Fig. 3. Chromatin structure of CatD regulatory regions and gene expression. A – Comparison of DNase I hypersensitivity of CatD gene in the 3 cell lines, untreated or treated with hormone (E2). B - Mapping of the DNase I hypersensitive sites in the 5’-flanking sequences of CatD gene. C– Influence of estradiol (E2) and two anti-estrogens (ICI and OH-Tam) on CatD mRNA expression in the 3 cell lines.hormone is present, there was only one hormone-independent DNase    Ihypersensitive site (pS2-HS2). This site maps immediately upstream of site pS2-HS1, and encompasses a region containing an AP1 binding site (Fig 2C). A synergistic effect of estrogens and IGFI on pS2 gene transcription has been described. We have compared the chromatin structure of pS2 gene in MCF7 cells untreated or treated with estradiol, IGFI or both. Estrogen or IGF treatment induced DNase I hypersensitive sites that are indistinguishable (pS2-HS 1 and pS2-HS4, Fig 2B). Treatment with both IGFI and estradiol did not increase the intensity of the hypersensitive sites, contrasting with the effect on RNA synthesis (Fig 2D). This is the first description of a DNase I hypersensitive site induced by a growth factor. The fact that hypersensitive sites induced by estradiol and IGFI are indistinguishable may be due to the recruitment of the partners from different signal transduction pathways in a large coactivator complex.

3.THE CatD GENE

We have performed the same chromatin structure analysis as for the pS2 gene in the three cell lines (Fig. 3). Contrasting with the results obtained for pS2 gene, there is no hormone-dependent DNase I hypersensitive site in the regulatory regions of CatD gene (Fig 3A), although RNA synthesis is induced by estradiol in the hormone dependent cell line MCF7 (Fig 3C). In HE5 cells the expression of estrogen receptor allows recovering the hormonal regulation of CatD while in the parental cell line MDA MB 231 CatD was constitutively expressed at a high level (Fig 3C). In these two cell lines the DNase I hypersensitivity profiles are indistinguishable. Both cell lines display an additional site compared to MCF7 cells (CatD-HS4, Fig. 3A). Similar studies in other hormone-independent cell lines are in progress to determine if the presence of this DNase I hypersensitive site is characteristic of the hormone-independent status, or of MDA MB 231 cells.

4. CONCLUSION

      The promoters of pS2 and CatD genes have been extensively studied with classical approaches. The comparison of chromatin structure changes in the regulatory regions of the two genes suggests that the mechanisms controlling their expression are different. The lack of expression of pS2 in cell lines MDA MB 231 and HE5 correlates with a closure of the hormone-dependent hypersensitive sites pS2-HS1 and pS2-HS4. As it should be expected from mRNA measurements, the presence of ERa in HE5 cells has no effect on   the chromatin structure. This indicates that in hormone-independent cells there is a closure of the chromatin over large domains (Fig. 1 - A), one of them containing the pS2 gene. Extinction of ERα gene in hormone-independent cells may obey to similar mechanisms. It has been established that factors other than ERα are involved in the progression of the cells towards hormone-independent growth. Such factors could also play a critical role in modulating chromatin structure. The situation for CatD gene is different. In hormone-dependent cells, CatD transcription is controlled by estrogens. However changes in transcription level are not accompanied by chromatin structure changes. Contrasting with pS2 gene, in hormone-independent cells, cathepsin D is constitutively expressed at a high level; in these cells, re-expression of ERα allows recovering the hormonal control of CatD gene expression. This indicates that in hormone-independent cells, it is the lack of ERα that results in an abnormally high expression of CatD. The chromatin structure of the regulatory regions of CatD does not undergo a closure in hormone-independent cells. DNase I hypersensitive sites are constitutive, allowing gene expression (actively transcribing chromatin). In the presence of unliganded ERαthere is a decrease in CatD expression. This decrease is not accompanied by detectable changes in chromatin structure probably because the chromatin being already open, proteins can fully interact with each other and regulate transcription. In this context, the exact role of histone acetyltransferases, histone deacetylases and/or chromatin remodelling complexes remain to elucidate.