Mammalian BET/fsh proteins

The BET family of proteins is a relatively small group of unique bromodomain-containing proteins with four members in human and mouse. The genes are designated BRD2/RING3, BRD3/ORFX, BRD4/HUNK/MCAP and BRD6/BRDT. As mentioned, all four proteins have two bromodomains in the N-terminal region of the respective protein and an ET domain in the C-terminal region (see figure 8).

BRD2/RING3 encodes a protein of 801 aa, BRD3/ORFX of 726 aa and BRD6/BRDT of 947 aa. BRD4 codes for two alternatively spliced variants: one denominated BRD4/HUNK with a length of 2169bp (722 aa) and the other BRD4/MCAP with 4203bp (1401 aa) (see figure 9).

All four mammalian BET proteins might be involved in some aspects of oncogenesis:

BRD2/RING3 showed elevated kinase activity in leukemic lymphoblasts of the B lineage (Denis,G.V. and Green,M.R., 1996), and BRD4 appears to play a role in cell cycle regulation and was found to be chromosomally translocated in certain carcinomas (Dey,A. et al., 2000; French,C.A. et al., 2001; French,C.A. et al., 2003).

Preliminary data suggest an overexpression of BRD6/BRDT in certain cancers (Scanlan,M.J. et al., 2000) and a possible involvement of BRD3/ORFX in DNA damage response (Levenson,V.V. et al., 1999). With the exception of BRD6/BRDT which seems to be mainly expressed in testis tissues (Jones,M.H. et al., 1997;

Shang,E. et al., 2004), the other BET proteins BRD2, BRD3 and BRD4 have been found to be ubiquitously expressed in many different tissues (Rhee,K. et al., 1998;

Dey,A. et al., 2000; Trousdale,R.K. and Wolgemuth,D.J., 2004; Shang,E. et al., 2004).

1.Introduction

Might BET protein functions be partially redundant? Some data indicate unique, differentiation stage-specific expression patterns for the mammalian BET proteins in reproductive tissues (Shang,E. et al., 2004) arguing in favour of distinct functions.

Other authors clearly demonstrate yeast bdf1 and bdf2 to be at least partially redundant, as a bdf1 bdf2 double-mutantion is lethal, but mutating either bdf1 or bdf2 is not (Chua,P. and Roeder,G.S., 1995). To date, the question of redundancy between BET proteins cannot be finally answered.

BRD2/RING3

BRD2/RING3 was first described in the early 1990s (Beck,S. et al., 1992) and the human BRD2 gene is located on the short arm of chromosome 6 (6p21.3) (Thorpe,K.L. et al., 1996). BRD2 is known to be highly expressed in both male and female reproductive tissues (Rhee,K. et al., 1998; Trousdale,R.K. and Wolgemuth,D.J., 2004), in established leukemic cell lines and in lymphocytes from patients with leukaemia (Denis,G.V. and Green,M.R., 1996). BRD2 protein localises to the nuclei of proliferating cells and also of cells that undergo apoptosis (Crowley,T.E. et al., 2002). In contrast, BRD2 protein has been shown to be cytoplasmic in resting neuronal cells (Crowley,T. et al., 2004). Together, these data are in line with a role for BRD2 during development. Kanno et al. were recently able, using fluorescence resonance energy transfer, to demonstrate a selective interaction of BRD2 protein with acetylated lysine 12 on Histone H4 (Kanno,T. et al., 2004).

Further, BRD2 protein has been shown to activate cell cycle regulatory promoters probably via an interaction with transcription factors of the E2F family (Denis,G.V. et al., 2000). Lymphocyte specific overexpression of BRD2 in a Eµ-BRD2 transgenic mouse resulted in the development of B cell lymphoma and leukaemia (Greenwald,R.J. et al., 2004).

Our group identified the BRD2 protein to interact with KSHV LANA-1 by yeast-two-hybrid assay (Platt,G.M. et al., 1999). Further, KSHV LANA-1 was shown to be phosphorylated in the presence of BRD2 protein (Platt,G.M. et al., 1999). Whether BRD2 is itself a kinase or recruits another cellular kinase is still controversial.

Whereas Denis et al. originally purified the BRD2 protein as a nuclear kinase with mitogen-stimulated autophosphorylation activity (Denis,G.V. and Green,M.R., 1996;

Ostrowski,J. et al., 1998), our group identified BRD2-associated kinase activity which most likely reflects the activity of an associated kinase and not of BRD2 itself

1.Introduction

(Platt,G.M. et al., 1999), Rhee and coworkers did not identify any kinase activity associated with the BRD2 protein (Rhee,K. et al., 1998). BRD2/RING3 is the only human BET protein with an identified ATP-binding site (Denis,G.V. and Green,M.R., 1996).

BRD4/HUNK and BRD4/MCAP

The human BRD4 gene is located on chromosome 19 (19p13.1) and codes for two putative transcripts: firstly, BRD4/HUNK encompassing 11 exons and secondly, BRD4/MCAP (mitotic chromosome associated proteins) encompassing 19 exons, out of which the first 10 exons are identical to BRD4/HUNK (see figure 9). For the MCAP transcript an alternative splice site and an alternative exon 11 (11’) is used, removing the last 5 amino acids near the C-terminus of HUNK (see figure 9). In different mouse tissues, BRD4 transcripts were found to be ubiquitously expressed (Shang,E. et al., 2004). Most functional studies performed so far have characterised the murine BRD4/MCAP. Targeted mutagenesis of the murine BRD4 gene has revealed that it is an essential gene for normal cell proliferation since BRD4 deficient mouse embryos die shortly after implantation and are compromised in their ability to maintain an inner cell mass in vitro (Houzelstein,D. et al., 2002). Further, the murine BRD4/MCAP has been shown to bind to mitotic chromosomes, mainly in euchromatic regions by binding to di- or tetraacetylated Histone H4 and diacetylated H3 (Dey,A. et al., 2003).

exon

ATGbp 1

STOPbp 2169 1 2 3 4 5 6 7 8 9 10 11 BRD4/HUNK

ATGbp 1

STOPbp 4203 1 2 3 4 5 6 7 8 9 10 11‘

BRD4/MCAP

exon

12 13

14 15

16

17 19

18

Figure 9: Exon-intron structure of the human BRD4 gene.

The human BRD4 gene codes for two putative transcripts. The shorter variant BRD4/HUNK consists of eleven exons and encompasses an open reading frame (orf) of 2169 bp which codes for a protein of 722 aa. BRD4/MCAP consists of 19 exons out of which exons 1-10 are identical to BRD4/HUNK.

BRD4/MCAP uses an alternative exon 11 (11’). Exons 11’-19 are unique to the longer splice variant BRD4/MCAP. The BRD4/MCAP transcript encompasses an orf of 4203 bp coding for a protein of 1401 aa.

1.Introduction

Ectopic overexpression of murine BRD4/MCAP in cell lines inhibited cell cycle progression from G1 to S phase, whereas the injection of BRD4 specific antibodies blocked cells in G2 to M phase (Dey,A. et al., 2000; Maruyama,T. et al., 2002; Dey,A.

et al., 2003). Two studies point at a possible role for BRD4 in different types of carcinomas (French,C.A. et al., 2001; French,C.A. et al., 2003). Recently, Farina et al. provided evidence that BRD4 may be linked to the GTPase-dependent mitogenic signaling pathway, as they found BRD4 to interact with the signal-induced proliferation associated protein 1 (SPA-1), which is a Rap GTPase activating protein (GAP) (Farina,A. et al., 2004).

BRD4/MCAP has been found to interact with the E2 proteins of bovine papillomavirus 1 (BPV 1) and human papillomavirus 16 (HPV 16) thereby tethering papillomaviral genomes to chromosomes in dividing cells (You,J. et al., 2004). BRD4 therefore represents the long sought cellular factor responsible for the chromatin binding function of E2.

BRD3/ORFX

The BRD3 gene has been mapped to the long arm of chromosomes 9 (9q34). In contrast to BRD6/BRDT, BRD3 has been found, similar to BRD2/RING3, to be expressed ubiquitously in human adult as well as fetal tissues (Thorpe,K.L. et al., 1997). Further, Thorpe et al. suggest that BRD2 and BRD3 might have arisen from an ancient duplication in a common ancestral chromosome.

BRD6/BRDT

The BRD6 gene is located on the short arm of chromosome 1 (1p). BRD6/BRDT (for bromodomain, testis specific) was given its name due to its expression mainly in testis tissue in the normal adult human (Jones,M.H. et al., 1997; Shang,E. et al., 2004). However, it seems to be expressed in some cancer tissues. Scanlan and coworkers detected BRDT transcripts by RT-PCR in 12 of 47 cases of non-small cell lung cancer and single cases of both squamous cell carcinoma of the head and neck (1/12) and esophagus (1/12) but not in melanoma or in cancers of the colon, breast, kidney and bladder (Scanlan,M.J. et al., 2000).

1.Introduction