Abstract
BackgroundProtein exchange kinetics correlate with the level of chromatin condensation and, in many cases, with the level of transcription. We used fluorescence recovery after photobleaching (FRAP) to analyse the kinetics of 18 proteins and determine the relationships between nuclear arrangement, protein molecular weight, global transcription level, and recovery kinetics. In particular, we studied heterochromatin-specific heterochromatin protein 1β (HP1β) B lymphoma Mo-MLV insertion region 1 (BMI1), and telomeric-repeat binding factor 1 (TRF1) proteins, and nucleolus-related proteins, upstream binding factor (UBF) and RNA polymerase I large subunit (RPA194). We considered whether the trajectories and kinetics of particular proteins change in response to histone hyperacetylation by histone deacetylase (HDAC) inhibitors or after suppression of transcription by actinomycin D.ResultsWe show that protein dynamics are influenced by many factors and events, including nuclear pattern and transcription activity. A slower recovery after photobleaching was found when proteins, such as HP1β, BMI1, TRF1, and others accumulated at specific foci. In identical cells, proteins that were evenly dispersed throughout the nucleoplasm recovered more rapidly. Distinct trajectories for HP1β, BMI1, and TRF1 were observed after hyperacetylation or suppression of transcription. The relationship between protein trajectory and transcription level was confirmed for telomeric protein TRF1, but not for HP1β or BMI1 proteins. Moreover, heterogeneity of foci movement was especially observed when we made distinctions between centrally and peripherally positioned foci.ConclusionBased on our results, we propose that protein kinetics are likely influenced by several factors, including chromatin condensation, differentiation, local protein density, protein binding efficiency, and nuclear pattern. These factors and events likely cooperate to dictate the mobility of particular proteins.
Highlights
Protein exchange kinetics correlate with the level of chromatin condensation and, in many cases, with the level of transcription
Cellular patterns of proteins studied In this study, we analysed the dynamics of a select group of proteins, which displayed the following cellular patterns: subtypes of heterochromatin protein 1 (HP1a and heterochromatin protein 1b (HP1b)) preferentially accumulated at foci that colocalised with centromeric clusters, called chromocentres
The B lymphoma Mo-MLV insertion region 1 (BMI1) protein is a member of the protein regulator of cytokinesis 1 (PRC1) protein complex, which accumulated at Polycomb bodies (PcG), but were observed outside of PcG bodies (Figure 1b)
Summary
Protein exchange kinetics correlate with the level of chromatin condensation and, in many cases, with the level of transcription. Chromatin consists of histones wrapped with DNA and the many proteins that are directly responsible for proper nuclear functions, such as replication, transcription, splicing, and DNA repair. The fluorescence recovery kinetics of several RNA pol I subunits in the G1 and S phases of the cell cycle displayed biphasic behaviour, characterised by fast fluorescence increase followed by a gentle fluorescence recovery phase [19]. These authors showed that the RNA polymerase I recovery kinetics decrease as cell cycle-dependent transcription increases. Actinomycin D is a useful agent that enables the study of gene silencing and corresponding epigenetic events
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