SUMMARY High Gleason grade prostate carcinomas are aggressive, poorly differentiated tumors that exhibit diminished estrogen receptor β (ERβ) expression. We report that a key function of ERβ and its specific ligand 5α-androstane-3β,17β-diol (3β-adiol) is to maintain an epithelial phenotype and repress mesenchymal characteristics in prostate carcinoma. Stimuli (TGF-β and hypoxia) that induce an epithelial-mesenchymal transition (EMT) diminish ERβ expression, and loss of ERβ is sufficient to promote an EMT. The mechanism involves ERβ-mediated destabilization of HIF-1α and transcriptional repression of VEGF-A. The VEGF-A receptor neuropilin-1 drives the EMT by promoting Snail1 nuclear localization. Importantly, this mechanism is manifested in high Gleason grade cancers, which exhibit significantly more HIF-1α and VEGF expression, and Snail1 nuclear localization compared to low Gleason grade cancers.

Recent work with mouse models and human leukemic samples has shown that gain-of-function mutation(s) in Notch1 is a common genetic event in T-cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a γ-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus, where it forms part of a transcriptional activator complex. To identify Notch1 target genes in leukemia, we developed mouse T-cell leukemic lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel, direct, and critical Notch1 target gene in T-cell leukemia. c-myc mRNA levels are increased in primary mouse T-cell tumors that harbor Notch1 mutations, and Notch1 inhibition decreases c-myc mRNA levels and inhibits leukemic cell growth. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with γ-secretase inhibitor treatment or Notch1 inhibition. Consistent with these findings, retroviral insertional mutagenesis screening of our T-cell leukemia mouse model revealed common insertions in either notch1 or c-myc genes. These studies define the Notch1 molecular signature in mouse T-ALL and importantly provide mechanistic insight as to how Notch1 contributes to human T-ALL.

Mutations in NOTCH1 are frequently detected in patients with T-cell acute lymphoblastic leukemia (T-ALL) and in mouse T-ALL models. Treatment of mouse or human T-ALL cell lines in vitro with ␥-secretase inhibitors (GSIs) results in growth arrest and/or apoptosis. These studies suggest GSIs as potential therapeutic agents in the treatment of T-ALL. To determine whether GSIs have antileukemic activity in vivo, we treated near-endstage Tal1/Ink4a/Arf ϩ/Ϫ leukemic mice with vehicle or with a GSI developed by Merck (MRK-003). We found that GSI treatment significantly extended the survival of leukemic mice compared with vehicle-treated mice. Notch1 target gene expression was repressed and increased numbers of apoptotic cells were observed in the GSItreated mice, demonstrating that Notch1 inhibition in vivo induces apoptosis. T-ALL cell lines also exhibit PI3K/mTOR pathway activation, indicating that rapamycin may also have therapeutic benefit. When GSIs are administered in combination with rapamycin, mTOR kinase activity is ablated and apoptosis induced. Moreover, GSI and rapamycin treatment inhibits human T-ALL growth and extends survival in a mouse xenograft model. This work supports the idea of targeting NOTCH1 in T IntroductionT-cell acute lymphoblastic leukemia (T-ALL) is associated with the misexpression of the basic helix-loop-helix protein TAL1/SCL and LIM-domain only proteins LMO1 and LMO2. [1][2][3][4][5][6][7][8] These oncogenes are found misexpressed in greater than 60% of human T-ALL patients. 3,9 Mouse models of T-ALL recapitulate the disease through ectopic expression of Tal1 in the thymus. [10][11][12] These mice develop respiratory distress due to the presence of large thymic masses and have detectable T-cell blasts in peripheral blood lymphocytes (PBLs), spleen, liver, and kidney. 10 Misexpression of Tal1 results in perturbed thymocyte development by interfering with the basic-helix-loop-helix (bHLH) heterodimer E47/HEB that regulates the expression of genes critical for thymocyte differentiation including Rag1/2, CD4, CD3, and TCR␣/␤. 13,14 Consistent with this finding, loss of the E2A gene that encodes the E47 and E12 bHLH protein is associated with human B-progenitor ALL. 15 Mutations in the Notch1 receptor have been frequently detected in mouse T-ALL models [16][17][18][19] and importantly in 54% of T-ALL patients. 20 These mutations cluster in the heterodimerization domain (HD) 20 and the juxtamembrane (JME) region, 21 or result in truncation of PEST regulatory sequences. 18,20 Mutations in the HD domain result in increased susceptibility to cleavage by the gamma-secretase complex; JME mutations may facilitate metalloprotease cleavage, whereas deletion of PEST regulatory sequences is thought to result in increased Notch1 stability. [21][22][23] Treatment of mouse Tal1 leukemic cell lines in vitro with ␥-secretase inhibitors (GSIs) results in cell cycle arrest and apoptosis, revealing that Notch1 signaling is required for leukemic growth/survival. 18Notch1-mediated leukemic growth in mouse and ...

Gene expression requires the recruitment of chromatin remodeling activities and general transcription factors (GTFs) to promoters. Whereas the role of activators in recruiting chromatin remodeling activities has been clearly demonstrated, the contributions of the transcription machinery have not been firmly established. Here we demonstrate that the remodeling of the RNR3 promoter requires a number of GTFs, mediator and RNA polymerase II. We also show that remodeling is dependent upon the SWI/SNF complex, and that TFIID and RNA polymerase II are required for its recruitment to the promoter. In contrast, Gcn5p-dependent histone acetylation occurs independently of TFIID and RNA polymerase II function, and we provide evidence that acetylation increases the extent of nucleosome remodeling, but is not required for SWI/SNF recruitment. Thus, the general transcription machinery can contribute to nucleosome remodeling by mediating the association of SWI/SNF with promoters, thereby revealing a novel pathway for the recruitment of chromatin remodeling activities.[Keywords: Chromatin remodeling; SWI/SNF; TAFs; RNR3; SAGA; TFIID] Supplemental material is available at http://www.genesdev.org.

Background: FSP27 depletion increases both basal and stimulated lipolysis. Results: FSP27 interacts with ATGL via amino acids 120 -220 to regulate lipolysis and triglyceride storage in human adipocytes. Conclusion: FSP27 inhibits ATGL-mediated lipolysis and protects adipocytes against free fatty acid-impaired insulin signaling. Significance: The novel lipolytic regulation shown here may lead to new treatments for insulin resistance.

Insulin-like growth factor II (IGF-II) mRNA binding protein 3 (IMP3) is emerging as a useful indicator of the progression and outcome of several cancers. IMP3 expression is associated with triple-negative breast carcinomas (TNBCs), which are aggressive tumors associated with poor outcome. In this study, we addressed the hypothesis that signaling pathways, which are characteristic of TNBCs, impact the expression of IMP3 and that IMP3 contributes to the function of TNBCs. The data obtained reveal that IMP3 expression is repressed specifically by estrogen receptor β (ERβ) and its ligand 3βA-diol but not by ERα. EGF receptor (EGFR) signaling and consequent activation of the MAP kinase pathway induce IMP3 transcription and expression. Interestingly, we discovered that the EGFR promoter contains an imperfect estrogen response element and that ERβ represses EGFR transcription. These data support a mechanism in which ERβ inhibits IMP3 expression indirectly by repressing the EGFR. This mechanism relates to the biology of TNBC, which is characterized by diminished ERβ and increased EGFR expression. We also demonstrate that IMP3 contributes to the migration and invasion of breast carcinoma cells. Given that IMP3 is an mRNA binding protein, we determined that it binds several key mRNAs that could contribute to migration and invasion including CD164 (endolyn) and MMP9. Moreover, expression of these mRNAs is repressed by ERβ and enhanced by EGFR signaling, consistent with our proposed mechanism for the regulation of IMP3 expression in breast cancer cells. Our findings show that IMP3 is an effector of EGFR-mediated migration and invasion and they provide the first indication of how this important mRNA binding protein is regulated in cancer.

DNA microarray and genetic studies of Saccharomyces cerevisiae have demonstrated that histone deacetylases (HDACs) are required for transcriptional activation and repression, but the mechanism by which they activate transcription remains poorly understood. We show that two HDACs, RPD3 and HOS2, are required for the activation of DNA damage-inducible genes RNR3 and HUG1. Using mutants specific for the Rpd3L complex, we show that the complex is responsible for regulating RNR3. Furthermore, unlike what was described for the GAL genes, Rpd3L regulates the activation of RNR3 by deacetylating nucleosomes at the promoter, not at the open reading frame. Rpd3 is recruited to the upstream repression sequence of RNR3, which surprisingly does not require Tup1 or Crt1. Chromatin remodeling and TFIID recruitment are largely unaffected in the ⌬rpd3/⌬hos2 mutant, but the recruitment of RNA polymerase II is strongly reduced, arguing that Rpd3 and Hos2 regulate later stages in the assembly of the preinitiation complex or facilitate multiple rounds of polymerase recruitment. Furthermore, the histone H4 acetyltransferase Esa1 is required for the activation of RNR3 and HUG1. Thus, reduced or unregulated constitutive histone H4 acetylation is detrimental to promoter activity, suggesting that HDAC-dependent mechanisms are in place to reset promoters to allow high levels of transcription.Although the correlation between histone modifications and gene expression was established many years ago (2), the underlying mechanism by which they affect transcription is still largely unknown. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) function in an antagonistic manner to regulate the balance of histone acetylation and gene activity (25,37,42). It is widely accepted that histone acetylation by HAT correlates with gene expression, while histone deacetylation by HDACs is associated with gene repression (25,37,42).Histone deacetylases catalyze the removal of acetyl groups from the amino-terminal tails of the core histones, making chromatin inaccessible to the transcriptional machinery (11,25,42). Two families of HDACs are found in yeast (Saccharomyces cerevisiae), and each family is classified based upon sequence homology among its members. Five HDACs, namely Hda1, Rpd3, Hos1, Hos2, and Hos3, belong to one family, and the other includes Sir2 and Hst1 to Hst4 (25). Two mechanisms have been proposed for how HDACs regulate transcription: targeted and nontargeted. The targeting mechanism involves the direct recruitment of HDACs to promoters by DNA binding proteins or corepressors, such as Ume6 or Tup1, respectively (21,22,23,36,38,46,47). This mechanism results in targeted deacetylation, spanning approximately two nucleosomes over the DNA binding site (22,38,47). The second mechanism is poorly understood and results in untargeted, genome-wide histone deacetylation (26,44,47), including deacetylation within coding regions. The mechanism of targeted versus global deacetylation was illuminated partly by the discovery of two Rpd3-cont...

Background: FSP27 is a lipid droplet-associated protein.Results: Expression of FSP27 in human adipocytes reversely correlates with ATGL levels. Mechanistically, FSP27 increases the inhibitory effect of Egr1 on the ATGL promoter. Conclusion: FSP27 controls lipolysis by regulating ATGL transcription. Significance: Our study provides a new model of regulation of lipolysis in adipocytes. Current epidemics of metabolic diseases, such as type 2 diabetes, cardiac dysfunction, hypertension, hepatic steatosis, etc., are largely caused by widespread obesity. Although obesity can affect human health via several different mechanisms (1), the best established connection between obesity and metabolic disease is abnormal levels of circulating fatty acids (FA). 3 FA play important physiological roles in energy production and the synthesis of most lipids; nonetheless, their oversupply is highly detrimental as it leads to insulin resistance, oxidative stress, and other pathophysiological effects via mechanisms that are currently under intense investigation (1-5). Lipolysis in fat tissueNormally, dietary FA are partitioned into adipose tissue, converted into triglycerides, and stored in lipid droplets (LDs) that represent dynamic intracellular organelles consisting of a core of triglycerides and cholesterol esters, surrounded by a monolayer of phospholipids. Several proteins are associated with this monolayer, notably the PAT family proteins, PLIN 1-5 (6, 7), and fat-specific protein 27 (FSP27, also known as CIDEC) (8 -10). The latter protein plays an essential role in the regulation of LD morphology. Depletion of FSP27 in adipocytes leads to fragmentation of LDs (11,12), whereas overexpression of FSP27 increases the size of LDs while decreasing their number (8, 9, 12) by promoting LD fusion (13) and exchanging lipids from one droplet to another (14).It has also been demonstrated by us and others that FSP27 has anti-lipolytic activity (8,9,12,(15)(16)(17)(18). Lipolysis in adipose tissue is the major source of circulating . Correspondingly, unrestricted lipolysis in adipose tissue represents a serious metabolic defect and a causative factor of insulin resistance, diabetes mellitus, and other metabolic diseases (3,(25)(26)(27). As the mechanism of the anti-lipolytic activity of FSP27 is not completely clear, we decided to focus on this problem.Our recent study showed that FSP27 directly interacts with ATGL and regulates its lipase activity (18). Another study predicted that FSP27-mediated fusion of LDs might limit the access of intracellular lipases to the LD surface due to decreased surface area of larger LDs (17). As ATGL represents a major lipolytic enzyme (28), reducing its contact with LDs may suppress lipolysis.Here, we report another mechanism of the anti-lipolytic action of FSP27 in human adipocytes. We have found that FSP27 inhibits expression of ATGL at the level of transcription by stimulating the effect of its transcriptional repressor Egr1. EXPERIMENTAL PROCEDURESCell Culture-Human preadipocytes were procured from the ...