA.
The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) is a genome editing tool widely used in biological research and clinical therapeutics. Natural human genetic variations, through altering the sequence context of CRISPR-Cas9 target regions, can significantly affect its DNA repair outcomes and ultimately lead to different editing efficiencies. However, these effects have not been systematically studied, even as CRISPR-Cas9 is broadly applied to primary cells and patient samples that harbor such genetic diversity.
View Article and Find Full Text PDFGenome-wide and cell-type-selective profiling of in vivo small noncoding RNA:target RNA interactions by chimeric RNA sequencing.
Cell Rep Methods
August 2024
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address:
Small noncoding RNAs (sncRNAs) regulate biological processes by impacting post-transcriptional gene expression through repressing the translation and levels of targeted transcripts. Despite the clear biological importance of sncRNAs, approaches to unambiguously define genome-wide sncRNA:target RNA interactions remain challenging and not widely adopted. We present CIMERA-seq, a robust strategy incorporating covalent ligation of sncRNAs to their target RNAs within the RNA-induced silencing complex (RISC) and direct detection of in vivo interactions by sequencing of the resulting chimeric RNAs.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!