Abstract
Regulatory signal that activates eukaryotic transcription factors can originate from a very distant source in the body. For instance, hormones released into the circulatory system by an organ that is part of the endocrine system travel through the circulation to essentially all parts of the body. The endocrine system can thus serve as a regulator to coordinate changes in transcription in cells of many different tissues. Some hormones are small molecules that, because of their lipid-solubility properties, can directly pass through the plasma membrane of the cell – like steroid hormones, such as glucocorticoid, testosterone, and estrogen. In the cell, steroid hormones bind to and regulate specific transcription factors in the nucleus. In metazoans they are called nuclear receptors (NRs). These are transcription factors that can regulate the expression of their target genes in a ligand dependent manner.
Our studies concentrated on the dynamic nature of transcriptional regulation. The retinoic x receptor (RXR), as a central molecule of nuclear receptor action, was in the focus of our work. The general concept was to apply methods with different temporal (and spatial) resolution and gain insight to various sides of the involved mechanisms. In contrast with the majority of earlier investigations of the field, the used microscopy methods could be operated in live cells.
We characterized and compared the nuclear dynamics of RXR and retinoic acid receptor (RAR) during activation in single cells on the sub-second scale using live-cell imaging methods. By applying FRAP and fluorescence correlation spectroscopy (FCS), techniques with different temporal resolution, a highly dynamic behaviour could be shown, which is best described by a two-state model of receptor mobility. In the unliganded state most NRs belonged to the fast population. Upon agonist treatment, the ratio of the slow population increased to as a result of an immediate redistribution. Coactivator binding appears to be indispensable for redistribution and has a major contribution to chromatin association.
A comparison of the FCS results gained from the RXR and from the RAR studies revealed differences on the behaviour of these two molecules: RXR appeared to be more dynamic. It changed its mobility at a larger scale upon ligand activation. The redistribution detected by FCS was confirmed and refined by single plane illumination microscopy (SPIM-FCS). In addition, it was revealed that the distribution of populations was rather homogenous, as no nuclear architecture related pattern was recognized either before or after activation.
Investigation of activation dependent changes showed that the occupancy of RXR’s genomic binding regions increased, but no significant change in the number of sites was revealed by ChIP-Seq.
The relationship of the RXR and other nuclear receptors during activation is an interesting question of the field. How the available binding sites, the ligands and the dimer partners determine and regulate the formation of different RXR heterodimers might now be answered. Another interesting topic is the relationship of the chromatin, the transcription factors and the actual active sites of transcriptions. Newer applications partially related to these methods are already available; such as SPIM-FCCS (SPIM - Fluorescence Cross- Correlation Spectroscopy) for the nuclear map of protein-protein interactions, or the GROseq (Global Run-On sequencing) for the detection of active RNA-production on a global scale. When the aim is to describe a mechanism, the key might still be the combined use of methods with different ways of targeting.