DNA elements tether canonical Polycomb Repressive Complex 1 to human genes

Development of multicellular animals requires epigenetic repression by Polycomb group proteins. The latter assemble in multi-subunit complexes, of which two kinds, Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2), act together to repress key developmental genes. How PRC1 and PRC2 recognize specific genes remains an open question. Here we report the identification of several hundreds of DNA elements that tether canonical PRC1 to human developmental genes. We use the term tether to describe a process leading to a prominent presence of canonical PRC1 at certain genomic sites, although the complex is unlikely to interact with DNA directly. Detailed analysis indicates that sequence features associated with PRC1 tethering differ from those that favour PRC2 binding. Throughout the genome, the two kinds of sequence features mix in different proportions to yield a gamut of DNA elements that range from those tethering predominantly PRC1 or PRC2 to ones capable of tethering both complexes. The emerging picture is similar to the paradigmatic targeting of Polycomb complexes by Polycomb Response Elements (PREs) of Drosophila but providing for greater plasticity. [GRAPHICS

Resistance to butyrate impairs bile acid-induced apoptosis in human colon adenocarcinoma cells via up-regulation of Bcl-2 and inactivation of Bax

AbstractA critical risk factor in colorectal carcinogenesis and tumor therapy is the resistance to the apoptotic effects of different compounds from the intestinal lumen, among them butyrate (main regulator of colonic epithelium homeostasis). Insensitivity to butyrate-induced apoptosis yields resistance to other agents, as bile acids or chemotherapy drugs, allowing the selective growth of malignant cell subpopulations. Here we analyze bile acid-induced apoptosis in a butyrate-resistant human colon adenocarcinoma cell line (BCS-TC2.BR2) to determine the mechanisms that underlay the resistance to these agents in comparison with their parental butyrate-sensitive BCS-TC2 cells. This study demonstrates that DCA and CDCA still induce apoptosis in butyrate-resistant cells through increased ROS production by activation of membrane-associated enzymes and subsequent triggering of the intrinsic mitochondrial apoptotic pathway. Although this mechanism is similar to that described in butyrate-sensitive cells, cell viability is significantly higher in resistant cells. Moreover, butyrate-resistant cells show higher Bcl‐2 levels that confer resistance to bile acid-induced apoptosis sequestering Bax and avoiding Bax-dependent pore formation in the mitochondria. We have confirmed that this resistance is reverted using the Bcl-2 inhibitor ABT-263, thus demonstrating that the lower sensitivity of butyrate-resistant cells to the apoptotic effects of bile acids is mainly due to increased Bcl-2 levels

PTE, a novel module to target Polycomb Repressive Complex 1 to the human cyclin D2 (CCND2) oncogene

Polycomb group proteins are essential epigenetic repressors. They form multiple protein complexes of which two kinds, PRC1 and PRC2, are indispensable for repression. Although much is known about their biochemical properties, how mammalian PRC1 and PRC2 are targeted to specific genes is poorly understood. Here, we establish the cyclin D2 (CCND2) oncogene as a simple model to address this question. We provide the evidence that the targeting of PRC1 to CCND2 involves a dedicated PRC1-targeting element (PTE). The PTE appears to act in concert with an adjacent cytosine-phosphate-guanine (CpG) island to arrange for the robust binding of PRC1 and PRC2 to repressed CCND2. Our findings pave the way to identify sequence-specific DNA-binding proteins implicated in the targeting of mammalian PRC1 complexes and provide novel link between polycomb repression and cancer

Key role of the N-terminus of chicken annexin A5 in vesicle aggregation

Annexins are calcium-dependent phospholipid-binding proteins involved in calcium signaling and intracellular membrane trafficking among other functions. Vesicle aggregation is a crucial event to make possible the membrane remodeling but this process is energetically unfavorable, and phospholipid membranes do not aggregate and fuse spontaneously. This issue can be circumvented by the presence of different agents such as divalent cations and/or proteins, among them some annexins. Although human annexin A5 lacks the ability to aggregate vesicles, here we demonstrate that its highly similar chicken ortholog induces aggregation of vesicles containing acidic phospholipids even at low protein and/or calcium concentration by establishment of protein dimers. Our experiments show that the ability to aggregate vesicles mainly resides in the N-terminus as truncation of the N-terminus of chicken annexin A5 significantly decreases this process and replacement of the N-terminus of human annexin A5 by that of chicken switches on aggregation; in both cases, there are no changes in the overall protein structure and only minor changes in phospholipid binding. Electrostatic repulsions between negatively charged residues in the concave face of the molecule, mainly in the N-terminus, seem to be responsible for the impairment of dimer formation in human annexin A5. Taking into account that chicken annexin A5 presents a high sequence and structural similarity with mammalian annexins absent in birds, as annexins A3 and A4, some of the physiological functions exerted by these proteins may be carried out by chicken annexin A5, even those that could require calcium-dependent membrane aggregation.Peer reviewe

Histone deacetylase inhibitors upregulate MMP11 gene expression through Sp1/Smad complexes in human colon adenocarcinoma cells

AbstractMMP-11 (stromelysin-3) is a matrix metalloproteinase associated with tumor progression and poor prognosis. Its expression was initially described exclusively in stromal cells surrounding tumors, but more recently it has also been detected in macrophages and hepatocarcinoma cells. Here we show MMP-11 expression in human epithelial colon adenocarcinoma cell lines (Caco-2, HT-29 and BCS-TC2). Treatment of BCS-TC2 cells with butyrate and trichostatin A (TSA) (histone deacetylase inhibitors) increases MMP11 promoter activity and protein expression. Using electrophoretic mobility shift assay (EMSA) and supershift assays, we demonstrate for the first time that Sp1 is able to bind to the GC-boxes within the MMP11 proximal promoter region; this binding has been confirmed by chromatin immunoprecipitation. Sp1 is involved in MMP11 basal expression and it is essential for the upregulation of transcription by histone deacetylase inhibitors as deduced from mutant constructs lacking the Sp1 sites and by inhibition of its binding to the promoter with mithramycin. This regulation requires the formation of Sp1/Smad2 heterocomplexes, which is stimulated by an increase in the acetylation status of Smad after butyrate or TSA treatments. We have also found that ERK1/2-mitogen-activated protein kinase (MAPK), but not p38-MAPK or JNK, is involved in the upregulation of MMP11 by HDAC inhibitors

Inhibition of pSTAT1 by tofacitinib accounts for the early improvement of experimental chronic synovitis

Background: In order to gain insight into the early effects drawn by JAK inhibitors on intra-joint JAK/STAT-dependent signaling, we sought synovial activation of STATs and their end-products, along with their modification with tofacitinib (TOFA), at flare-up in antigen induced arthritis (AIA). New Zealand rabbits were randomly assigned to four groups –healthy controls, AIA, TOFA-treated AIA, or TOFA-treated controls–. AIA was induced with 4 weekly intra-articular ovalbumin injections in sensitized animals. TOFA (10 mg·kg− 1·day− 1) was administered for the last 2 weeks. Animals were euthanized 24 h after the last injection. Results: AIA animals showed high-grade synovitis, which was partially improved by TOFA. No effects of the treatment were found on serum C-reactive protein or on the synovial macrophage infiltration at this stage. Synovial MMP-1,-3 and -13 expression levels in treated AIA rabbits were found to drop to those of controls, while a downregulation of IL6, IFNγ and TNF was evident in treated versus untreated AIA rabbits. Concurrently, a reduction in pSTAT1 and SOCS1, but not in pSTAT3, SOCS3 or active NFκB-p65, was noted with TOFA. Conclusions: Studying the mechanism of action of immunomodulatory drugs represents a major challenge in vivo, since drug-dependent decreases in inflammation very likely mask direct effects on disease mechanisms. This study design allowed us to prevent any confounding effect resulting from reductions in the overall inflammatory status, hence assessing the true pharmacological actions of TOFA in a very severe synovitis. Our findings point to pSTAT1 and MMPs as early molecular readouts of response to this JAK inhibitor

4F2hc-silencing impairs tumorigenicity of HeLa cells via modulation of galectin-3 and β-catenin signaling, and MMP-2 expression

Abstract4F2hc is a type-II glycoprotein whose covalent-bound association with one of several described light chains yields a heterodimer mainly involved in large neutral amino acid transport. Likewise, it is well known that the heavy chain interacts with β-integrins mediating integrin-dependent events such as survival, proliferation, migration and even transformation. 4F2hc is a ubiquitous protein whose overexpression has been related to tumor development and progression. Stable silencing of 4F2hc in HeLa cells using an artificial miRNA impairs in vivo tumorigenicity and leads to an ineffective proliferation response to mitogens. 4F2hc colocalizes with β1-integrins and CD147, but this interaction does not occur in lipid rafts in HeLa cells. Moreover, silenced cells present defects in integrin- (FAK, Akt and ERK1/2) and hypoxia-dependent signaling, and reduced expression/activity of MMP-2. These alterations seem to be dependent on the inappropriate formation of CD147/4F2hc/β1-integrin heterocomplexes on the cell surface, arising when CD147 cannot interact with 4F2hc. Although extracellular galectin-3 accumulates due to the decrease in MMP-2 activity, galectin-3 signaling events are blocked due to an impaired interaction with 4F2hc, inducing an increased degradation of β-catenin. Furthermore, cell motility is compromised after protein silencing, suggesting that 4F2hc is related to tumor invasion by facilitating cell motility. Therefore, here we propose a molecular mechanism by which 4F2hc participates in tumor progression, favoring first steps of epithelial–mesenchymal transition by inhibition of β-catenin proteasomal degradation through Akt/GSK-3β signaling and enabling cell motility