Phase Separation Mediated Sub-Nuclear Compartmentalization of Androgen Receptors

Abstract

The androgen receptor (AR), a member of the nuclear steroid hormone receptor family of transcription factors, plays a crucial role not only in the development of the male phenotype but also in the development and growth of prostate cancer. While AR structure and AR interactions with coregulators and chromatin have been studied in detail, improving our understanding of AR function in gene transcription regulation, the spatio-temporal organization and the role of microscopically discernible AR foci in the nucleus are still underexplored. This review delves into the molecular mechanisms underlying AR foci formation, focusing on liquid-liquid phase separation and its role in spatially organizing ARs and their binding partners within the nucleus at transcription sites, as well as the influence of 3D-genome organization on AR-mediated gene transcription.

Keywords: gene transcription regulation; liquid–liquid phase separation; nuclear receptors; prostatic neoplasms; subnuclear compartmentalization.

Figure 1

AR and its coregulators: schematic representation of the AR’s mode-of-action starting from hormonal stimulation (A), followed by DNA-binding at AR-binding sites (ARBS) containing androgen-responsive elements (AREs) at CIS-regulatory elements (CISs) as either monomer (not shown) or homodimer (B). Subsequently, AR interacts with various coregulators (C) to eventually initiate gene transcription by phosphorylating serine 5 of RNA polymerase II of the preinitiation complex (PIC) via the mediator complex (D).

Figure 2

Relationship between AR structural status and LLPS: (A) Confocal image of a cell expressing testosterone-stimulated EGFP-AR. Arrows indicate the location of some well-defined AR foci as example. (B) AR full-length wild type consists of an N-terminal domain (NTD), DNA-binding domain (DBD) and Ligand-binding domain (LBD). Upon stimulation by testosterone and other (synthetic) derivates, the AR wild type forms intranuclear foci whereas AR truncated mutants lacking either the NTD, DBD or the LBD (e.g., ARv7) are not able to form foci. Biophysical measurements using fluorescence recovery after photobleaching (FRAP) revealed that AR WT diffuses the slowest over time whereas the AR truncated mutants lacking the NTD, DBD or the LBD are significantly faster diffusing.

Figure 3

Three-dimensional chromatin regulation in context of AR biology. ARs are located at transcriptionally active and inactive regions, which are topologically organized in well-separated chromatin domain (TADs) by the cohesin complex, mainly located at euchromatic regions. Active and inactive TADs are characterized by presence of transcriptionally initiating or repressing epigenetic marks. ARs play role during Intra-TAD interactions, such as promotor-enhancer or promotor-insulator interactions, by potentially recruiting mainly cohesin-STAG2 complexes at enhancers. Outcome of this regulation depends on the type of CIS-regulatory elements (enhancer or insulator) present in the (intra-)TAD region, alongside the epigenetic status and chromatin compaction of the locus.

Figure 4

AR condensation in live cells. (A) schematic illustration showing protein condensation process known as LLPS. Proteins consisting intrinsically disorder regions (IDR), such as AR, are able to form membrane-less compartments in the nuclear environment together with cofactors and other biomolecules such as DNA and RNAs. (B) Mobile, highly diffusive ARs in N/C conformation are not yet nucleated on the DNA upon testosterone stimulation. (C,D) Shortly afterwards, AR condensation takes place, forming either transcriptionally active condensates (C) (containing coactivators such as HATs) or transcriptionally repressive condensates (D) (consisting corepressors such as HDACs) which both consists of confined molecules with a low diffusivity as consequence of direct DNA-binding and weak protein-protein interactions.