RNA Is an Integral Component of Chromatin that Contributes to Its Structural Organization
Figure 1
Purified chicken liver chromatin contains RNA.
A) Chicken liver chromatin was prepared by micrococcal nuclease digestion of purified nuclei and then subjected to sedimentation through a linear 5%–30% sucrose gradients. After centrifugation, 1ml fractions were collected, subjected to total nucleic acids extraction and analyzed by electrophoresis in 1% agarose-TBE gels (left panel). In parallel, chromatin fractions were subjected to RNA extraction using Ultraspec™ RNA Isolation System (Biotecx) and analyzed by Northern blotting in a glyoxal-1% agarose gel using high molecular weight genomic chicken DNA as a probe (right panel). Fraction numbers are indicated. Lanes M correspond to molecular weight markers. B) Chromatin fractions 7 and 10 of the gradient shown in A) were subjected to RNA extraction as indicated above, treated with RNase A (lanes 3 and 4) or not (lanes 1 and 2), and analyzed by Northern blotting as in A). Lane M, corresponds to molecular weight markers. Lane P, corresponds to the probe used. C) Chromatin from a mixture of fractions 7, 8 and 9 of the gradient shown in A) were subjected to RNA extraction and either untreated (lane 2), treated with RNase A in the absence (lane 1) or in the presence of anti-RNase (Ambion) (lane 3), or treated with DNase I (Roche) (lane 4). After phenol extraction and isopropanol precipitation, samples were 32P-labeled by reverse transcription with Omniscript® RT Kit (Qiagen) (2 h at 37°C) using a mixture of hexanucleotides of random sequence. Samples were then analyzed in a 1% agarose-TBE gel, blotted and the membrane directly exposed. Lane M, corresponds to molecular weight markers. D) Chromatin-associated RNA was purified and incubated with oligo-dT immobilized resin (Oligotex™ mRNA Purification System, QIAGEN). After elution, bound (lane 2) and unbound material (lane1) were analyzed in a glyoxal-1% agarose, 10 mM sodium phosphate (pH 6,8) gel. Lanes M correspond to molecular weight markers.