Brd4 is a double bromodomain-containing protein that binds preferentially to acetylated chromatin. It belongs to the BET (bromodomains and extraterminal) family that includes mammalian Brd2, Brd3, Brd4, Brdt, Drosophila Fsh, yeast Bdf1, Bdf2, and corresponding homologues in other species. Brd4 is essential for cellular growth and has been implicated in cell cycle control, DNA replication, and gene rearrangement found in t(15;19)-associated carcinomas. Recently, Brd4 has been found in several transcription complexes, including the general cofactor Mediator and the P-TEFb elongation factor, and is capable of stimulating HIV-1 transcription in a Tat-independent manner. In addition, Brd4 is used as a cellular adaptor by some animal and human papillomaviruses (HPV) for anchoring viral genomes to mitotic chromosomes. This tethering, mediated by Brd4 interaction with virus-encoded E2 protein, facilitates viral genome segregation during mitosis. Interestingly, Brd4 is also identified in a transcriptional silencing complex assembled by HPV E2 and turns out to be the long sought cellular corepressor that inhibits the expression of HPV-encoded E6 and E7 oncoproteins that antagonize p53 and pRB tumor suppressor activity, respectively. The dual role of Brd4 in gene activation and repression illustrates how a dynamic chromatin-binding adaptor is able to recruit distinct transcriptional regulators to modulate promoter activity through cell cycle progression. Brd4 and BET Family ProteinsBromodomain-containing protein 4 (Brd4) 2 is a member of the BET family that in yeast and animals contains two tandem bromodomains (BDI and BDII) and an extraterminal (ET) domain (1). The bromodomain is a conserved region of ϳ110 amino acids that structurally forms 4 ␣-helices (␣ z , ␣ A , ␣ B , and ␣ C ) and 2 loops, linking ␣ z and ␣ A (ZA loop) and ␣ B and ␣ C (BC loop), capable of binding acetyl-lysine residues in histones and many other proteins (2). In humans, four BET proteins (Brd2, Brd3, Brd4, and Brdt) exhibit similar gene arrangements, domain organizations, and some functional properties. Brd2, formerly named RING3 (really interesting new gene 3) or Fshrg1 (female sterile homeotic related gene 1), is a nuclear serine/threonine kinase possessing chromatin binding activity with preference for acetylated lysine 12 on histone H4 and transcription activity via its association with transcriptional regulators such as E2F1 (3, 4). Brd3 (also called ORFX or Fshrg2) and Brdt (for bromodomain, testis-specific) are less well characterized although mouse Brdt has been reported to induce global chromatin reorganization in an acetylation-dependent manner (5). Brd4, originally named MCAP (mitotic chromosomeassociated protein; Ref. 6) but also called Fshrg4 or Hunk1, is a chromatin binding factor with preference for acetylated Lys-14 on histone H3 and Lys-5/12 on H4 (7). Except for Brdt, which is expressed specifically in testis and ovary, Brd2, Brd3, and Brd4 are widely distributed (8, 9). Interestingly, the chromosomal locations of these Brd genes a...

Triple negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy1-3. BET bromodomain inhibitors, which have shown efficacy in several models of cancer4-6, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyllysine recognition modules, leading to inhibition of oncogenic transcriptional programs7-9. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.

The SIRT1 deacetylase inhibits fat synthesis and stimulates fat oxidation in response to fasting, but the underlying mechanisms remain unclear. Here we report that SREBP-1c, a key lipogenic activator, is an in vivo target of SIRT1. SIRT1 interaction with SREBP-1c was increased during fasting and decreased upon feeding, and consistently, SREBP-1c acetylation levels were decreased during fasting in mouse liver. Acetylated SREBP-1c levels were also increased in HepG2 cells treated with insulin and glucose to mimic feeding conditions, and down-regulation of p300 by siRNA decreased the acetylation. Depletion of hepatic SIRT1 by adenoviral siRNA increased acetylation of SREBP-1c with increased lipogenic gene expression. Tandem mass spectrometry and mutagenesis studies revealed that SREBP-1c is acetylated by p300 at Lys-289 and Lys-309. Mechanistic studies using acetylation-defective mutants showed that SIRT1 deacetylates and inhibits SREBP-1c transactivation by decreasing its stability and its occupancy at the lipogenic genes. Remarkably, SREBP-1c acetylation levels were elevated in dietinduced obese mice, and hepatic overexpression of SIRT1 or treatment with resveratrol, a SIRT1 activator, daily for 1 week decreased acetylated SREBP-1c levels with beneficial functional outcomes. These results demonstrate an intriguing connection between elevated SREBP-1c acetylation and increased lipogenic gene expression, suggesting that abnormally elevated SREBP-1c acetylation increases SREBP-1c lipogenic activity in obese mice. Reducing acetylation of SREBP-1c by targeting SIRT1 may be useful for treating metabolic disorders, including fatty liver, obesity, and type II diabetes.The NAD ϩ -dependent SIRT1 (sirtuin 1) deacetylase plays a critical role in cellular metabolism, stress responses, and possibly aging by modulating the activity of transcription factors and cofactors by protein deacetylation (1-4). In response to low nutritional availability, SIRT1 functions as a master switch to maintain lipid and glucose homeostasis and energy balance by regulating important metabolic regulators, such as PGC-1␣ (PPAR␥ coactivator ␣), Foxo-1, and liver X receptor (1, 5-7). We recently identified the nuclear bile acid receptor, farnesoid X receptor (FXR), 3 as an important in vivo target of SIRT1 in the regulation of hepatic lipid metabolism (8). Of these reported regulators, the function of SIRT1 in deacetylating and enhancing the activity of PGC-1␣ has been well established (1,5,9,10).

Summary The nuclear bile acid receptor FXR is critical for regulation of lipid and glucose metabolism. Here we report that FXR is a target of SIRT1, a deacetylase that mediates nutritional and hormonal modulation of hepatic metabolism. Lysine 217 of FXR is the major acetylation site targeted by p300 and SIRT1. Acetylation of FXR increases its stability but inhibits heterodimerization with RXRα, DNA binding, and transactivation activity. Down-regulation of hepatic SIRT1 increased FXR acetylation with deleterious metabolic outcomes. Surprisingly, in mouse models of metabolic disease, FXR interaction with SIRT1 and p300 was dramatically altered, FXR acetylation levels were elevated, and overexpression of SIRT1 or resveratrol treatment reduced acetylated FXR levels. Our data demonstrate that FXR acetylation is normally dynamically regulated by p300 and SIRT1 but is constitutively elevated in metabolic disease states. Small molecules that inhibit FXR acetylation by targeting SIRT1 or p300 may be promising therapeutic agents for metabolic disorders.