Agonist-controlled competition of RAR and VDR nuclear receptors for heterodimerization with RXR is manifested in their DNA binding.

We found previously that nuclear receptors (NRs) compete for heterodimerization with their common partner, retinoid X receptor (RXR), in a ligand-dependent manner. To investigate potential competition in their DNA binding, we monitored the mobility of retinoic acid receptor (RAR) and vitamin D receptor (VDR) in live cells by fluorescence correlation spectroscopy. First, specific agonist treatment and RXR coexpression additively increased RAR DNA binding, while both agonist and RXR were required for increased VDR DNA binding, indicating weaker DNA binding of the VDR/RXR dimer. Second, coexpression of RAR, VDR, and RXR resulted in competition for DNA binding. Without ligand, VDR reduced the DNA-bound fraction of RAR and vice versa, i.e., a fraction of RXR molecules was occupied by the competing partner. The DNA-bound fraction of either RAR or VDR was enhanced by its own and diminished by the competing NR's agonist. When treated with both ligands, the DNA-bound fraction of RAR increased as much as due to its own agonist, whereas that of VDR increased less. RXR agonist also increased DNA binding of RAR at the expense of VDR. In summary, competition between RAR and VDR for RXR is also manifested in their DNA binding in an agonist-dependent manner: RAR dominates over VDR in the absence of agonist or with both agonists present. Thus, side effects of NR-ligand-based (retinoids, thiazolidinediones) therapies may be ameliorated by other NR ligands and be at least partly explained by reduced DNA binding due to competition. Our results also complement the model of NR action by involving competition both for RXR and for DNA sites.

Agonist binding directs dynamic competition among nuclear receptors for heterodimerization with retinoid X receptor.

Retinoid X receptor (RXR) plays a pivotal role as a transcriptional regulator and serves as an obligatory heterodimerization partner for at least 20 other nuclear receptors (NRs). Given a potentially limiting/sequestered pool of RXR and simultaneous expression of several RXR partners, we hypothesized that NRs compete for binding to RXR and that this competition is directed by specific agonist treatment. Here, we tested this hypothesis on three NRs: peroxisome proliferator-activated receptor gamma (PPARγ), vitamin D receptor (VDR), and retinoic acid receptor alpha (RARα). The evaluation of competition relied on a nuclear translocation assay applied in a three-color imaging model system by detecting changes in heterodimerization between RXRα and one of its partners (NR1) in the presence of another competing partner (NR2). Our results indicated dynamic competition between the NRs governed by two mechanisms. First, in the absence of agonist treatment, there is a hierarchy of affinities between RXRα and its partners in the following order: RARα > PPARγ > VDR. Second, upon agonist treatment, RXRα favors the liganded partner. We conclude that recruiting RXRα by the liganded NR not only facilitates a stimulus-specific cellular response but also might impede other NR pathways involving RXRα.

Simultaneous Mapping of Molecular Proximity and Comobility Reveals Agonist-Enhanced Dimerization and DNA Binding of Nuclear Receptors.

Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)mobility by fluorescence (cross-)correlation spectroscopy (F(C)CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation and facilitates comapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed, and an extension of the molecular switch model of the nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR-RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. The RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.

Arginine Methyltransferase PRMT8 Provides Cellular Stress Tolerance in Aging Motoneurons.

Aging contributes to cellular stress and neurodegeneration. Our understanding is limited regarding the tissue-restricted mechanisms providing protection in postmitotic cells throughout life. Here, we show that spinal cord motoneurons exhibit a high abundance of asymmetric dimethyl arginines (ADMAs) and the presence of this posttranslational modification provides protection against environmental stress. We identify protein arginine methyltransferase 8 (PRMT8) as a tissue-restricted enzyme responsible for proper ADMA level in postmitotic neurons. Male PRMT8 knock-out mice display decreased muscle strength with aging due to premature destabilization of neuromuscular junctions. Mechanistically, inhibition of methyltransferase activity or loss of PRMT8 results in accumulation of unrepaired DNA double-stranded breaks and decrease in the cAMP response-element-binding protein 1 (CREB1) level. As a consequence, the expression of CREB1-mediated prosurvival and regeneration-associated immediate early genes is dysregulated in aging PRMT8 knock-out mice. The uncovered role of PRMT8 represents a novel mechanism of stress tolerance in long-lived postmitotic neurons and identifies PRMT8 as a tissue-specific therapeutic target in the prevention of motoneuron degeneration.SIGNIFICANCE STATEMENT Although most of the cells in our body have a very short lifespan, postmitotic neurons must survive for many decades. Longevity of a cell within the organism depends on its ability to properly regulate signaling pathways that counteract perturbations, such as DNA damage, oxidative stress, or protein misfolding. Here, we provide evidence that tissue-specific regulators of stress tolerance exist in postmitotic neurons. Specifically, we identify protein arginine methyltransferase 8 (PRMT8) as a cell-type-restricted arginine methyltransferase in spinal cord motoneurons (MNs). PRMT8-dependent arginine methylation is required for neuroprotection against age-related increased of cellular stress. Tissue-restricted expression and the enzymatic activity of PRMT8 make it an attractive target for drug development to delay the onset of neurodegenerative disorders.