The position and length of the steroid-dependent hypersensitive region in the mouse mammary tumor virus long terminal repeat are invariant despite multiple nucleosome B frames

Abstract

Stimulation of the mouse mammary tumor virus with steroids results in the generation of a DNase I-hypersensitive region (HSR) spanning the hormone responsive element (HRE) in the long terminal repeat. Restriction enzymes were used to characterize the accessibility of various sites within the HSR of mouse mammary tumor virus long terminal repeat-reporter constructions in four different cell lines. The glucocorticoid-dependent HSR was found to span minimally 187 bases, a stretch of DNA longer than that associated with histones in the core particle. Although the 5'-most receptor binding site within the HRE is downstream of -190, hypersensitive sites were found further upstream to at least -295. The relationship in the accessibility between pairs of sites in the vicinity of the HSR was further examined in one cell line by a two-enzyme restriction access assay. In the uninduced state, the accessibilities at these sites were found to be independent of each other. In contrast, when stimulated with hormone, the accessibilities at these sites were observed to become linked. That is, once a distinct promoter was activated, all of the sites within the HSR of that molecule became accessible. The HSR formed along an invariant stretch of DNA sequence despite the multiplicity of nucleosome frames in the nucleosome B region, where the HRE is located. The results indicate that the macroscopic length of the HSR does not arise from core length-remodeling events in molecules containing Nuc-B in alternative positions.

FIG. 1

Time course of accessibility in the HSR. (A) Nuclei were prepared from 3134 cells treated with dexamethasone for the times indicated. Aliquots containing a 10-μg equivalent of DNA were treated with either SacI (lanes 1 to 8) or HaeIII (lanes 9 to 16) at 1,000 U/ml and processed as described in Materials and Methods. Primer extension reactions were conducted with oligonucleotide 669 (lanes 1 to 8) or 670 (lanes 9 to 16), and the products were resolved in a sequencing gel. The times of dexamethasone treatment are shown above the lanes; the bands corresponding to the SacI and HaeIII cleavage sites are indicated (the doublet appearance is presumably due to the action of endogenous nucleases on the sticky ends). The upper band in each lane corresponds to the DpnII (lanes 1 to 8) or NlaIII (lanes 9 to 16) secondary cuts. (B) Cleavage at the SacI and HaeIII sites was quantitated and normalized to obtain the fractional cleavage, F(x). Duplicate data points from the first 5 h of a plot of F(x) as a function of time of dexamethasone treatment are plotted for the SacI and HaeIII sites, as indicated. (C) The change in fractional cleavage relative to time zero, ΔF(x), is plotted as a function of time. The means of the duplicates shown in panel B were used in this plot.

FIG. 2

Analysis of the accessibility and hypersensitivity of the LTR. (A) Positions of the restriction sites on the LTR. Fo, FokI; Hp, HphI; Ap, ApoI; Ha, HaeIII; Av, AvaII; Nl, NlaIII; Al, AlwNI; Sa, SacI. The ApoI site shown at −74 is found in the C3H strain of the LTR; the GR strain has a HinfI site at this position. In the −370 region, there are two sites for NlaIII, at −378 and at −362; there is also one site for RsaI at −365. The low-resolution positions of nucleosomes A through F in the LTR are denoted by ellipses. (B) Primer extension analysis of cleavage at various sites of the LTR (GR strain) in untreated (−) or 60-min-dexamethasone-treated (+) 19g11.2 cells. The cuts by the various enzymes, indicated above each pair of lanes, are marked by an asterisk to the right of each pair of lanes; the nucleotide position of the recognition site is shown at the bottom. The top band in each lane represents the secondary enzyme cut used for quantitation. The primers used for the analysis shown were (from left to right) 756, 764, 767, 767, 761, 761, 669, 669, 669, 766, and 766. (C) The fractional cleavage, F(x), for each site in the uninduced cells is shown for the 3134, 1471.1, 1361.5, and 19g11.2 cell lines, as indicated at the bottom. The LTR of the GR strain (in the 19g11.2 cells) lacks sites for NlaIII at −252, HaeIII at −225, and HphI at +21; the low signal from HphI at −972 could not be quantitated in this cell line because of high background in the assays. (D) the ΔF(x) after a 60-min dexamethasone treatment is shown; sites and cell lines are as in panel C.

FIG. 3

AlwNI-SacI two-enzyme digestion of 3134 cell nuclei. (A) aliquots of nuclei (5 μg of DNA) from uninduced or induced 3134 cells were digested in a volume of 50 μl with SacI at 2,500 U/ml and AlwNI at 0 (lanes 1 and 8), 10 (lanes 2 and 9), 25 (lanes 3 and 10), 50 (lanes 4 and 11), 100 (lanes 5 and 12), 250 (lanes 6 and 13), and 500 (lanes 7 and 14) U/ml. After digestion and extraction of the DNA, the MspII site at +103 was cut and quantitation was performed with oligonucleotide 765 for primer extension. The electrophoretic analysis of the extension products shows, from the bottom of the gel, the bands corresponding to cuts at the AlwNI, SacI, and MspII sites (indicated to the right); lanes 1 to 7, uninduced cells; lanes 8 to 14, dexamethasone-induced cells. The image is overexposed to highlight the SacI and AlwNI bands. This does not affect the phosphorimager quantitation but gives rise to the appearance that SacI cuts more than we report in panel C (e.g., over 50% by inspection of lane 8, in contrast to a quantitated value of 37%). (B) The F(SACI) in uninduced cells is plotted against the F(AlwNI) (squares); the broken line indicates the least-squares line through the data. The F(SACI) expected for independent (I), mutually exclusive (ME), and fully linked (L) outcomes was computed from equation (4), with the appropriate values of k, as described in the text. (C) The F(SACI) in induced cells is plotted against the F(AlwNI) (squares). The computed family of solutions differing in the value of the partition parameters w_SacI and w_AlwNI for the independent (I), mutually exclusive (ME), and fully linked (L) outcomes of F(SACI) versus F(AlwNI) are shaded. The boundary of the set of linked outcomes closest to the data has w_SacI and w_AlwNI values of 1; the line shown was computed with values of 0.8.

FIG. 4

FokI-SacI two-enzyme digestion of 3134 cell nuclei. (A) Aliquots of nuclei (10 μg of DNA) from uninduced or induced 3134 cells were digested in a volume of 100 μl with SacI at 2,500 U/ml and FokI at 0, 5, 10, 20, 30, 40, 50, 100, and 250 U/ml; the ApoI site at −74 was subsequently cut for the quantitation with oligonucleotide 730. The F(SACI) in uninduced cells is plotted against F(FokI) (squares). The computed F(SACI) outcomes expected from independent (I), mutually exclusive (ME), and fully linked (L) relations with AlwNI are indicated. The broken line indicates the best line through the data. (B) The F(SACI) in induced cells is plotted against the F(FokI) (squares). The family of solutions computed for each outcome of F(SACI) versus F(FokI) is shaded; the line through the data was computed with w_SacI and w_FokI values of 0.85; other details are as in Fig. 3.

FIG. 5

Linkage analysis of sites outside the HSR. (A) Aliquots of nuclei from uninduced or induced 3134 cells were digested with 2,500 U of RsaI per ml and increasing amounts of AlwNI, as in Fig. 3A; a HinfI site at −758 was subsequently cut for the quantitation with oligonucleotide 670. The F(RSAI) in uninduced cells is plotted against F(AlwNI) (squares). The computed F(RSAI) outcomes expected from independent (I), mutually exclusive (ME), and fully linked (L) relations with AlwNI are indicated. The best line through the data coincided with the independent outcome. (B) The F(RSAI) in induced cells is plotted against the F(AlwNI) (squares). The family of solutions computed for each outcome of F(RSAI) versus F(AlwNI) is shown shaded; the line shown was computed with w_RsaI and w_AlwNI of 0.25; details are as in Fig. 3.

FIG. 6

The decrease in F(A) is attributed to secondary cleavage interfering with detection. (A) Scheme used to assess the effect of cutting the SacI site on the accessibility of the AlwNI site. Induced and uninduced 3134 nuclei were digested with AlwNI at 1,000 U/ml and SacI at the concentrations shown in panels B and C. The extracted DNA was restricted with RsaI and extracted, and aliquots were taken for analysis by primer extension with either oligonucleotide 752 or 669. (B) F(AlwNI) plotted as a function of the SacI concentration with oligonucleotide 752 for analysis; triangles (Dex), induced nuclei; squares (Cont), uninduced nuclei. (C) F(ALWNI) plotted as a function of SacI concentration with oligonucleotide 669 for analysis; other details are as in panel B.

FIG. 7

Two alternative classes of models for the generation of the steroid-dependent HSR of MMTV. (A) Disturbance in the chromatin structure is restricted to a Nuc-B nucleosomal repeat. Binding of the glucocorticoid receptor (GR) to Nuc-B (left) triggers a series of changes such as decreased interactions between histone H1 and Nuc-B and/or altered contacts between the core histones and the adjacent linker-DNA. Such changes, in combination with differences between the higher-order chromatin structures of the steroid receptor-bound and unbound states, result in an increased accessibility of the entire Nuc-B-containing nucleosomal repeat (right, delimited by arrows). The newly accessible linker DNA could be either 5′, 3′, or both 5′ and 3′ of the glucocorticoid-responsive Nuc-B frame according to the mechanistic details. A very small set of adjacent Nuc-B frames, perhaps just one, responds to the steroid receptor. (B) Highly localized disturbance in the vicinity of a chromatin-bound transcription factor. cis elements are organized in various molecules in different chromatin configurations, in Nuc-B, Nuc-C, and the B/C linker, due to the existence of multiple nucleosome frames (top). GR binds to its cognate sites and further recruits additional trans activators. The chromatin in the immediate vicinity of various factors is disrupted. The end result of multiple localized disruptions is increased accessibility throughout the Nuc-C/Nuc-B chromatin stretch containing the bound trans activators, perhaps delimited by the bound GR (bottom).