Distinct Mechanisms Determine Transposon Inheritance and Methylation via Small Interfering RNA and Histone Modification (original) (raw)
Figure 2
Inheritance of Transposon Modification
Reverse-transcribed cDNA (A), ChIP (B), and McrBC-digested genomic DNA (C) were amplified by PCR using primers from five retroelements and one DNA transposon in mutant (m/m) and backcrossed plants (m/+). Primers corresponded to transcribed ORFs for each element except for AtMu1 ChIP, which was done on the terminal inverted repeat (TIR). For ATLANTYS2, the larger product is ATLANTYS2-1 and smaller product is ATLANTYS2-2. Input RNA was normalized for each genotype using actin primers.
(A) Mock RT–PCR was performed without reverse transcriptase (−RT) using primers specific for the Cen180 repeat, which can detect trace amounts of contaminating DNA due to its high-copy number.
(B) ChIP was performed with antibodies recognizing dimethyl lysine-9 (K9) and dimethyl lysine-4 (K4) of histone H3 along with no antibody (na) and total (T) DNA controls. ChIP analysis for AtMu1 and ATCOPIA4 was performed using reduced cycles of PCR and Southern blotting (see Materials and Methods).
(C) McrPCR was carried out on untreated (−) and McrBC-treated (+) DNA (see Materials and Methods).