Destabilization of nucleosomes by an unusual DNA conformation adopted by poly(dA) small middle dotpoly(dT) tracts in vivo

Abstract

Poly(dA) small middle dotpoly(dT) tracts are common and often found upstream of genes in eukaryotes. It has been suggested that poly(dA) small middle dotpoly(dT) promotes transcription in vivo by affecting nucleosome formation. On the other hand, in vitro studies show that poly(dA) small middle dotpoly(dT) can be easily incorporated into nucleosomes. Therefore, the roles of these tracts in nucleosome organization in vivo remain to be established. We have developed an assay system that can evaluate nucleosome formation in yeast cells, and demonstrated that relatively longer tracts such as A(15)TATA(16) and A(34) disrupt an array of positioned nucleosomes, whereas a shorter A(5)TATA(4) tract is incorporated in positioned nucleosomes of yeast minichromosomes. Thus, nucleosomes are destabilized by poly(dA) small middle dotpoly(dT) in vivo in a length-dependent manner. Furthermore, in vivo UV footprinting revealed that the longer tracts adopt an unusual DNA structure in yeast cells that corresponds to the B' conformation described in vitro. Our results support a mechanism in which a unique poly(dA) small middle dot poly(dT) conformation presets chromatin structure to which transcription factors are accessible.

Fig. 1. Chromatin structure of TALS minichromosomes in yeast α cells and DNA inserts used in this study. TALS contains the α2 operator (indicated by the hatched box) from the STE6 promoter at the EcoRI site of TRP1/ARS1 plasmid, and the location of nucleosomes in TALS was determined previously (Roth et al., 1990). DNA inserts were cloned into the SacI site of TALS, corresponding to near the pseudo-dyad axis of nucleosome IV, or into the DraIII site corresponding to the linker region between nucleosomes III and IV.

Fig. 2. Locations of the positioned nucleosome IV, α2 operator and poly(dA)⋅poly(dT) inserts in TALS and its derivative minichromosomes as described (Shimizu et al., 1991; Kladde and Simpson, 1994). The SacI and DraIII sites are at 1460 and 1412 m.u. (map units), respectively, in nucleosome IV as shown by the shaded ellipse. The α2 operator and poly(dA)⋅poly(dT) tracts are shown as hatched and shaded boxes, respectively. The polylinker attached with the A₁₅TATA₁₆ insert is indicated by white boxes (see Materials and methods).

Fig. 3. Primer extension analysis of MNase cleavage sites in the nucleosome IV region of TALS and its derivative minichromosomes. Plasmids pTS703, pOM801, pTS713, pOM802 and pTS711 contain A₁₅TATA₁₆, A₃₄, A₇TATA₈, A₅TATA₄ and GC(GAGCTCGC)₆, respectively, at the SacI site of TALS. Plasmid pTS704 contains A₁₅TATA₁₆ at the DraIII site. Chromatin (in isolated nuclei) (lanes labeled C) and naked DNA (lanes labeled D) were digested at 37°C for 10 min with various concentrations of micrococcal nuclease as follows: 2.5 U/ml (lanes 1, 4, 7, 10, 13, 16 and 19), 1.25 U/ml (lanes 3, 6, 9, 12,15,18 and 21) and 0.05 U/ml (lanes 2, 5, 8, 11, 14, 17 and 20), as described (Shimizu et al., 1991). The nucleosome IV regions are shown on the left of each gel by brackets. The locations of nucleosomes II to IV and the α2 operator are schematically shown to the left of the gel.

Fig. 4. Chromatin structure of TALS and its derivatives analyzed by indirect end-labeling. Plasmids pTS703, pOM801, pTS713, pOM802 and pTS711 contain A₁₅TATA₁₆, A₃₄, A₇TATA₈, A₅TATA₄ and GC(GAGCTCGC)₆, respectively, at the SacI site of TALS. Plasmid pTS704 contains A₁₅TATA₁₆ at the DraIII site. Chromatin (in isolated nuclei) (lanes labeled C) and naked DNA (lanes labeled D) were digested at 37°C for 10 min with micrococcal nuclease as follows: 2.5 U/ml (odd-numbered lanes) and 0.05 U/ml (even-numbered lanes). The samples were digested with EcoRV and resolved by electrophoresis on 1.2% agarose gel, then the MNase cleavage sites were detected by indirect end-labeling using the EcoRV–HindIII fragment (385–615 m.u. in TALS; see Figure 1) as a probe (Roth et al., 1990). The locations of nucleosomes I to VII and the α2 operator are shown to the left of the gel. The four cleavage sites of TALS in naked DNA, but not in chromatin, are indicated by *a, *b, *c and *d.

Fig. 5. Nucleosome repeat analysis of TALS and its derivative minichromosomes. Plasmids pTS703, pOM801, pTS713, pOM802 and pTS711 contain A₁₅TATA₁₆, A₃₄, A₇TATA₈, A₅TATA₄ and GC(GAGCTCGC)₆, respectively, at the SacI site of TALS. Chromatin (in isolated nuclei) was digested at 37°C for 10 min with micrococcal nuclease: 2.5 U/ml (odd-numbered lanes) and 1.25 U/ml (even-numbered lanes). The MNase digest samples were resolved by 1.3% agarose gel electrophoresis. (A) Southern blots of MNase-digested chromatin were probed using a ³²P-labeled oligonucleotide with a complementary sequence adjacent to the SacI site in nucleosome IV (proximal probe, top of panel; see Materials and methods). (B) After stripping the proximal probe off, the blots were re-probed using a ³²P-labeled oligonucleotide with a sequence complementary to the nucleosome IX region (distal probe, top of panel; see Materials and methods).

Fig. 5. Nucleosome repeat analysis of TALS and its derivative minichromosomes. Plasmids pTS703, pOM801, pTS713, pOM802 and pTS711 contain A₁₅TATA₁₆, A₃₄, A₇TATA₈, A₅TATA₄ and GC(GAGCTCGC)₆, respectively, at the SacI site of TALS. Chromatin (in isolated nuclei) was digested at 37°C for 10 min with micrococcal nuclease: 2.5 U/ml (odd-numbered lanes) and 1.25 U/ml (even-numbered lanes). The MNase digest samples were resolved by 1.3% agarose gel electrophoresis. (A) Southern blots of MNase-digested chromatin were probed using a ³²P-labeled oligonucleotide with a complementary sequence adjacent to the SacI site in nucleosome IV (proximal probe, top of panel; see Materials and methods). (B) After stripping the proximal probe off, the blots were re-probed using a ³²P-labeled oligonucleotide with a sequence complementary to the nucleosome IX region (distal probe, top of panel; see Materials and methods).

Fig. 6. In vivo UV photofootprinting of the T₃₄ and T₁₆ATAT₁₅ strands in pOM801 and pTS703 minichromosomes. Intact cells (lanes labeled C) and naked DNA (lanes labeled D) were irradiated at 254 nm with different UV dosages as follows: 500 mJ (lanes 1, 4, 7 and 10), 750 mJ (lanes 3, 6, 9 and 12) and 60 mJ (lanes 2, 5, 8 and 11), as described (Shimizu et al., 1997, 1998). The nucleosome IV region and the α2 operator are shown by brackets on the left of the gel. The sequences of T₃₄ and T₁₆ATAT₁₅ tracts and the α2 operator are shown to the side of gel. + indicates the sites protected from UV irradiation in cells.