title: Direct chromatin PCR (DC-PCR): hypotonic conditions allow differentiation of chromatin states during thermal cycling.
abstract: Current methods to study chromatin configuration are not well suited for high throughput drug screening since they require large cell numbers and multiple experimental steps that include centrifugation for isolation of nuclei or DNA. Here we show that site specific chromatin analysis can be achieved in one step by simply performing direct chromatin PCR (DC-PCR) on cells. The basic underlying observation was that standard hypotonic PCR buffers prevent global cellular chromatin solubilization during thermal cycling while more loosely organized chromatin can be amplified. Despite repeated heating to >90 °C, 41 of 61 tested 5' sequences of silenced genes (CDKN2A, PU.1, IRF4, FOSB, CD34) were not amplifiable while 47 could be amplified from expressing cells. Two gene regions (IRF4, FOSB) even required pre-heating of cells in isotonic media to allow this differentiation; otherwise none of 19 assayed sequences yielded PCR products. Cells with baseline expression or epigenetic reactivation gave similar DC-PCR results. Silencing during differentiation of CD34 positive cord blood cells closed respective chromatin while treatment of myeloma cells with an IRF4 transcriptional inhibitor opened a site to DC-PCR that was occupied by RNA polymerase II and NFκB as determined by ChIP. Translation into real-time PCR can not be achieved with commercial real-time PCR buffers which potently open chromatin, but even with simple ethidium bromide addition to standard PCR mastermix we were able to identify hits in small molecules screens that suppressed IRF4 expression or reactivated CDKN2A in myeloma cells using densitometry or visual inspection of PCR plates under UV light. While need in drug development inspired this work, application to genome-wide analysis appears feasible using phi29 for selective amplification of open cellular chromatin followed by library construction from supernatants since such supernatants yielded similar results as gene specific DC-PCR.
authors.affiliation: Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, The Cleveland Clinic Foundation, Cleveland, Ohio, United States of America.
authors.forename: Sergei
authors.initials: S
authors.lastname: Vatolin
authors.forename: Shahper N
authors.initials: SN
authors.lastname: Khan
authors.forename: Frederic J
authors.initials: FJ
authors.lastname: Reu
mesh_terms: D018952:Antigens, CD34 / Q000096:biosynthesis / Q000737:chemistry; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005312:Fetal Blood / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D020869:Gene Expression Profiling; D020868:Gene Silencing; D006358:Hot Temperature; D006801:Humans; D050835:Interferon Regulatory Factors / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D013696:Temperature; D000099218:Interferon Regulatory Factor-4
chemical_list: D018952:Antigens, CD34; D002843:Chromatin; D050835:Interferon Regulatory Factors; D016328:NF-kappa B; D016271:Proto-Oncogene Proteins c-myc; D012319:RNA Polymerase II; D000099218:Interferon Regulatory Factor-4