Estrogen-induced and TAFII30-mediated gene repression by direct recruitment of the estrogen receptor and co-repressors to the core promoter and its reversal by tamoxifen.

Estradiol (E2) acts through the estrogen receptor (ER) to downregulate many genes, and tamoxifen (Tam) largely reverses this repression but the underlying mechanisms are unclear. Repression of the folate receptor (FR)-alpha P4 core promoter by ER is enhanced by E2 and reversed by Tam. This effect was unaffected by inhibition of new protein synthesis and required the E/F and the DNA-binding domains of ER without direct binding of ER to DNA. The repression by E2/ER was not specific for either Sp1 or TATA elements but was loosely selective for the initiator and flanking sequence. Insertion of a response element or a relatively strong Sp1 cluster to recruit ER upstream of the core promoters caused a switch to activation by E2/ER that was inhibited by Tam. In nuclear extracts, association of ER with a biotinylated core promoter fragment was promoted by E2 but Tam blocked this effect. Repression/de-repression of the P4 promoter and endogenous FR-alpha expression by E2/Tam required SMRT and/or NCoR. ER associated with the chromosomal P4 promoter and SMRT and NCoR associated with it in an ER-dependent manner; these associations were favored by E2 but disrupted by Tam, in the short term, without changes in ER expression. TAFII30 was required for optimal P4 promoter activity and for the repressive association of ER. E2 may thus maintain a low transcriptional status of genes by favoring direct TAFII30-dependent association of ER with the core promoter in a co-repressor complex containing SMRT and/or NCoR; this repression is overridden in target genes containing an upstream element that strongly recruits ER. In addition to suppressing the activation of classical E2 target genes, Tam may upregulate genes by passively dissociating the ER co-repressor complex.

Evidence for segregation of heterologous GPI-anchored proteins into separate lipid rafts within the plasma membrane.

Cholesterol and glycosphingolipid-rich membrane rafts, which are rich in GPI-anchored proteins and are distinct from caveolae, are believed to serve as platforms for signal transduction events and protein recycling. GPI-anchored proteins with diverse functions as well as caveolin may be recovered in a membrane fraction insoluble in cold non-ionic detergent. This study tests for possible heterogeneity in the protein composition of the lipid rafts and detergent-insoluble membrane complexes by examining the two GPI-anchored homologous human folate receptors (FR)-alpha and -beta, the GPI-anchored human placental alkaline phosphatase (PLAP), and caveolin (control) in transfected CHO cells. Both FR and PLAP showed the equal distribution of cell-surface vs. sequestered (recycling) protein typical of GPI-proteins. Quantitative affinity purification of detergent-insoluble complexes using biotinylated folate or specific antibodies demonstrated a strong association of the homologous FR-alpha and FR-beta in the same detergent-insoluble complex and separate complexes containing either PLAP or caveolin. Immunogold localization experiments using antibody crosslinking to produce larger aggregates of GPI-anchored proteins for visualization by electron microscopy also showed a clear separation between FR- and PLAP-rich membrane microdomains. Thus, even though functionally diverse and heterologous GPI-anchored proteins are known to share endocytic and recycling vesicles, they may be segregated in distinct lipid rafts on the basis of their ecto(protein) domains facilitating clustering, compartmentalization and homotypic protein interactions.