Global Nature of Dynamic Protein-Chromatin Interactions In Vivo: Three-Dimensional Genome Scanning and Dynamic Interaction Networks of Chromatin Proteins

Abstract

Genome structure and gene expression depend on a multitude of chromatin-binding proteins. The binding properties of these proteins to native chromatin in intact cells are largely unknown. Here, we describe an approach based on combined in vivo photobleaching microscopy and kinetic modeling to analyze globally the dynamics of binding of chromatin-associated proteins in living cells. We have quantitatively determined basic biophysical properties, such as off rate constants, residence time, and bound fraction, of a wide range of chromatin proteins of diverse functions in vivo. We demonstrate that most chromatin proteins have a high turnover on chromatin with a residence time on the order of seconds, that the major fraction of each protein is bound to chromatin at steady state, and that transient binding is a common property of chromatin-associated proteins. Our results indicate that chromatin-binding proteins find their binding sites by three-dimensional scanning of the genome space and our data are consistent with a model in which chromatin-associated proteins form dynamic interaction networks in vivo. We suggest that these properties are crucial for generating high plasticity in genome expression.Organization of DNA into higher-order chromatin structure serves to accommodate the genome within the spatial confines of the cell nucleus and acts as an important regulatory mechanism (22,36,46,60). Establishment, maintenance, and alterations of global and local chromatin states are modulated by the combined action of a multitude of chromatin-binding proteins. The nucleosome, containing histone proteins, acts as a structural scaffold and as an entry point for regulatory mechanisms (60, 63). Nonhistone proteins, including the HMG proteins, further contribute to the structural maintenance and regulation of chromatin regions (6, 61). In heterochromatin, specific factors such as HP1 convey a transcriptionally repressed state, possibly by influencing higher-order chromatin structure (19,27). Histone-modifying enzymes such as histone acetyl-and methyltransferases are instrumental in generating epigenetic marks on chromatin domains (60). Chromatin remodeling factors act on specific sites to facilitate access to regulatory DNA elements. Once accessible, transcriptional activators bind specific sequences on DNA and recruit the basal transcription machinery (37,44,46). All of these steps involve binding of proteins to chromatin.Due to their functional significance, chromatin-associated proteins have been extensively characterized-mostly by biochemical extraction and in vitro binding assays. Little is known about the dynamics of how chromatin proteins bind to their target sites in native chromatin in living cells. In vivo microscopy techniques are providing novel tools to study chromatin proteins in living cells (32,39,41,50). Qualitative analysis of photobleaching experiments has revealed a wide range of dynamic behavior for chromatin-associated proteins. The bulk of core histones is immobile on DNA, whereas the linker histone H1...

Chromatin Remodeling Complexes Interact Dynamically with a Glucocorticoid Receptor–regulated Promoter

“…The dynamic exchange of remodeling proteins on the MMTV array are consistent with our in vitro results obtained from rapid UV laser crosslinking where purified SWI/SNF binds to and is displaced from purified MMTV chromatin (Fletcher et al, 2002;Nagaich et al, 2004). Because the FRAP recovery kinetics of chromatin proteins are directly related to their chromatinbinding properties (Lefebvre et al, 1991;Fragoso et al, 1998;Lever et al, 2000;Kimura and Cook, 2001;Hager et al, 2002;Kimura et al, 2002;Maruvada et al, 2003;Phair et al, 2004;Becker et al, 2005;Chen et al, 2005), we conclude that the remodeling proteins with the slowest exchange rate reside longest on the MMTV promoter and associate most strongly with MMTV chromatin. A comparison of the kinetic properties of chromatin-remodeling complexes revealed that BRG1 was more strongly associated with the MMTV array than BRM ( Figure 6D).…”

“…In fact, this modulation is one of the key mechanisms essential for the functional role of nuclear hormone receptors (Stenoien et al, 2001;Schaaf and Cidlowski, 2003;Stavreva et al, 2004;Elbi et al, 2004b;Farla et al, 2005;Rayasam et al, 2005). In vivo FRAP has been used to study the dynamic properties of chromatin proteins and that the FRAP recovery kinetics of chromatin proteins are directly related to their chromatin-binding properties (Lefebvre et al, 1991;Fragoso et al, 1998;Lever et al, 2000;Kimura and Cook, 2001;Kimura et al, 2002;Maruvada et al, 2003;Phair et al, 2004;Becker et al, 2005;Chen et al, 2005). Because BRG1 and BRM were selectively recruited to the MMTV array in response to hormone (Figures 2 and 3), we used FRAP to study the binding kinetics of chromatin-remodeling complexes at the amplified MMTV target in vivo.…”

Integrase, LEDGF/p75 and HIV replication

“…Finally, the reality for p75 may be dynamically complex. Many chromatin-associated proteins are highly mobile, such that they rapidly and continuously exchange among chromatin binding sites in a stop-and-go manner [20,21]. The inferred competition between endogenous p75 and IBD fusion proteins would be consistent with a situation whereby p75 interacts in such a manner with both the PIC and chromatin [96,97].…”

“…Similarly, chromatin is compositionally and structurally intricate. Furthermore, protein-protein and protein-DNA interactions are highly dynamic within chromatin [20,21]. They are also likely to evolve within the PIC as it transits the nucleus and completes the integration process [22].…”

Mechanisms of nuclear reprogramming by eggs and oocytes: a deterministic process?

“…In general, core histones are relatively immobile in the chromatin of cultured cells when compared with other chromatin components (such as linker histones, high mobility group (HMG) proteins and HP1) [33][34][35][36] . In the case of the core histone H3, only the variant H3.3 is loaded onto chromatin within the first day following nuclear transfer.…”