Potential mechanism of UPS and ERα cofactor network

So far, we showed significantly decreased estrogen-induced long-range interactions after proteasome inhibition using Bortezomib. Next, the underlying mechanism has to be analyzed.

We hypothesize that either ERα cofactors and/or other “looping factors” play a role in facilitating the formation of 3D interactions.

In a recent study using a murine embryonic stem cell system, mediator and cohesin were described as such “looping factors” which mediate looping formations between enhancers and gene promoters. It was shown that the transcriptional coactivator mediator; cohesin, the complex of proteins that holds sister chromatids together; and the cohesin-loading factor Nipbl co-occupy enhancer and core promoters of different genes, mediate DNA looping between the tested enhancers and promoters and thereby mediate gene transcription (Kagey et al., 2010). Cohesin has been reported to bind to CCCTC-binding factor (CTCF) binding sites and thereby mediates transcriptional insulation (Wendt et al., 2008). Importantly, an independent recent study using ChIP-Seq revealed that cohesin indeed colocalizes with the ERα on estrogen responsive genes in MCF-7 cells, in an estrogen-dependent and CTCF-independent manner. Further, combining these ChIP-Seq data with former genome-wide chromosomal ERα interaction data revealed that cohesin preferentially binds to interacting EREs which indicates to a function of cohesin in mediating long-range interactions

Discussion

102 between distal and proximal EREs and thereby regulating target gene transcription (Schmidt et al., 2010). The potential involvement of mediator and cohesin in our system as well as their regulation by proteasome activity will be examined.

Our previous work revealed two cofactors CLIM/LDB1/NLI and RLIM/RNF12 which regulate the activity of ERα. Both cofactors not only directly interact with ERα but are also present on the EREs of estrogen responsive genes. While CLIM inhibits ERα transcriptional activity, the ubiquitin ligase RLIM enhances transcription of ERα target genes (Johnsen et al., 2009). Since RLIM ubiquitinates CLIM and thereby causes its degradation, we think that inhibiting the proteasome destabilizes this network. Further, CLIM has already been implicated in long-range interaction and was shown to facilitate a loop structure between the ß-globin locus control region (LCR) and the ß-globin gene (Song et al., 2007). Since proteasome inhibition decreased ERα-dependent long-range interactions, we hypothesize that the effects after proteasomal inhibition on the spatial organization of ERα target genes may be due at least in part to the stabilization of CLIM. Based on this hypothesis we propose two possible models:

In model 1 (Figure 34 A), CLIM binds to the ERα dimer at the EREs, of e.g. CXCL12 gene and thus inhibits its transcriptional activity by recruiting the distal ERα enhancer to an unknown site “locus X” and thereby preventing the long-range intrachromosomal interactions between the distal and proximal EREs/enhancers. In the presence of estrogen the E3 ligase RLIM is recruited, ubiquitinates CLIM which in turn is degraded by the proteasome.

Subsequently, the cleared distal and proximal enhancers can come into close proximity via long-range interaction and thereby facilitate the expression of the CXCL12 gene. When proteasomal activity is blocked, e.g. by Bortezomib, CLIM is not degraded and therefore stays in contact with the distal ERE (enhancer site 1). Thus, looping formation between these sites is prevented and the negative regulation of ERα-mediated transcription by CLIM is maintained.

In model 2 (Figure 34 B), CLIM does not translocate the distal enhancer to an unknown site but rather directly prevents the binding of one or rather multiple unknown “looping factors Y”

such as a cohesin and/or mediator which promote long-range interactions between EREs. In the absence of proteasome inhibitor, RLIM causes the ubiquitination and degradation of CLIM and thus clears the binding sites for the “looping factor Y”. Upon binding of “factor Y”

intrachromosomal loopings are formed which bring the distal and proximal EREs into close proximity and thereby induce target gene transcription. In case of a blocked proteasome, the

Discussion

103 association between CLIM and ERα at the EREs is maintained, looping formation is prevented and thus ERα transcriptional activity is inhibited.

These two hypothetical models will be tested in the near future. Initially, 3C analyses will be performed in MCF-7 cells either depleted of CLIM and RLIM (via siRNA-mediated knockdown) or by overexpressing either factor in order to examine if these two ERα cofactors are in fact involved in estrogen-induced enhancer interactions. If that is the case, we will further combine e.g. knockdown and proteasome inhibitor conditions in 3C experiments. For example, we would expect a blockage of the negative effects of Bortezomib on the formation of long-range interactions in cells depleted of CLIM which in our model is the principal protein involved in preventing estrogen-induced looping events.

Collectively, this study reveals more insight into the complex mechanism by which the ubiquitin-proteasome system regulates nuclear hormone receptor-mediated transcription. Our data demonstrate that the UPS influences ERα transcriptional activity at various layers which exceed by far the simple receptor turnover.

Most results were obtained in an ERα-positive breast cancer cell line model and studies should be extended to other nuclear hormone receptor model systems such as the GR or AR as well as to normal human mammary epithelial cells. It may also be interesting to see if these interactions display a tissue- (i.e., mammary epithelial vs. endometrial) or ligand- (i.e., Tamoxifen vs. Raloxifene) specific effects. By understanding these mechanisms better it may be possible to develop even more specific anti-ERα (or anti-AR) treatments for breast (or prostate) cancer which specifically target the looping function of the nuclear hormone receptor/s.

Discussion

104

Figure 34: Models of CLIM/RLIM-mediated long-range interactions at CXCL12 locus. (A) Model 1. CLIM binds to the ERα dimer at ERE binding sites and prevents long-range interactions between enhancer site 1 and the proximal ERE site 3 by recruiting the enhancer to a unknown locus with the help of a factor “X”. ERα transcriptional activity is inhibited. In the presence of estrogen, the ubiquitin ligase RLIM ubiquitinates CLIM which in turn is proteolytically degraded. The freed distal and proximal EREs can interact via looping formation and CXCL12 gene expression is induced. Blockade of proteasomal activity by Bortezomib inhibits the degradation of CLIM which stays associated with ERα dimer and prevents CXCL12 transcription. For simplicity, CLIM is shown bound to only one ERE, although the mechanism may apply to both EREs. (B) Model 2. CLIM associates with the ERα dimer and thereby prevents the binding of a “looping factor Y” which is needed for long-range interaction formation between interaction sites 1 and 3. After estrogen induction, RLIM causes the ubiquitination and degradation of CLIM. The “looping factor Y” can bind to the ERE and induces looping formation and CXCL12 gene expression.

Appendix

105

6 Appendix