In the present study we analyze the functional effects concerning of SENP induced PR deSUMOylation in detail. Our results indicate that on a compound promoter, SENP1 enhances transcription in a dose dependent manner, but this requires full length PR. However enhanced transcription is independent of PR DNA binding specificity or the PR S294 phosphorylation site. By deSUMOylating PR, SENP increases PR sensitivity to hormone. The histone deacety lase inhibitor Trichostatin A has a marked biphasic effect. At high concentrations, which promote global his tone hyperacetylation and modify many proteins, TSA strongly suppresses transcription and this is reversed by the coactivator SRC 1. However, low TSA concentra tions upregulate PR dependent transcription.

This effect of TSA is uncoupled from inhibition by SUMOylation indicating that HDAC activity is not involved in transcrip tional synergy controlled by SENP1. Results SENP and PR deSUMOylation SUMOylation and the promoter context of PR transcriptional synergy Figure 1A is a schematic of PR B and PR A showing location of the single ��KxE SUMO conjugation motif centered at K388 of PR B. Also shown are 3 hormone dependent serine and multiple other N terminal phosphorylation sites, and a hinge domain KxKK acetylation site. We previously showed that SUMOylation at K388 is hormone dependent and suppresses PR B and PR A regulated transcription of an exogenous promoter containing two or more palindromic PREs but not a single PRE. To assess the generality of this, we used the MMTV LTR, which contains 1 palindromic PRE and 3 PRE half sites.

In contrast to GRs that prefer the palin drome, the half sites are preferentially used by PRs, possibly as monomers. To examine the role of PR SUMOylation on transcriptional synergy involving PRE half sites, HeLa cells were transfected with 5 1000 ng of DNA encoding wild type PR B or the SUMOylation defi cient K388R PR B mutant, together with the PRE2 Luc or MMTV Luc reporters, in the presence of the progestin R5020. PR B were tested since they are more potent transactivators of the MMTV LTR than PR A. On PRE2 Luc, wild type PR B were transcriptionally active, and mutation of their K388 SUMOylation motif synergistically raised transcription further as receptor concentrations were increased between 5 and 100 ng DNA. High PR concentrations led to a decrement in transcription likely due to transcription factor squelching.

Wild type PR B dependent transcription on MMTV LTR showed a similar dose dependent increase. However, absolutely no tran scriptional synergy was observed with the K388R PR B mutant suggesting that SUMOylation does not control synergy on PRE half sites. Most of the studies below Brefeldin_A use PRE2 Luc DeSUMOylation by SENP The K388R PR mutant is an artificial construct while proteins are naturally deSUMOylated by SENPs in vivo.