Reciprocal recruitment of DRIP/mediator and p160 coactivator complexes in vivo by estrogen receptor

Two functionally distinct classes of coactivators are recruited by liganded estrogen receptor, the DRIP/Mediator complex and p160 proteins, although the relative dynamics of recruitment is unclear. Previously, we have shown a direct, estradiol-dependent interaction between the DRIP205 subunit of the DRIP complex and the estrogen receptor (ER) AF2 domain. Here we demonstrate the in vivo recruitment of other endogenous DRIP subunits to ER in response to estradiol treatment in MCF-7 cells. To explore the relationship between DRIP and p160 coactivators, we examined the kinetics of coactivator recruitment to the ER target promoter, pS2, by chromatin immunoprecipitation. We observed a cyclic association and dissociation of coactivators with the promoter, with recruitment of p160s and DRIPs occurring in opposite phases, suggesting an exchange between these coactivator complexes at the target promoter.     

Differential role of two VDR coactivators, DRIP205 and SRC-3, in keratinocyte proliferation and differentiation

Cell programs such as proliferation and differentiation involve the selective activation and repression of gene expression. The vitamin D receptor (VDR), through 1,25(OH)(2)D(3), controls the proliferation and differentiation of keratinocytes. Previously, we have identified two VDR binding coactivator complexes. In proliferating keratinocytes VDR bound preferentially to the DRIP complex, whereas in differentiated keratinocytes the SRC complex was preferred. We proposed that different coactivators are required for sequential gene regulation in the transition from proliferation to differentiation. Here we examined the roles of DRIP205 and SRC-3 in this transition. Silencing of DRIP205 and VDR caused hyperproliferation of keratinocytes, demonstrated by increased XTT and BrdU incorporation. SRC-3 silencing, on the other hand, did not have an effect on proliferation. In contrast, SRC-3 as well as DRIP205 and VDR silencing blocked keratinocyte differentiation as shown by decreased expression of keratin 1 and filaggrin. These results are consistent with the differential localization of DRIP205 and SRC-3 in skin. These results indicate that DRIP205 is required for keratinocyte proliferation. Both DRIP205 and SRC-3 are required for the keratinocyte differentiation. These results support the concept that the selective use of coactivators by VDR underlies the selective regulation of gene expression in keratinocyte proliferation and differentiation.     

Vitamin D-interacting protein 205 (DRIP205) coactivation of estrogen receptor alpha (ERalpha) involves multiple domains of both proteins

Vitamin D-interacting protein 205 (DRIP205) is a mediator complex protein that anchors the complex to the estrogen receptor (ER) and other nuclear receptors (NRs). In ZR-75 breast cancer cells treated with 17beta-estradiol (E2) and transfected with a construct containing three tandem estrogen responsive elements (pERE(3)), DRIP205 coactivates ERalpha-mediated transactivation. DRIP205Delta587-636 is a DRIP205 mutant in which both NR boxes within amino acids 587-636 have been deleted and, in parallel transfection studies, DRIP205Delta587-636 also coactivates ERalpha. Moreover, both wild-type and variant DRIP205 also colocalize with ERalpha in the nuclei of transfected cells. Extensive deletion analysis of DRIP205 shows that multiple domains of this protein play a role in coactivation of ERalpha and in interactions with ERalpha. Coactivation of ERalpha by DRIP205 does not require NR boxes, and variants with deletion of N-terminal (amino acids 1-639) and C-terminal (amino acids 576-1566) significantly coactivate ERalpha. DRIP205 resembles p160 coactivators that also interact with multiple regions of ERalpha; however, unlike p160 coactivators, DRIP205 coactivation of ERalpha does not require NR boxes.