Steroid receptors regulate gene expression in a ligand-dependent manner by binding specific DNA sequences. Ligand binding also changes the conformation of the ligand binding domain (LBD), allowing interaction with coregulators via LxxLL motifs. Androgen receptors (ARs) preferentially interact with coregulators containing LxxLL-related FxxLF motifs. The AR is regulated at an extra level by interaction of an FQNLF motif in the N-terminal domain with the C-terminal LBD (N/C interaction). Although it is generally recognized that AR coregulator and N/C interactions are essential for transcription regulation, their spatiotemporal organization is largely unknown. We performed simultaneous fluorescence resonance energy transfer and fluorescence redistribution after photobleaching measurements in living cells expressing ARs double tagged with yellow and cyan fluorescent proteins. We provide evidence that AR N/C interactions occur predominantly when ARs are mobile, possibly to prevent unfavorable or untimely cofactor interactions. N/C interactions are largely lost when AR transiently binds to DNA, predominantly in foci partly overlapping transcription sites. AR coregulator interactions occur preferentially when ARs are bound to DNA.

Proteins involved in chromatin-interacting processes, like gene transcription, DNA replication, and DNA repair, bind directly or indirectly to DNA, leading to their immobilisation. However, to reach their target sites in the DNA the proteins have to somehow move through the nucleus. Fluorescence recovery after photobleaching (FRAP) has been shown to be a strong approach to study exactly these properties, i.e. mobility and (transient) immobilisation of the proteins under investigation. Here, we provide and discuss detailed protocols for some of the FRAP procedures that we have used to study protein behaviour in living cell nuclei. In addition, we provide examples of their application in the investigation of the androgen receptor (AR), a hormone-inducible transcription factor, and of two DNA-maintenance factors, the telomere binding proteins TRF1 and TRF2. We also provide protocols for qualitative FRAP analysis and a general scheme for computer modelling of the presented FRAP procedures that can be used to quantitatively analyse experimental FRAP curves.

Telomeric proteins have an essential role in the regulation of the length of the telomeric DNA tract and in protection against end-to-end chromosome fusion. Telomere organization and how individual proteins are involved in different telomere functions in living cells is largely unknown. By using green fluorescent protein tagging and photobleaching, we investigated in vivo interactions of human telomeric DNA-binding proteins with telomeric DNA. Our results show that telomeric proteins interact with telomeres in a complex dynamic fashion: TRF2, which has a dual role in chromosome end protection and telomere length homeostasis, resides at telomeres in two distinct pools. One fraction (ϳ73%) has binding dynamics similar to TRF1 (residence time of ϳ44 s). Interestingly, the other fraction of TRF2 binds with similar dynamics as the putative end-protecting factor hPOT1 (residence time of ϳ11 min). Our data support a dynamic model of telomeres in which chromosome end-protection and telomere length homeostasis are governed by differential binding of telomeric proteins to telomeric DNA.

The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa 53 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA.