SV40 minichromosomes have a nuclease-hypersensitive region within a nucleosome-free domain. Mordechai Choder, Susan Bratosin and Yosef Aloni.
The EMBO Journal vol.3 no. 12 pp.2929-2936, 1984
A direct analysis of transcribed minichromosomes: all transcribed SV40 minichromosomes have a nuclease-hypersensitive region within a nucleosome-free domain
Mordechai Choder, Susan Bratosin and Yosef Aloni Department of Genetics, Weizmann Institute of Science, Rehovot 76100, Israel Communicated by Y.Aloni
A direct quantitative method was developed for detecting a small fraction of minichromosomes active in transcription and for analyzing their chromatin structure. For this, SV40 minichromosomes were incubated in vitro in the presence of [o-32P]UTP to allow elongation of the in vivo pre-initiated nascent RNA and thus labeling of the in vivo transcriptionally active molecules. Subsequently the minichromosomes were treated with Sarkosyl to remove most of the proteins bound to the viral DNA while leaving the 32P-labeled RNA-DNApolymerase ternary complexes intact. Following agarose gel electrophoresis, the bulk SV40 DNA was identified by staining with ethidium bromide and the labeled ternary complexes by autoradiography, although they are not well separated in the gel. When the viral minichromosomes were cleaved with certain restrction enzymes or mildly with DNase I prior to the in vitro labeling and Sarkosyl treatment, analysis by gel electrophoresis showed that all transcriptionally active minichromosomes were cleaved, whereas a large fraction of the bulk minichromosomes was resistant to cleavage. In all molecules the cleavage occurred within the transcriptional regulatory region. In addition, analysis by electron microscopy revealed that SV40 minichromosomes having a nudeosome-free region (gap) are also more sensitive to the initial cleavage by both DNase I and BglI restriction enzyme than non-gapped molecules and that the initial DNase I cleavage occurs within the gap. Thus, at least in SV40, the nuclease-hypersensitive region is positively correlated with the existence of a gap. Key words: chromatin structure/minichromosome-EM/ nuclease hypersensitivity/SV40 minichromosomes/transcribed minichromosomes
Introduction It is generally assumed that the higher order chromatin structure of actively transcribed genes differs from that of nontranscribed genes (for review, see Mathis et al., 1980; IgoKemenes et al., 1982). The evidence for a unique chromatin structure of actively transcribed genes has been provided mainly through the use of various nucleases (Weintraub and Groudine, 1976; Wu, 1980; Stadler et al., 1980; Weintraub, 1983; McGhee et al., 1981; Elgin, 1981). Using this method it has been found that the unique chromatin structure is associated with enhanced sensitivity to DNase I over a considerable region and with hypersensitivity to DNase I over a smaller region located in most cases near the 5' end of the gene (Weintraub and Groudine, 1976; Wu, 1980; Stadler et al., 1980; Weintraub, 1983; McGhee et al., 1981; Elgin, 1981). However, several exceptions have been noted and they have led to the conclusion that the existence of the DNase 1-hyper© IRL Press Limited, Oxford, England.
sensitive sites at the 5'-flanking region of a gene is neither a requirement for expression nor is it evidence that the gene is being expressed (Elgin, 1981; Groudine and Weintraub, 1982; Weintraub et al., 1982). With this limitation in mind it is still important to determine whether the nuclease-hypersensitive sites in a given gene are present and if they play any role during transcription. A common procedure for detecting the actively transcribed gene in a cell involves hybridization to complementary DNA or RNA probes (Mathis et al., 1980; Igo-Kemenes et al., 1982). A serious limitation of such an approach is that one is not sure that in every cell that is harvested the gene in question is actually in the process of transcription. This is so because transcription of specific genes depends on transcription factors whose activity can fluctuate according to the cell cycle or according to the growth state of the cells (Heintz et al., 1983; Heintz and Roeder, 1984). Since we have been interested in analyzing the chromatin architecture of a gene undergoing transcription per se we chose to analyze chromatin in which the nascent (unfinished) transcripts are still attached by their 3' ends to the template together with active RNA polymerase. SV40 DNA is found in infected cells in the form of a minichromosome. It is complexed with cellular histones very similar to cellular chromatin and uses the cellular machinery for transcription (Tooze, 1981; Aloni, 1982). A restricted transcriptional regulatory region of -400 bp involved in late transcription of the virus has been shown to possess, similarly to cellular genes, increased sensitivity to endogenous nuclease in 30%o of the minichromosomes (Scott and Wigmore, 1978; Waldeck et al., 1978), to DNase I in
