Summary Twist is a key transcription activator of epithelial-mesenchymal transition (EMT). It remains unclear how Twist induces gene expression. Here we reported a mechanism by which Twist recruits BRD4 to direct WNT5A expression in basal-like breast cancer (BLBC). Twist contains a “histone H4 mimic” GK-X-GK motif that is di-acetylated by Tip60. The di-acetylated Twist binds the second bromodomain of BRD4, whose first bromodomain interacts with acetylated H4, thereby constructs an activated Twist/BRD4/P-TEFb/RNA-PolII complex at the WNT5A promoter and enhancer. Pharmacologic inhibition of the Twist-BRD4 association reduced WNT5A expression and suppressed invasion, cancer stem cell (CSC)-like properties, and tumorigenicity of BLBC cells. Our study indicates that the interaction with BRD4 is critical for the oncogenic function of Twist in BLBC.

Mg(OH) 2 nanocrystallines with rod-, tube-, needle-, or lamella-like morphologies have been synthesized by a hydrothermal reaction using different magnesium precursors and solvents as the reactants. The products appeared to have narrow size distributions with a monodisperse nature. Subsequent thermal decomposition at 450 °C gave nanosized MgO, which preserved well the morphological features of the Mg(OH) 2 samples. The specific surface areas of the MgO samples were determined by the BET technique, which gave a feature of high surface area generally larger than 100 m 2 /g. The channels formed in the thermal dehydroxylation process may account for this feature of the MgO nanocrystallines. CM000607E

Niemann-Pick type C disease (NPC) is a lysosomal storage disorder causing accumulation of unesterified cholesterol in lysosomal storage organelles. Recent studies have shown that hydroxypropyl-β-cyclodextrin injections in npc1 −/− mice are partially effective in treating this disease. Using cultured fibroblasts, we have investigated the cellular mechanisms responsible for reduction of cholesterol accumulation. We show that decreased levels of cholesterol accumulation are maintained for several days after removal of cyclodextrin from the culture medium. This suggests that endocytosed cyclodextrin can reduce the cholesterol storage by acting from inside endocytic organelles rather than by removing cholesterol from the plasma membrane. To test this further, we incubated both NPC1 and NPC2 mutant cells with cholesterol-loaded cyclodextrin for 1 h, followed by chase in serum-containing medium. Although the cholesterol content of the treated cells increased after the 1-h incubation, the cholesterol levels in the storage organelles were later reduced significantly. We covalently coupled cyclodextrin to fluorescent dextran polymers. These cyclodextrin-dextran conjugates were delivered to cholesterol-enriched lysosomal storage organelles and were effective at reducing the cholesterol accumulation. We demonstrate that methyl-β-cyclodextrin is more potent than hydroxypropyl-β-cyclodextrin in reducing both cholesterol and bis(monoacylglycerol) phosphate accumulation in NPC mutant fibroblasts. Brief treatment of cells with cyclodextrins causes an increase in cholesterol esterification by acyl CoA:cholesterol acyl transferase, indicating increased cholesterol delivery to the endoplasmic reticulum. These findings suggest that cyclodextrin-mediated enhanced cholesterol transport from the endocytic system can reduce cholesterol accumulation in cells with defects in either NPC1 or NPC2.acyl CoA:cholesterol acyl transferase | cholesterol accumulation | lysosomal storage organelles | bis(monoacylgycerol)phosphate, pinocytosis

SUMMARYTranscription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity. Here, we demonstrate that BRD4, a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers, regulates the self-renewal ability and pluripotency of ESCs. BRD4 inhibition resulted in induction of epithelial-tomesenchymal transition (EMT) markers and commitment to the neuroectodermal lineage while reducing the ESC multidifferentiation capacity in teratoma as-says. BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation. Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation.

Metastatic melanoma remains a mostly incurable disease. Although newly approved targeted therapies are efficacious in a subset of patients, resistance and relapse rapidly ensue. Alternative therapeutic strategies to manipulate epigenetic regulators and disrupt the transcriptional program that maintains tumor cell identity are emerging. Bromodomain and extraterminal domain (BET) proteins are epigenome readers known to exert key roles at the interface between chromatin remodeling and transcriptional regulation. Here, we report that BRD4, a BET family member, is significantly upregulated in primary and metastatic melanoma tissues compared with melanocytes and nevi. Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. RNA sequencing of BET inhibitor–treated cells followed by Gene Ontology analysis showed a striking impact on transcriptional programs controlling cell growth, proliferation, cell-cycle regulation, and differentiation. In particular, we found that, rapidly after BET displacement, key cell-cycle genes (SKP2, ERK1, and c-MYC) were downregulated concomitantly with the accumulation of cyclin-dependent kinase (CDK) inhibitors (p21 and p27), followed by cell-cycle arrest. Importantly, BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status, opening the possibility of using these small-molecule compounds to treat patients for whom no effective targeted therapy exists. Collectively, our study reveals a critical role for BRD4 in melanoma tumor maintenance and renders it a legitimate and novel target for epigenetic therapy directed against the core transcriptional program of melanoma.

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BRD4, characterized by two acetyl-lysine binding bromodomains and an extra-terminal (ET) domain, is a key chromatin organizer that directs gene activation in chromatin through transcription factor recruitment, enhancer assembly, and pause release of the RNA polymerase II complex for transcription elongation. BRD4 has been recently validated as a new epigenetic drug target for cancer and inflammation. Our current knowledge of the functional differences of the two bromodomains of BRD4, however, is limited, hindered by the lack of selective inhibitors. Here, we report our structure-guided development of diazobenzene-based small molecule inhibitors for the BRD4 bromodomains that have over 90% sequence identity at the acetyl-lysine binding site. Our lead compound MS436, through a set of water-mediated interactions, exhibits low nanomolar affinity (estimated Ki of 30–50 nM) with preference for the first bromodomain over the second. We demonstrated that MS436 effectively inhibits BRD4 activity in NF-κB-directed production of nitric oxide and pro-inflammatory cytokine interleukin-6 in murine macrophages. MS436 represents a new class of bromodomain inhibitors and will facilitate further investigation of the biological functions of the two bromodomains of BRD4 in gene expression.

The RNA polymerase inhibitor tiacumicin B is currently undergoing phase III clinical trial for treatment of Clostridium difficile associated diarrhea with great promise. To understand the biosynthetic logic and to lay a foundation for generating structural analogues via pathway engineering, the tiacumicin B biosynthetic gene cluster was identified and characterized from the producer Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085. Sequence analysis of a 110,633 bp DNA region revealed the presence of 50 open reading frames (orfs). Functional investigations of 11 orfs by in vivo inactivation experiments, preliminarily outlined the boundaries of the tia-gene cluster and suggested that 31 orfs were putatively involved in tiacumicin B biosynthesis. Functions of a halogenase (TiaM), two glycosyltransferases (TiaG1 and TiaG2), a sugar C-methyltransferase (TiaS2), an acyltransferase (TiaS6), and two cytochrome P450s (TiaP1 and TiaP2) were elucidated by isolation and structural characterization of the metabolites from the corresponding gene-inactivation mutants. Accumulation of 18 tiacumicin B analogues from 7 mutants not only provided experimental evidence to confirm the proposed functions of individual biosynthetic enzymes, but also set an example of accessing microbial natural product diversity via genetic approach. More importantly, biochemical characterization of the FAD-dependent halogenase TiaM reveals a sequentially acting dihalogenation step tailoring tiacumicin B biosynthesis.