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.

We describe a novel nuclear factor called mitotic chromosome-associated protein (MCAP), which belongs to the poorly understood BET subgroup of the bromodomain superfamily. Expression of the 200-kDa MCAP was linked to cell division, as it was induced by growth stimulation and repressed by growth inhibition. The most notable feature of MCAP was its association with chromosomes during mitosis, observed at a time when the majority of nuclear regulatory factors were released into the cytoplasm, coinciding with global cessation of transcription. Indicative of its predominant interaction with euchromatin, MCAP localized on mitotic chromosomes with exquisite specificity: (i) MCAP-chromosome association became evident subsequent to the initiation of histone H3 phosphorylation and early chromosomal condensation; and (ii) MCAP was absent from centromeres, the sites of heterochromatin. Supporting a role for MCAP in G 2 /M transition, microinjection of anti-MCAP antibody into HeLa cell nuclei completely inhibited the entry into mitosis, without abrogating the ongoing DNA replication. These results suggest that MCAP plays a role in a process governing chromosomal dynamics during mitosis.

On entry into mitosis, many transcription factors dissociate from chromatin, resulting in global transcriptional shutdown. During mitosis, some genes are marked to ensure the inheritance of their expression in the next generation of cells. The nature of mitotic gene marking, however, has been obscure. Brd4 is a double bromodomain protein that localizes to chromosomes during mitosis and is implicated in holding mitotic memory. In interphase, Brd4 interacts with P-TEFb and functions as a global transcriptional coactivator. We found that throughout mitosis, Brd4 remained bound to the transcription start sites of many M/G1 genes that are programmed to be expressed at the end of, or immediately after mitosis. In contrast, Brd4 did not bind to genes that are expressed at later phases of cell cycle. Brd4 binding to M/G1 genes increased at telophase, the end phase of mitosis, coinciding with increased acetylation of histone H3 and H4 in these genes. Increased Brd4 binding was accompanied by the recruitment of P-TEFb and de novo M/G1 gene transcription, the events impaired in Brd4 knockdown cells. In sum, Brd4 marks M/G1 genes for transcriptional memory during mitosis, and upon exiting mitosis, this mark acts as a signal for initiating their prompt transcription in daughter cells. INTRODUCTIONThe states of gene expression, either active or silenced, are inherited through generations of somatic cells, providing a basis for stable cellular functions (Ringrose and Paro, 2004;Egli et al., 2008;Ng and Gurdon, 2008). With respect to inheritance of gene expression, mitosis poses a mystery, because most transcription factors dissociate from chromosomes during that time (Delcuve et al., 2008;Egli et al., 2008). The massive dissociation of transcription factors accompanies global cessation of transcription, which likely erases gene expression patterns established before mitosis (Martinez-Balbas et al., 1995;Gottesfeld and Forbes, 1997;Delcuve et al., 2008). Transcription factors that dissociate from mitotic chromosomes include RNA polymerase II, Oct1,2, Sp1,3, Pax 3, E2F1, Brg1, Brm, TFIIB, and TFIID among others. Mitotic nuclei are thus thought to represent a transcriptionally uncommitted state. Consistent with this idea, mitotic nuclei serve as better donors of genome transfer compared with interphase nuclei (Egli et al., 2008). The transcriptionally inert state is reversed at the end of mitosis, when RNA polymerase II (Pol II) and other transcription factors are sequentially reloaded onto chromosomes, leading to the initiation of transcription at telophase (Prasanth et al., 2003). Although the prior modes of transcription established in parental cells would be erased upon entry into mitosis, some "memory" remains during mitosis, allowing daughter cells to reproduce an inherited pattern of gene expression after mitosis. Relevant to this memory, some core histones retain their acetylation mark during mitosis, although histone acetylation is generally reduced during mitosis (Kruhlak et al., 2001;Nishiyama et al., 2006). Particular...

Bromodomain protein 4 (BRD4) is a chromatin-binding protein implicated in cancer and autoimmune diseases that functions as a scaffold for transcription factors at promoters and super-enhancers. Whereas chromatin de-compaction and transcriptional activation of target genes are associated with BRD4 binding, the mechanism(s) involved are unknown. We report that BRD4 is a novel histone acetyltransferase (HAT) that acetylates histones H3 and H4 with a pattern distinct from other HAT’s. Both mouse and human BRD4 demonstrate intrinsic HAT activity. Importantly, BRD4 acetylates H3K122, a residue critical for nucleosome stability, resulting in nucleosome eviction and chromatin de-compaction. Nucleosome clearance by BRD4 occurs genome-wide, including at its targets MYC, FOS and AURKB (Aurora B kinase), resulting in increased transcription. Since BRD4 regulates transcription, these findings lead to a model where BRD4 actively links chromatin structure and transcription: It mediates chromatin de-compaction by acetylating and evicting nucleosomes of target genes, thereby activating their transcription.

Brd4 is a bromodomain protein that binds to acetylated chromatin. It regulates cell growth, although the underlying mechanism has remained elusive. Brd4 has also been shown to control transcription of viral genes, whereas its role in transcription of cellular genes has not been fully elucidated. Here we addressed the role of Brd4 in cell growth and transcription using a small hairpin (sh) RNA approach. The Brd4 shRNA vector stably knocked down Brd4 protein expression by ϳ90% in NIH3T3 cells and mouse embryonic fibroblasts. Brd4 knockdown cells were growth impaired and grew more slowly than control cells. When synchronized by serum starvation and released, Brd4 knockdown cells were arrested at G 1 , whereas control cells progressed to S phase. In microarray analysis, although numerous genes were up-regulated during G 1 in control cells, many of these G 1 genes were not up-regulated in Brd4 knockdown cells. Reintroduction of Brd4 rescued expression of these G 1 genes in Brd4 knockdown cells, allowing cells to progress toward S phase. Chromatin immunoprecipitation analysis showed that Brd4 was recruited to the promoters of these G 1 genes during G 0 -G 1 progression. Furthermore, Brd4 recruitment coincided with increased binding of Cdk9, a component of P-TEFb and RNA polymerase II to these genes. Brd4 recruitment was low to absent at genes not affected by Brd4 shRNA. The results indicate that Brd4 stimulates G 1 gene expression by binding to multiple G 1 gene promoters in a cell cycle-dependent manner.Brd4 is a ubiquitously expressed 200-kDa nuclear protein that belongs to the BET family (1, 2). Proteins of this family carry two tandem bromodomains through which they interact with acetylated histones (3-6). Bromodomains are also present in other chromatin-binding proteins such as histone acetylases and chromatin remodeling factors. They also bind to acetylated histones and are involved in transcriptional regulation of many genes. Recent structural analysis indicates that the bromodomain of Brd2, a factor closely related to Brd4, forms a dimmer to bind to acetyl residues of the histone H4 tails (7). Binding of Brd4 and Brd2 to acetylated chromatin persists even during mitosis as well as meiosis when chromatin is highly condensed and transcription is interrupted (2,5,6,8).Evidence indicates that BET family proteins are multifunctional and regulate cell growth and transcription (3, 4, 9, 10). In line with this evidence, there are reports indicating that Brd4 is involved in cell growth regulation; Brd4 Ϫ/Ϫ embryos fail to grow and die early at around the time of implantation (11). Similarly, Brd4Ϫ/Ϫ embryonic stem cells do not grow in culture (12). Moreover, in some malignant cells, Brd4 is fused to the NUT gene, and the fusion protein exhibits a growth regulatory activity (13,14). In addition, overexpression of Brd4 in cultured cells is shown to alter their growth properties, in part due to the interaction of Brd4 with growth regulatory proteins such as RFC140 or Sipa1 (15, 16). The reports that Brd4 facilitates parti...

Major histocompatibility complex (MHC) class I-deficient cell lines were used to demonstrate that the MHC class II transactivator (CIITA) can induce surface expression of MHC class I molecules. CIITA induces the promoter of MHC class I heavy chain genes. The site alpha DNA element is the target for CIITA-induced transactivation of class I. In addition, interferon-gamma (IFNgamma)-induced MHC class I expression also requires an intact site alpha. The G3A cell line, which is defective in CIITA induction, does not induce MHC class I antigen and promoter in response to IFNgamma. Trans-dominant-negative forms of CIITA reduce class I MHC promoter function and surface antigen expression. Collectively, these data argue that CIITA has a role in class I MHC gene induction.