Differentiation of embryonic stem (ES) cells from a pluripotent to a committed state involves global changes in genome expression patterns. Gene activity is critically determined by chromatin structure and interactions of chromatin binding proteins. Here, we show that major architectural chromatin proteins are hyperdynamic and bind loosely to chromatin in ES cells. Upon differentiation, the hyperdynamic proteins become immobilized on chromatin. Hyperdynamic binding is a property of pluripotent cells, but not of undifferentiated cells that are already lineage committed. ES cells lacking the nucleosome assembly factor HirA exhibit elevated levels of unbound histones, and formation of embryoid bodies is accelerated. In contrast, ES cells, in which the dynamic exchange of H1 is restricted, display differentiation arrest. We suggest that hyperdynamic binding of structural chromatin proteins is a functionally important hallmark of pluripotent ES cells that contributes to the maintenance of plasticity in undifferentiated ES cells and to establishing higher-order chromatin structure.

The linker histone H1 is believed to be involved in chromatin organization by stabilizing higher-order chromatin structure. Histone H1 is generally viewed as a repressor of transcription as it prevents the access of transcription factors and chromatin remodelling complexes to DNA. Determining the binding properties of histone H1 to chromatin in vivo is central to understanding how it exerts these functions. We have used photobleaching techniques to measure the dynamic binding of histone H1-GFP to unperturbed chromatin in living cells. Here we show that almost the entire population of H1-GFP is bound to chromatin at any one time; however, H1-GFP is exchanged continuously between chromatin regions. The residence time of H1-GFP on chromatin between exchange events is several minutes in both euchromatin and heterochromatin. In addition to the mobile fraction, we detected a kinetically distinct, less mobile fraction. After hyperacetylation of core histones, the residence time of H1-GFP is reduced, suggesting a higher rate of exchange upon chromatin remodelling. These results support a model in which linker histones bind dynamically to chromatin in a stop-and-go mode.

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...

H1 linker histones stabilize the nucleosome, limit nucleosome mobility and facilitate the condensation of metazoan chromatin. Here, we have combined systematic mutagenesis, measurement of in vivo binding by photobleaching microscopy, and structural modeling to determine the binding geometry of the globular domain of the H1(0) linker histone variant within the nucleosome in unperturbed, native chromatin in vivo. We demonstrate the existence of two distinct DNA-binding sites within the globular domain that are formed by spatial clustering of multiple residues. The globular domain is positioned via interaction of one binding site with the major groove near the nucleosome dyad. The second site interacts with linker DNA adjacent to the nucleosome core. Multiple residues bind cooperatively to form a highly specific chromatosome structure that provides a mechanism by which individual domains of linker histones interact to facilitate chromatin condensation.

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The ability of regulatory factors to access their nucleosomal targets is modulated by nuclear proteins such as histone H1 and HMGN (previously named HMG-14/-17 family) that bind to nucleosomes and either stabilize or destabilize the higherorder chromatin structure. We tested whether HMGN proteins affect the interaction of histone H1 with chromatin. Using microinjection into living cells expressing H1-GFP and photobleaching techniques, we found that wild-type HMGN, but not HMGN point mutants that do not bind to nucleosomes, inhibits the binding of H1 to nucleosomes. HMGN proteins compete with H1 for nucleosome sites but do not displace statically bound H1 from chromatin. Our results provide evidence for in vivo competition among chromosomal proteins for binding sites on chromatin and suggest that the local structure of the chromatin fiber is modulated by a dynamic interplay between nucleosomal binding proteins.

To identify functional differences among non-allelic variants of the mammalian H1 linker histones a system for the overexpression of individual H1 variants in vivo was developed. Mouse 3T3 cells were transformed with an expression vector containing the coding regions for the H1c or H10 variant under the control of an inducible promoter. Stable, single colony transformants, in which the normal stoichiometry of H1 variants was perturbed, displayed normal viability, unaltered morphology and no long-term growth arrest. However, upon release from synchronization at different points in the cell cycle transformants significantly overproducing H10 exhibited transient inhibition of both G1 and S phase progression. Overexpression of H1c to comparable levels had no effect on cell cycle progression. Analysis of transcript levels for several cell cycle-regulated and housekeeping genes indicated that overexpression of H10 resulted in significantly reduced expression of all genes tested. Surprisingly, overexpression of H1c to comparable levels resulted in either a negligible effect or, in some cases, a dramatic increase in transcript levels. These results support the suggestion that functional differences exist among H1 variants.

Accurate casts can be made with either stock trays or custom trays. An impression made from polyether should be poured only once and within 24 hours after impression making, because of the distortion of the material over time. Silicone impression material has better dimensional stability than polyether. .