The MMTV LTR adopts a specific nucleoprotein organization when introduced into cells. This structure involves the positioning of six nucleosomes (A-F) over the 1300 base pair LTR. A specific chromatin transition is induced by the binding of steroid receptors to the B nucleosome. By comparing the activity of transient MMTV reporter constructs (which are not organized in specific chromatin structures) with identical sequence elements that have replicated (and manifest the phased nucleosome array), we have shown that the chromatin transition is mechanistically important in transactivation, and that steroid receptors function on replicated genes by relieving chromatin repression. Our previous work showed that each phased nucleosome corresponds to a family of octamer cores positioned in that region. Thus, the low resolution phasing pattern results from the frequency- biased occupancy of a subset of these frames. We developed a new assay, the linked restriction enzyme assay, that permitted us to examine the nature of the nucleoprotein transition. We showed that the chromatin alteration does not correspond to a single nucleosome event, but involves both the B and C nucleosome families.We have now developed in vitro assembly systems that support the reconstitution of MMTV promoter DNA into ordered nucleosome arrays. Our results indicate that the addition of purified glucocorticoid receptor to the assembled arrays induces a chromatin transiition that is directly analogous to that observed in vivo. These findings indicate that the receptor induced nucleoprotein transition is reproduced in vitro, and are a first step to a system that will permit the analysis of the highly complex chromatin remodeling events that are now known to be fundamental to gene activation. - mammary gland, molecular interactions, Oncogenes, Proto-oncogenes, receptors, Retroviruses, acetylation,
Showing the most recent 10 out of 26 publications
