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.
Highlights
The epigenetic activity of genes is determined by the net balance of activating and repressing histone modifications, methylation of regulatory CpG islands and binding of regulatory proteins and RNAs [1]
Direct Chromatin PCR (DC-PCR) Development During experiments aimed at streamlining site-specific chromatin analysis for application in drug discovery we included PCR of unprocessed cells as one of the controls using cells grown in RMPI 1640 media diluted at 1:25 in standard hypotonic PCR buffer supplemented with protease inhibitors
To investigate whether direct chromatin-PCR (DC-PCR) yields consistent results in settings other than reactivation of tumor suppressor genes by epigenetic agents, which directly act on chromatin, we studied CD34 positive cord blood cells that are known to grow and differentiate in vitro, gradually loosing CD34 expression, under the influence of cytokines [13]
Summary
The epigenetic activity of genes is determined by the net balance of activating and repressing histone modifications, methylation of regulatory CpG islands and binding of regulatory proteins and RNAs [1]. It offers the advantage over ChIP that it does not require assessment of activating and repressing modifications to assess net chromatin activity, it, like ChIP, is done on nuclear extracts posing a major challenge to high throughput drug screening applications. Nucleosome-free regions are enriched in active chromatin and susceptible to enzymatic cleavage but protocols require nuclear extraction [7,8]. Another way to obtain nucleosome-free DNA is by crosslinking histone-bound DNA followed by mechanical fragmentation through sonication [9] but here separation of DNA fragments makes centrifugation necessary and application in drug screens cumbersome. Key to the success of this procedure is the observation that standard hypotonic PCR buffers prevent universal heat solubilization of cellular chromatin during PCR while areas with weaker DNAprotein bonds can be amplified
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