In this study, we have demonstrated that translocated in liposarcoma (TLS), also termed FUS, is an interacting molecule of the p65 (RelA) subunit of the transcription factor nuclear factor B (NF-B) using a yeast two-hybrid screen. We confirmed the interaction between TLS and p65 by the pull-down assay in vitro and by a coimmunoprecipitation experiment followed by Western blot of the cultured cell in vivo. TLS was originally identified as part of a fusion protein with CHOP arising from chromosomal translocation in human myxoid liposarcomas. TLS has been shown to be involved in TFIID complex formation and associated with RNA polymerase II. However, the role of TLS in transcriptional regulation has not yet been clearly elucidated. We found that TLS enhanced the NF-B-mediated transactivation induced by physiological stimuli such as tumor necrosis factor ␣, interleukin-1␤, and overexpression of NF-B-inducing kinase. TLS augmented NF-B-dependent promoter activity of the intercellular adhesion molecule-1 gene and interferon-␤ gene. These results suggest that TLS acts as a coactivator of NF-B and plays a pivotal role in the NF-B-mediated transactivation. Nuclear factor B (NF-B)1 is an inducible cellular transcription factor that regulates a wide variety of cellular and viral genes including cytokines, cell adhesion molecules and human immunodeficiency virus (1-5). The members of the NF-B family in mammalian cells include the proto-oncogene c-Rel, RelA (p65), RelB, NFkB1 (p50/105), and NFkB2 (p52/p100). These proteins share a conserved 300-amino acid region known as the Rel homology domain, which is responsible for DNA binding, dimerization, and nuclear translocation of NF-B (1, 2, 4, 5). In most cells, Rel family members form hetero-and homodimers with distinct specificities in various combinations. p65, RelB, and c-Rel are transcriptionally active members of the NF-B family, whereas p50 and p52 primarily serve as DNA binding subunits (1, 2, 4, 5). These proteins play fundamental roles in immune and inflammatory responses and in the control of cell proliferation (4, 6 -9). A common feature of the regulation of NF-B is the sequestration in the cytoplasm as an inactive complex with a class of inhibitory molecules known as IBs (2, 10). Treatment of cells with a variety of inducers such as phorbol esters, interleukin-1 (IL-1), and tumor necrosis factor ␣ (TNF-␣) results in phosphorylation, ubiquitination, and degradation of the IB proteins (5, 11, 12). The degradation of IB proteins exposes the nuclear localization sequence in the remaining NF-B dimers, followed by the rapid translocation of NF-B to the nucleus where it activates the target genes by binding to the DNA regulatory element (1, 2, 4, 5).The protein regions responsible for the transcriptional activation (called "transactivation domain") of p65, RelB, and c-Rel have been mapped in their unique C-terminal regions. p65 contains at least two independent transactivation domains within its C-terminal 120 amino acids (Fig. 1A) (13-16). One of these transactivation doma...

The amino-terminal enhancer of split (AES) encodes a 197-amino acid protein that is homologous to the NH 2 -terminal domain of the Drosophila Groucho protein but lacks COOH-terminal WD40 repeats. Although the Drosophila Groucho protein and its mammalian homologs, transducin-like enhancer of split proteins, are known to act as non-DNA binding corepressors, the role of the AES protein remains unclarified. Using the yeast twohybrid system, we have identified the protein-protein interaction between AES and the p65 (RelA) subunit of the transcription factor nuclear factor B (NF-B), which activates various target genes involved in inflammation, apoptosis, and embryonic development. The interaction between AES and p65 was confirmed by in vitro glutathione S-transferase pull down assay and by in vivo co-immunoprecipitation study. In transient transfection assays, AES repressed p65-driven gene expression. AES also inhibited NF-B-dependent gene expression induced by tumor necrosis factor ␣, interleukin-1␤, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1, which is an upstream kinase for NF-B activation. These data indicate that AES acts as a corepressor for NF-B and suggest that AES may play a pivotal role in the regulation of NF-B target genes.Nuclear factor B (NF-B) 1 is an inducible cellular transcription factor that regulates a wide variety of cellular and viral genes including several cytokines, cell adhesion molecules, and human immunodeficiency virus (HIV) (1-4). The members of the NF-B family in mammalian cells include the proto-oncogene c-Rel, Rel A (p65), Rel B, NF B1 (p50/105), and NF B2 (p52/p100). These proteins share a conserved 300-amino acids region known as the Rel homology domain, which is responsible for DNA binding, dimerization, and nuclear translocation of NF-B (1-4). In most cells, Rel family members form heteroand homodimers with distinct specificities in various combinations. p65, Rel B, and c-Rel are transcriptionally active members of the NF-B family, whereas p50 and p52 primarily serve as mere DNA binding subunits (1-4). The transactivation domains of p65, Rel B, and c-Rel have been mapped in their unique COOH-terminal regions. p65 was shown to contain at least two independent transactivation domains within its COOH-terminal 120 amino acids (5-8). One p65 activation domain, TA1, is confined to the COOH-terminal 30 amino acids of p65. The second domain TA2 is contained within the NH 2 -terminally adjacent 90 amino acids.A common feature of the regulation of NF-B family is their sequestration in the cytoplasm as inactive complexes with a class of inhibitory molecules known as I Bs (1-4). Treatment of cells with a variety of inducers such as phorbol esters, interleukin-1, and tumor necrosis factor (TNF) results in phosphorylation, ubiquitination, and degradation of the I B proteins (4, 9, 10). The degradation of I B proteins exposes the nuclear localization sequence in the remaining NF-B dimers, leading to nuclear translocation and subsequent binding of NF...

RNA helicase A (RHA), a member of DNA and RNA helicase family containing ATPase activity, is involved in many steps of gene expression such as transcription and mRNA export. RHA has been reported to bind directly to the transcriptional coactivator, CREB-binding protein, and the tumor suppressor protein, BRCA1, and links them to RNA Polymerase II holoenzyme complex. Using yeast twohybrid screening, we have identified RHA as an interacting molecule of the p65 subunit of nuclear factor jB (NF-jB). The interaction between p65 and RHA was confirmed by glutathione-S transferase pull-down assay in vitro, and by coimmunoprecipitation assay in vivo. In transient transfection assays, RHA enhanced NF-jB dependent reporter gene expression induced by p65, tumor necrosis factor-a, or NFjB inducing kinase. The mutant form of RHA lacking ATPbinding activity inhibited NF-jB dependent reporter gene expression induced by these activators. Moreover, depletion of RHA using short interfering RNA reduced the NF-jB dependent transactivation. These data suggest that RHA is an essential component of the transactivation complex by mediating the transcriptional activity of NF-jB.

NF-B is an inducible transcriptional factor for the expression of multiple genes involved in immunoinflammatory responses, cell proliferation, and survival, thus playing crucial roles in the pathogenesis of many diseases, including cancer, leukemia, and autoimmune diseases (1-4). NF-B primarily exists as the heterodimer consisting of p65 and p50 in the cytoplasm and is sequestered by binding to its inhibitory protein IB (5). Upon signaling, such as by tumor necrosis factor ␣ (TNF␣), IB is phosphorylated and is followed by proteasomemediated degradation, which liberates NF-B to the nucleus thereby activating the target genes.PKA exists in the cytoplasm as an inactivated tetramer holoenzyme composed of dimer catalytic subunits and dimer regulatory subunits, which dissociate upon elevation of cAMP (6 -10). PKAc is also predominantly involved in the IB-NF-B complex in the cytoplasm, and IB sequesters PKAc by masking its catalytic domain (11). Following a variety of extracellular stimuli such as TNF␣, IB is phosphorylated by IKK 2 and degraded by the 26 S proteasome (12, 13). As a consequence, the p65/p50 heterodimer complex is liberated, and the catalytic center of PKAc is exposed, enabling activated PKAc to phosphorylate p65 at 15). This PKA-dependent phosphorylation of p65 facilitates the recruitment of transcription coactivator CBP and DNA binding activity of p65; therefore, activation of PKA augments NF-B-dependent gene expression, and PKAc signaling is considered to up-regulate . With regard to the role of PKAc in NF-B signaling, some claimed that cAMP-dependent PKA activation down-regulated NF-B-dependent transcription by changing its DNA binding ability (18,19), modifying the transactivation domain of p65 (20), or blocking the degradation of IB proteins (21,22).In our previous report, we identified AKIP1 as a novel p65-interacting protein (23). AKIP1 was initially found in breast cancer cells (24) in which it facilitated the nuclear translocation of PKAc (25). We found that AKIP1 appears to serve as a molecular bridge between p65 and PKAc, promoting their interaction and subsequent p65 phosphorylation at Ser-276, thus enhancing NF-B signaling (23). Because NF-B is constitutively activated in some breast cancer cells, endowing them with resistance to apoptosis (26 -28), we hypothesized that the effect of PKA in NF-B cascade is associated with AKIP1 and could be modulated by AKIP1 in a cell type-dependent fashion.In this study, we further investigate the role of PKA in regulating NF-B-dependent transcription in various cell lines with different expression levels of endogenous AKIP1. We provide evidence that in minimal AKIP1-expressing cell lines, elevation of cAMP decreased p65-PKA binding and p65 phosphorylation at Ser-276, which eventually leads to down-regulation of the NF-B-dependent transcription. In contrast, in breast cancer cell lines MDA-MB231 and MCF7 with high AKIP1 expression, the PKA-activating agents increased p65-PKA binding and its phosphorylation and up-regulated the NF-B-dependent transcriptio...

A number of studies have recently demonstrated that super-enhancers, which are large cluster of enhancers typically marked by a high level of acetylation of histone H3 lysine 27 and mediator bindings, are frequently associated with genes that control and define cell identity during normal development. Super-enhancers are also often enriched at cancer genes in various malignancies. The identification of such enhancers would pinpoint critical factors that directly contribute to pathogenesis. In this study, we performed enhancer profiling using primary leukemia samples from adult T-cell leukemia/lymphoma (ATL), which is a genetically heterogeneous intractable cancer. Super-enhancers were enriched at genes involved in the T-cell activation pathway, including , and in both ATL and normal mature T cells, which reflected the origin of the leukemic cells. Super-enhancers were found at several known cancer gene loci, including , and, in multiple ATL samples, but not in normal mature T cells, which implicated those genes in ATL pathogenesis. A small-molecule CDK7 inhibitor, THZ1, efficiently inhibited cell growth, induced apoptosis, and downregulated the expression of super-enhancer-associated genes in ATL cells. Furthermore, enhancer profiling combined with gene expression analysis identified a previously uncharacterized gene, , that was associated with super-enhancers in all ATL samples, but not in normal T cells. Knockdown of induced apoptosis in ATL cell lines, whereas overexpression of this gene promoted cell growth. Our study provides a novel strategy for identifying critical cancer genes.