Nuclease hypersensitivity of endogenous murine leukemia virus-related gene sequences in isolated nuclei

Inbred strains of mouse contain 40-60 copies of DNA sequences homologous to murine leukemia virus (MuLV) in various chromosomal locations; and, the majority of those sequences belong to the non-ecotropic and non-xenotropic MuLv-related proviral sequences or the polytropic classes. MuLv-related specific RNA transcripts were detected in the liver and kidney of RFM/Un and NFS/N mice. In order to investigate how many and which members of MuLV-related proviral sequences are responsible for the expression of the RNA transcripts, I investigated the nuclease hypersensitivity of MuLv-related proviral sequences in the nuclear chromatin of a few tissues of these mouse strains. The indirect end-labeling method using DNaseI was able to detect several strong DNasel-hypersensitive sites in the LTRs (long terminal repeats) of MuLv-related proviral sequences. Hypersensitive sites were mapped to immediately upstream and downstream of the 14-bp repeat negative regulatory element of the 5' and 3' LTRs, cap (transcription initiation) site and the "CAAT" box of the 5’ LTR, and the U3 region immediately upstream of the IS (insertion sequence) of the 3' LTR. Sensitivity to restriction endonuclease PstI was also studied in the isolated nuclei because MuLV and related proviral sequences contain a common PstI site in the LTR and DNasel-hypersensitive sites are located near the PstI site. The PstI site within the LTR of certain proviral sequences in the nuclei was sensitive to cleavage by this restriction endonuclease while the PstI sites in the structural gene region of the same proviral sequences were not. In addition, the PstI sensitivity was presented mainly in a minor population of MuLv-related proviral sequences, which contain sequence deletion(s) in the structural gene regions. It is known that chromatin structure involved in gene regulation is also associated with DNA methylation. The Smal site in the LTR partially overlaps with the Kpnl site, therefore methylation status at the Smal site can be detected by comparing the intensity of double digestion fragments between Pstl/Smal and Pstl/KpnI. Most of MuLV-related proviral sequences were methylated; however, in the liver the minor population of proviral LTR that were relatively sensitive to PstI digestion in the nuclei was digested by Smal, suggesting that these LTRs were relatively undermethylated in the DNA. In the test is, no such correlation between PstI hypersensitivity and Smal digestion was seen, suggesting that the proviral LTRs were relatively methylated, regardless of their nuclease hypersensitivity.