The novel mammalian protein, Bromodomain-containing protein 4 (Brd4), is a ubiquitously expressed 200-kDa nuclear protein which plays a vital role in the regulation of cell growth. Brd4 is expressed broadly in many tissues and is induced in response to growth stimuli. Brd4 belongs to the poorly understood BET (bromodomain and extraterminal domain) family. Proteins of this family usually carry two tandem bromodomains (BDI and BDII) and an extra terminal (ET) domain both of which are conserved from yeasts to humans, including Saccharomyces cerevisiae Bdf1 and Bdf2 proteins, Drosophila melanogaster female sterile homeotic and the mammalian Brd2/Ring3, Brd3/Orfx, Brd4/MCAP, and Brdt proteins. Bromodomain is a conserved region of approx 110 amino acids that structurally forms four alpha helices and two loops, capable of binding acetyl-lysine residues in histones and is involved in transcriptional regulation of many genes. Bromodomain also interacts with non-histone proteins to regulate transcription, DNA replication, cell cycle progression and other cellular activities (1, 2, 3).
Brd4, first identified in mouse as MCAP (mitotic chromosome-associated protein but also called Fshrg4 or Hunk1), is a chromatin-binding factor with preference for acetylated K14 on histone H3 and K5/12 on H4. The protein associates predominantly with the regions of chromosomes containing euchromatin and less with heterochromatin. Brd4-chromosome association became visible at a time when transcription factors are displaced from chromatin, an event characteristic of mitosis in higher eukaryotes. Brd4 is usually mobile in the nucleus and transiently associate with acetylated chromatin. (4)
Brd4 has emerged as a key player
in transcription, DNA replication,
cell cycle control, oncogenesis, and
viral genome segregation and is clearly
an excellent working model for
many chromatin adaptors.
Functions of Brd4 has been well implicated in cellular growth, cell cycle control, DNA replication, and gene rearrangement found in t(15;19)-associated carcinomas. However recent studies have confirmed some of the additional roles of Brd4, which assigns the protein to be a dynamic chromatic-binding adaptor, able to recruit distinct transcriptional regulators to modulate promoter activity through cell cycle progression.
1. Brd4 in Cell Growth and Cell Cycle Progression
Brd4 regulates cell growth by multiple mechanisms acting at different stages of the cell cycle. Both over expression and under expression of Brd4 are detrimental to cell growth. Brd4 interacts with acetylated chromatin with a rapid "on and off" mode of binding in vivo. The association of Brd4 with chromatin persists throughout the cell cycle, which is vital in maintaining the global acetylation state of chromatin in the cell. Moreover during its transient association with chromatin, Brd4 also interacts with other proteins to exert multiple functions. For example, it was found to interact with a component of DNA replication machinery, RFC, thereby contributing to the regulation of G1/S transition, the entry phase of mitosis. Brd4 also regulates the G2/M transition by interacting with cyclin B-cdc2 complex, the key regulator of the onset and completion of mitosis. (1, 5)
2. Brd4 in selective forms of Mediator complexes
The detection of Brd4 in several transcription complexes, including the general cofactor Mediator, indicates its role in transcriptional control. Analysis of a mouse Mediator complex detects the presence of an uncharacterized sequence identical to human, Brd2 which is likely to be mouse Brd4 protein. (6)
3. Association of Brd4 with Distinct Forms of P-TEFb
Recently, Brd4 has been connected to transcriptional activation. It has been observed that Brd4 binds to the positive transcriptional elongation factor (P-TEFb) and displaces an inhibitory subunit, thus transforming P-TEFb into a transcriptionally active complex. The human P-TEFb complex is a heterodimer of cyclin-dependent kinase 9 (Cdk9) and cyclin T1, T2, or K, capable of phosphorylation of the largest subunit of RNA polymerase II (RNAPII) and induction of a productive transcriptional elongation. P-TEFb binds to HIV Tat, a viral transactivator, and facilitates viral transcription and replication. P-TEFb is also recruited to cellular promoters by interacting with a variety of transcription factors. Brd4 acts as a positive regulatory component of P-TEFb and regulates its activity in transcription. (1, 6, 7) |
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4. Brd4 Is a Component of an HPV Transcriptional
Silencing Complex
Brd4 is known to recruit human papillomavirus (HPV) E2, a sequence-specific DNA-binding protein involved in viral DNA replication, transcription, genome maintenance, and segregation. As a transcriptional regulator, HPV E2 mainly acts as a repressor to inhibit viral gene expression, including E6 and E7 oncoproteins that antagonize p53 and pRB tumor suppressor activity. The identification of Brd4 as a component in a dominant form of E2 complexes indicates that Brd4 may be the long sought cellular cofactor for HPV E2 repressor function. Brd4 also modulates E2 interactions with DNA and with general cofactors Mediator, TFIID, and positive cofactor 4 (PC4), as well as components of the general transcription machinery to fine-tune the efficiency of preinitiation complex assembly, correlating with its involvement in gene activation. This clearly confirms the dual role of Brd4 in gene activation and gene repression classifying it as an authentic transcriptional cofactor. (1, 8)
5. Role of Brd4 in Viral Genome Segregation
Brd4 is often used as a cellular adaptor by some animal and human papillomaviruses (HPV) for anchoring viral genomes to mitotic chromosomes. This tethering is mediated by Brd4 interaction with virus-encoded E2 protein thus facilitating viral genome segregation during mitosis. (1, 9)
6. Brd4 as a functional contributor to cancer
metastatic potential
Apart from its role in cell growth and cell cycle progression, Brd4 has recently been shown to be a functional contributor to cancer metastatic potential. Studies have confirmed that Brd4 is a modulator of metastatic progression either by virtue of its interaction with an invasiveness-suppressing GTPase activating protein, Sipa1 or independently through its pleiotropic cellular functionality. Sipa1, a GTPase activating protein (GAP) and identified as the first polymorphic metastasis efficiency gene in mice negatively regulates Rap-GTPases. Polymorphisms in human Sipa1 are associated with indicators of poor outcome in breast cancer. Sipa1 interacts both in vitro and in vivo with Brd4. This interaction modulates the enzymatic activity of Sipa1, with a consequent increase in the RAP1GAP activity. Brd4 is a modulator of metastasis given the apparent regulation of Sipa1. Moreover, deregulation of Brd4-pathway both as a consequence of somatic mutation, germ-line polymorphism, or epigenetic silencing, may both drive the expression of many of the genes present in breast cancer gene expression signatures and be a central event in tumor progression. (10)
Brd4 has emerged as a key player in transcription, DNA replication, cell cycle control, oncogenesis, and viral genome segregation and is clearly an excellent working model for many chromatin adaptors. However some of its potential role is yet to be defined. Studies indicate Brd4 dynamics to be a mechanism ensuring the recovery of cells from drug-induced mitotic arrest. (11) Understanding Brd4 dynamics may serve as a new paradigm to study the contribution of chromatin to mitotic stress responses. Beside the exact status of Brd4 in breast cancer, whether it is a proximal factor or is an intermediary molecule of some other inherited factor that drives the progression of breast cancer is still unclear. A lot of research needs to be done so as to elucidate the physiological and functional importance of Brd4 in this context.
IMGENEX has thus come up with an antibody against Brd4 to aid in the research work targeted towards Brd4.
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