4.4 Article

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using BrdU-ChIP-Slot-Western Technique

Journal

JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 107, Pages -

Publisher

JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/53647

Keywords

Molecular Biology; Issue 107; HDAC1, 2; Nascent DNA; DNA replication; Genome stability; Chromatin immunoprecipitation; Cancer; BrdU; Slot blot; Histones; Chromatin remodeler and HDAC inhibitors

Funding

  1. Dept. of Radiation Oncology and Huntsman Cancer Institute and the National Institute of Health [R01-CA188520]
  2. NATIONAL CANCER INSTITUTE [R01CA188520] Funding Source: NIH RePORTER

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Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA. Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

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