Enhancer sequences responsible for DNase I hypersensitivity in polyomavirus chromatin.

Abstract

DNase I preferentially cleaves polyomavirus minichromosomes at two sites in the enhancer, each of which comprises the sequence AAGCAPuPuAAG flanked by short inverted repeats. A tandem duplication of this sequence generates an additional hypersensitive locus. Mutations which alter either the AAGCAPuPuAAG or flanking repeats diminish hypersensitivity. This region must determine the chromatin conformation recognized by DNase I. The chromatin of the 5' flanking regions of many active or potentially active genes is unusually susceptible to nuclease attack (7). This 5' hypersensitivity probably reflects the accessibility of specific sequences in the DNA, but its physical nature and functional significance remain speculative (2,7,17). Polyomavirus is a useful system with which to study sequences conferring DNase I hypersensitivity to chromatin since its DNA can be manipulated and then reintroduced into mouse cells, in which it is packaged into minichromosomes. That region of the viral genome from the origin of replication to the sites of initiation of transcription of late mRNAs is hypersensitive to various nucleases and frequently lacks nucleosome morphology (3,14). These sequences are required both for DNA replication and early mRNA transcription and can serve as a transcriptional enhancer for cis-linked cellular genes (5,6,13,15,16,25,26).We examined the effect of mutations in this region on DNase I hypersensitivity. Our data indicated that altered chromatin structure is determined at the site of DNase I cleavage by the sequence AAGCAPuPuAAG flanked by small inverted repeats.Herbomel et al. (14) previously demonstrated that the enhancer region of the polyomavirus strain A2 contains two regions exhibiting DNase I hypersensitivity, but their analysis did not establish whether cleavage by DNase I was determined by sequences within or outside of the enhancer. To precisely map these DNase I-hypersensitivity sites, we used the technique of indirect end labeling (28) with the BamHI site as reference point (Fig. 1A).Mouse 3T6 cells were infected with polyomavirus strain A3 at 0.5 PFU per cell. After 24 h, the nuclei were isolated and digested with DNase I to produce approximately 1 cut per 10 kilobases. The DNA was purified, digested with BamHI, and fractionated by agarose gel electrophoresis, and then probed with a radiolabeled fragment extending from nucleotide 4659 to 5048. Of the polyomavirus minichromosomes cut with DNase I, most are cleaved in a region 470 to 570 base pairs (bp) from the BamHI site (Fig. 1C). DNase 1 digestion of purified DNA produces an entirely different * Corresponding author. t Present address: Genex Corp., Gaithersburg, MD 20877. pattern ( Fig. 2) and shows that specific cutting between the BamHI site and the site of origin of DNA replication is dependent on chromatin structure. Using densitometric scans, we established the two main locations of cutting to within 5 bp. The two hypersensitive sequences resemble each other in that cleavage occurs within a region containing the sequence AAGCAPuPu...

Nucleotides in the polyomavirus enhancer that control viral transcription and DNA replication.

“…This element is homologous to sequences in the transcriptional enhancers of the adenovirus 5 ElA gene (31) and the human p-interferon gene (26). Furthermore, it is duplicated in various laboratory isolates of polyomavirus (60,61) and is responsible for DNase I hypersensitivity of polyomavirus chromatin (8).…”

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

“…Base substitution mutations in the P (B) element result in their ability to allow Py to replicate in the mouse embryonal carcinoma cell line F9 (see reference 1 and references therein), presumably owing to increased enhancer function. DNase I-hypersensitive sites are located in both a (A) and P (B) regions (7,27), and nuclear proteins capable of binding to sites within the enhancer have been described (6,20,41,43). It has been speculated that the binding of specific proteins to the enhancer region modifies the interaction of the core ori with the replication complex (16).…”

A Detailed Analysis Of Duplications Appearing During Early, High Multiplicity Infections With Polyoma Virus

“…Moreover, an altered DNA conformation in the control region has been reported for SV40 using different approaches (Azorin & Rich, 1985;Deb et al, 1986;Iacono-Connors & Kowalski, 1986;Ostrander et al, 1988). Brian & Folk (1986) have defined sequence elements within the Py enhancer that are responsible for DNase I hypersensitivity; these elements are included in the duplicated region of the defective genomes. The rearrangement points on the late region side of the origin of replication defined by our experiments using MBN are close to this sequence and duplication of this region may enlarge the nucleosomefree gap.…”