Genetic Rearrangements Modify Nearby Chromatin Figure 6. Model for transcriptional regulation of epialleles. (A) The silent state is maintained by interplay of DNA hypermethylation, repressive histone modifications, lack of activating marks, presence of sRNAs and structure of the controlled gene, all mutually reinforcing the block of transcription. (B,C) Release of silencing can occur after structural rearrangements. (B) Even minor changes, like in cis-mutant RD4, can trigger a switch to high transcript levels, drastic changes of chromatin features and stable genetic transmission. (C) A major rearrangement like in cis-mutant RD1, although deleting the sRNA-producing region, does not necessarily result in stable switches. Transcript levels are lower; exchange of histone marks is incomplete and not stable in subsequent generations. This points to a significant role of sequence or gene structure, possibly by different secondary structure or nucleosome positioning, in stability of epialleles. doi:10.1371/journal.pgen.1002331.g006 blot analysis of total and poly(A) RNA, 5 mg of RNA were denatured with 15% glyoxal and 50% DMSO for 1 h at 50uC and separated using 1.5% agarose gels in 10 mM sodium phosphate buffer pH7 in a Sea2000 circular flow electrophoresis chamber (Elchrom Scientific). DNA and RNA gels were blotted onto Hybond N+ (Amersham) membranes overnight with 206 SSC, washed and UV-crosslinked using a Stratalinker (Stratagene). Hybridization was performed as described [51]. Radioactively labeled sequence-specific probes were synthesized from 25 ng of DNA using the Rediprime labeling kit (Amersham) and 50 mCi dCTP-a-32P (Amersham or Hartmann Analytic) and purified on G50 Probequant (Amersham) columns. Signals were detected with phosphoimager screens (Bio-Rad) and scanned with a Molecular Imager FX (Bio-Rad). Rapid Amplification of cDNA 39 Ends 39-RACE was performed with the SMART RACE cDNA Amplification Kit (Clontech) according to the instructions. Total RNA (700 ng) was treated with DNaseI (Fermentas), then reverse- transcribed with RevertAidRT (Fermentas) with 3-RACE A primer (5–AAGCAGTGGTATCAACGCAGAGTAC(T)30V N–3) in a 20 ml reaction. Two ml of cDNA reaction were used as template in 39-RACE PCR. For this, Advantage 2 PCR Kit (Clontech) was used according to instructions. A control primer (Actin, Act2F primer: 5- GCCATCCAAGCTGTTCTCTC-3) and gene-specific primers were used in combination with UniA_45 (5–CTAATACGACT- CACTATAGGGCAAGCAGTGGTATCAACGCAGAGT–3). Reverse Transcription PCR and Quantitative Real-Time PCR RNA samples were treated with DNase I (MBI Fermentas) for 30 min at 37uC to remove residual DNA contamination. The reaction was inactivated by addition of EDTA and incubation at 65uC for 10 min. Reverse transcription was performed on 1 mg of RNA with 0.2 mg of random hexamer primers (MBI Fermentas) using 1 U RevertAid H Minus M-MuLV-RTase (MBI Fermentas) in the presence of 20 U RiboLock Ribonuclease inhibitor at 42uC for 1.5 h. Real time PCR analysis was performed with the 26 SensiMix Plus SYBR & Fluorescein Kit (Quantace) protocol using an iQ5 Real-Time-PCR System (BioRad Laboratories). The obtained Ct values were analyzed with the iQ5 Optical System Software Version 2.0 (Bio-Rad), applying the mathematical model for relative quantification in Excel (Microsoft) as described [52]. All primer sequences are listed in Table S4. PLoS Genetics | www.plosgenetics.org 10 October 2011 | Volume 7 | Issue 10 | e1002331