Previous in vitro studies showed that the bromodomain binds to acetyllysines on histone tails, leading to the proposal that the domain is involved in deciphering the histone code. However, there is little in vivo evidence supporting the binding of bromodomains to acetylated chromatin in the native environment. Brd4 is a member of the BET family that carries two bromodomains. It associates with mitotic chromosomes, a feature characteristic of the family. Here, we studied the interaction of Brd4 with chromatin in living cells by photobleaching. Brd4 was mobile and interacted with chromatin with a rapid ''on and off'' mode of binding. This interaction required both bromodomains. Indicating a preferential interaction with acetylated chromatin, Brd4 became less mobile upon increased chromatin acetylation caused by a histone deacetylase inhibitor. Providing biochemical support, salt solubility of Brd4 was markedly reduced upon increased histone acetylation. This change also required both bromodomains. In peptide binding assays, Brd4 avidly bound to di-and tetraacetylated histone H4 and diacetylated H3, but weakly or not at all to mono-and unacetylated H3 and H4. By contrast, it did not bind to unacetylated H4 or H3. Further, Brd4 colocalized with acetylated H4 and H3 in noncentromeric regions of mitotic chromosomes. This colocalization also required both bromodomains. These observations indicate that Brd4 specifically recognizes acetylated histone codes, and this recognition is passed onto the chromatin of newly divided cells.A cetylation of lysines on histone tails is thought to form distinct histone codes that direct molecular processes important for transcription (1, 2). A bromodomain is a motif present in a number of chromatin-modifying proteins including histone acetylases of the GNAT family, CBP͞p300, general transcription factors including TAFII250, and chromatin remodeling factors of the SWI͞SNF family (3, 4). Structural analyses in vitro have shown that the bromodomain is composed of four ␣-helices and binds to acetylated lysines on histone H3 and H4, although with relatively low affinity (5-9). Based on these studies, the bromodomain has been proposed to act as a chromatin targeting module, deciphering histone acetylation codes (1, 2, 10). However, despite in vitro evidence, it has not been clear whether bromodomain proteins interact with acetylated chromatin in the native nuclear environment in vivo. Besides the question of in vivo interaction, it has not been clear whether differentially acetylated histones are distinguished by bromodomains. The latter question is of interest in view of the fact that bromodomains of different proteins have considerable structural diversity (3, 4). Furthermore, histone acetylation codes are likely to be diverse and translated into distinct processes, as individual lysines on histone H3 and H4 are acetylated in a highly specific and ordered fashion during transcription (11,12). In addition to transcription, histone acetylation codes may play a role in cell growth, as H3 and ...

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...

The general inhibition in transcriptional activity during mitosis abolishes the stress-inducible expression of the human hsp70 gene. Among the four transcription factors that bind to the human hsp70 promoter, the DNA-binding activities of three (C/EBP, GBP, and HSF1) were normal, while Sp1 showed reduced binding activity in mitotic cell extracts. In vivo footprinting and immunocytochemical analyses revealed that all of the sequence-specific transcription factors were displaced from promoter sequences as well as from bulk chromatin during mitosis. The correlation of transcription factor displacement with chromatin condensation suggests an involvement of chromatin structure in mitotic repression. However, retention of DNase I hypersensitivity suggests that the hsp70 promoter was not organized in a canonical nucleosome structure in mitotic chromatin. Displacement of transcription factors from mitotic chromosomes could present another window in the cell cycle for resetting transcriptional programs.