DNA‐binding domain as the minimal region driving RNA‐dependent liquid–liquid phase separation of androgen receptor

Abstract

Androgen receptor (AR) is a nuclear hormone receptor that regulates the transcription of genes involved in the development of testis, prostate and the nervous system. Misregulation of AR is a major driver of prostate cancer (PC). The primary agonist of full‐length AR is testosterone, whereas its splice variants, for example, AR‐v7 implicated in cancer may lack a ligand‐binding domain and are thus devoid of proper hormonal control. Recently, it was demonstrated that full‐length AR, but not AR‐v7, can undergo liquid–liquid phase separation (LLPS) in a cellular model of PC. In a detailed bioinformatics and deletion analysis, we have analyzed which AR region is responsible for LLPS. We found that its DNA‐binding domain (DBD) can bind RNA and can undergo RNA‐dependent LLPS. RNA regulates its LLPS in a reentrant manner, that is, it has an inhibitory effect at higher concentrations. As RNA binds DBD more weakly than DNA, while both RNA and DNA localizes into AR droplets, its LLPS depends on the relative concentration of the two nucleic acids. The region immediately preceding DBD has no effect on the LLPS propensity of AR, whereas the functional part of its long N‐terminal disordered transactivation domain termed activation function 1 (AF1) inhibits AR‐v7 phase separation. We suggest that the resulting diminished LLPS tendency of AR‐v7 may contribute to the misregulation of the transcription function of AR in prostate cancer.

Transcription Regulators and Membraneless Organelles Challenges to Investigate Them

“…Recent reports linked the regions that contain AFs to the LLPS phenomenon [ 81 ]. In steroid NR family representatives, the androgen receptor (AR), the estrogen receptor (ER), and the glucocorticoid receptor (GR), LBD (AF2) seems essential for this process [ 81 , 82 , 83 ]. It was found that, in a cellular model of prostate cancer, only full-length AR could phase separate on its own [ 84 ], where its splice variant lacking LBD, AR-v7, did not undergo LLPS [ 83 ].…”

“…In steroid NR family representatives, the androgen receptor (AR), the estrogen receptor (ER), and the glucocorticoid receptor (GR), LBD (AF2) seems essential for this process [ 81 , 82 , 83 ]. It was found that, in a cellular model of prostate cancer, only full-length AR could phase separate on its own [ 84 ], where its splice variant lacking LBD, AR-v7, did not undergo LLPS [ 83 ]. This indicates that a cooperative interaction between NTD and LBD of AR is important for the phase separation of this receptor [ 83 ].…”

“…It was found that, in a cellular model of prostate cancer, only full-length AR could phase separate on its own [ 84 ], where its splice variant lacking LBD, AR-v7, did not undergo LLPS [ 83 ]. This indicates that a cooperative interaction between NTD and LBD of AR is important for the phase separation of this receptor [ 83 ]. Additionally, in vitro analysis of AR NTD showed that it underwent LLPS at 100 μM concentration and the presence of the tumor suppressor speckle-type POZ protein (SPOP) lowered the concentration at which NTD of AR could phase separate [ 85 ].…”

“…Additionally, in vitro analysis of AR NTD showed that it underwent LLPS at 100 μM concentration and the presence of the tumor suppressor speckle-type POZ protein (SPOP) lowered the concentration at which NTD of AR could phase separate [ 85 ]. In vitro analysis of individual regions of AR showed that in the presence of RNA and DNA DBD could also undergo LLPS [ 83 ]. The process was downregulated by AF1 located in AR NTD.…”

“…Sensitivity to 1,6-hexanediol and also 1,2-pentanediol or 1,2-hexanediol is the characteristic for MLOs, for which contribution of hydrophobic interactions is observed. In the case where LLPS is driven by electrostatic interactions, 1,6-hexanediol has no effect [ 83 ]. However, Yuji Itoh and coworkers showed that 1,6-hexanediol removes water molecules around chromatin and locally condenses it.…”