Evidence that nucleosomes on the mouse mammary tumor virus promoter adopt specific translational positions

Abstract

We have previously demonstrated that an array of six nucleosomes are phased on the mouse mammary tumor virus (MMTV) long terminal repeat (1,2). In this study, we devised a new assay to measure the translational positions of specific nucleosomes on the MMTV promoter. Nucleosome core particles were purified and shown to contain A and B nucleosomal DNA by Taq polymerase primer extension with nucleosome-specific primers. The 5' and 3' boundaries of A and B nucleosomes were measured by extending to the end of the core DNA with internal primers. This approach yielded results consistent with major translational positions of -23 to +123 and -221 to -75 for A and B nucleosomes, respectively. The micrococcal nuclease cleavage patterns of A and B nucleosome regions in isolated nuclei are conserved at base-pair resolution in multiple murine cell lines containing either stable MMTV-reporter chimeras or endogenous proviruses. As the refined nucleosome positions place important transcription factor binding sites at the 3' edge of the B nucleosome and in the nucleosome A/B linker, we propose that linker histone depletion and chromatin unfolding may be required to expose these cis-elements during steroid hormone-induced transcription initiation.

Nucleosome positioning on the MMTV LTR results from the frequency-biased occupancy of multiple frames.

“…A periodic micrococcal nuclease (MNase) and methidiumpropyl-EDTA-iron [MPE-Fe(II)] digestion pattern consistent with six positioned nucleosomes (designated A-F) has been observed for the long terminal repeat (LTR) in a variety of contexts (Richard-Foy and Hager 1987). Chromatin in the promoter region, nucleosomes A (Nuc-A) and B (Nut-B), has been characterized further at high resolution (Bresnick et al 1992b;Truss et al 1993;Mymryk et al 1995); at base-pair resolution, the nucleoprotein structure in different cells is again found to be indistinguishable, even with the promoter fused to a variety of reporter genes, and the constructs present in different copy numbers.…”

“…Short fragments of the LTR, when reconstituted with histones into nucleosomes in vitro, will wrap on the histone octamer with the DNA in a rotationally fixed orientation (Perlmann and Wrange 1988;Pina et al 1990a, b;Bresnick et al 1991), leading to the suggestion that cores on the MMTV LTR, the B nucleosome in particular, may adopt a unique rotational and translational position (Truss et al 1992). Although our previous characterization of A-B region chromatin indicated a highly reproducible structure (Richard-Foy and Hager 1987;Bresnick et al 1992b), we have been unable to obtain evidence for unique rotational and translational positioning.…”

Steroid-selective Initiation of Chromatin Remodeling and Transcriptional Activation of the Mouse Mammary Tumor Virus Promoter Is Controlled by the Site of Promoter Integration

“…Recently evidence has been presented for a direct interaction of GR with the human SWI-SNF homolog, BRG1 (30). Activation of the MMTV promoter is also accompanied by a partial depletion of histone H1 from the template in vivo (31).…”

Papel de BRG1 e Brm, reguladores globais de transcrição, na reversão fenotípica de células ST1 pela ação de glicocorticóides

“…A ação de GR é acompanhada de alterações na estrutura da cromatina (Archer et al, 1991, Bresnick et al, 1992, sendo que estas alterações são provocadas por A linhagem celular C6, transformada e tumorigênica, foi isolada a partir de um glioma de rato, induzido quimicamente por metilnitrosouréia (Benda et al, 1968). Esta linhagem responde ao tratamento com hormônios glicocorticóides, com diminuição da proliferação e recuperação da resposta a fatores peptídicos de crescimento (Armelin et al, 1982;1983; e Freshney, 1984.…”

“…A ação de GR é acompanhada de alterações na estrutura da cromatina (Archer et al, 1991, Bresnick et al, 1992, sendo que estas alterações são provocadas por complexos protéicos co-ativadores transcricionais, como SWI/SNF. A ligação de GR a GRE foi capaz de estimular a desestruturação de nucleossomos, pelo complexo SWI/SNF, in vitro (Östlund et al, 1997).…”