Genome-wide analysis of transcription factor E2F1 mutant proteins reveals that N- and C-terminal protein interaction domains do not participate in targeting E2F1 to the human genome

Previous studies of E2F family members have suggested that protein-protein interactions may be the mechanism by which E2F proteins are recruited to specific genomic regions. We have addressed this hypothesis on a genome-wide scale using ChIP-seq analysis of MCF7 cell lines that express tagged wild type and mutant E2F1 proteins. First, we performed ChIP-seq for tagged WT E2F1. Then, we analyzed E2F1 proteins that lacked the N-terminal SP1 and cyclin A binding domains, the C-terminal transactivation and pocket protein binding domains, and the internal marked box domain. Surprisingly, we found that the ChIP-seq patterns of the mutant proteins were identical to that of WT E2F1. However, mutation of the DNA binding domain abrogated all E2F1 binding to the genome. These results suggested that the interaction between the E2F1 DNA binding domain and a consensus motif may be the primary determinant of E2F1 recruitment. To address this possibility, we analyzed the in vivo binding sites for the in vitro-derived consensus E2F1 motif (TTTSSCGC) and also performed de novo motif analysis. We found that only 12% of the ChIP-seq peaks contained the TTTSSCGC motif. De novo motif analysis indicated that most of the in vivo sites lacked the 5' half of the in vitro-derived consensus, having instead the in vivo consensus of CGCGC. In summary, our findings do not provide support for the model that protein-protein interactions are involved in recruiting E2F1 to the genome, but rather suggest that recognition of a motif found at most human promoters is the critical determinant.     

Ca2+-dependent GTPase, extra-large G protein 2 (XLG2), promotes activation of DNA-binding protein related to vernalization 1 (RTV1), leading to activation of floral integrator genes and early flowering in Arabidopsis

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, play important roles in plant development and cell signaling. In Arabidopsis, in addition to one prototypical G protein α subunit, GPA1, there are three extra-large G proteins, XLG1, XLG2, and XLG3, of largely unknown function. Each extra-large G (XLG) protein has a C-terminal Gα-like region and a ～400 amino acid N-terminal extension. Here we show that the three XLG proteins specifically bind and hydrolyze GTP, despite the fact that these plant-specific proteins lack key conserved amino acid residues important for GTP binding and hydrolysis of GTP in mammalian Gα proteins. Moreover, unlike other known Gα proteins, these activities require Ca(2+) instead of Mg(2+) as a cofactor. Yeast two-hybrid library screening and in vitro protein pull-down assays revealed that XLG2 interacts with the nuclear protein RTV1 (related to vernalization 1). Electrophoretic mobility shift assays show that RTV1 binds to DNA in vitro in a non-sequence-specific manner and that GTP-bound XLG2 promotes the DNA binding activity of RTV1. Overexpression of RTV1 results in early flowering. Combined overexpression of XLG2 and RTV1 enhances this early flowering phenotype and elevates expression of the floral pathway integrator genes, FT and SOC1, but does not repress expression of the floral repressor, FLC. Chromatin immunoprecipitation assays show that XLG2 increases RTV1 binding to FT and SOC1 promoters. Thus, a Ca(2+)-dependent G protein, XLG2, promotes RTV1 DNA binding activity for a subset of floral integrator genes and contributes to floral transition.     

MicroRNA-155 targets SMAD2 and modulates the response of macrophages to transforming growth factor-{beta}

Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine with important effects on processes such as fibrosis, angiogenesis, and immunosupression. Using bioinformatics, we identified SMAD2, one of the mediators of TGF-β signaling, as a predicted target for a microRNA, microRNA-155 (miR-155). MicroRNAs are a class of small non-coding RNAs that have emerged as an important class of gene expression regulators. miR-155 has been found to be involved in the regulation of the immune response in myeloid cells. Here, we provide direct evidence of binding of miR-155 to a predicted binding site and the ability of miR-155 to repress SMAD2 protein expression. We employed a lentivirally transduced monocyte cell line (THP1-155) containing an inducible miR-155 transgene to show that endogenous levels of SMAD2 protein were decreased after sustained overexpression of miR-155. This decrease in SMAD2 led to a reduction in both TGF-β-induced SMAD-2 phosphorylation and SMAD-2-dependent activation of the expression of the CAGA(12)LUC reporter plasmid. Overexpression of miR-155 altered the cellular responses to TGF-β by changing the expression of a set of genes that is involved in inflammation, fibrosis, and angiogenesis. Our study provides firm evidence of a role for miR-155 in directly repressing SMAD2 expression, and our results demonstrate the relevance of one of the two predicted target sites in SMAD2 3'-UTR. Altogether, our data uncover an important role for miR-155 in modulating the cellular response to TGF-β with possible implications in several human diseases where homeostasis of TGF-β might be altered.     

Response gene to complement 32 is essential for fibroblast activation in renal fibrosis

Response gene to complement 32 (RGC-32) is a downstream target of transforming growth factor-β (TGF-β). TGF-β is known to play a pathogenic role in renal fibrosis. In this study, we investigated RGC-32 function in renal fibrosis following unilateral ureteral obstruction (UUO) in mice, a model of progressive tubulointerstitial fibrosis. RGC-32 is normally expressed only in blood vessels of mouse kidney. However, UUO induces RGC-32 expression in renal interstitial cells at the early stage of kidney injury, suggesting that RGC-32 is involved in interstitial fibroblast activation. Indeed, expression of smooth muscle α-actin (α-SMA), an indicator of fibroblast activation, is limited to the interstitial cells at the early stage, and became apparent later in both interstitial and tubular cells. RGC-32 knockdown by shRNA significantly inhibits UUO-induced renal structural damage, α-SMA expression and collagen deposition, suggesting that RGC-32 is essential for the onset of renal interstitial fibrosis. In vitro studies indicate that RGC-32 mediates TGF-β-induced fibroblast activation. Mechanistically, RGC-32 interacts with Smad3 and enhances Smad3 binding to the Smad binding element in α-SMA promoter as demonstrated by DNA affinity assay. In the chromatin setting, Smad3, but not Smad2, binds to α-SMA promoter in fibroblasts. RGC-32 appears to be essential for Smad3 interaction with the promoters of fibroblast activation-related genes in vivo. Functionally, RGC-32 is crucial for Smad3-mediated α-SMA promoter activity. Taken together, we identify RGC-32 as a novel fibrogenic factor contributing to the pathogenesis of renal fibrosis through fibroblast activation.     

Protein-tyrosine phosphatases are involved in interferon resistance associated with insulin resistance in HepG2 cells and obese mice

Insulin resistance is a risk factor for non-response to interferon/ribavirin therapy in patients with chronic hepatitis C. The aim of this study was to determine the role played by protein-tyrosine phosphatases (PTPs) in the absence of interferon-α (IFNα) response associated with insulin resistance. We induced insulin resistance by silencing IRS-2 or by treating HepG2 cells with tumor necrosis factor-α (TNFα) and analyzed insulin response by evaluating Akt phosphorylation and IFNα response by measuring Stat-1 tyrosine phosphorylation and 2',5'-oligoadenylate synthase and myxovirus resistance gene expression. The response to IFNα was also measured in insulin-resistant obese mice (high fat diet and ob/ob mice) untreated and treated with metformin. Silencing IRS-2 mRNA induces insulin resistance and inhibits IFNα response. Likewise, TNFα suppresses insulin and IFNα response. Treatment of cells with pervanadate and knocking down PTP-1B restores insulin and IFNα response. Both silencing IRS-2 and TNFα treatment increase PTP and PTP-1B activity. Metformin inhibits PTP and improves IFNα response in insulin-resistant cells. Insulin-resistant ob/ob mice have increased PTP-1B gene expression and activity in the liver and do not respond to IFNα administration. Treatment with metformin improves this response. In HepG2 cells, insulin resistance provokes IFNα resistance, which is associated with an increased PTP-1B activity in the liver. Inhibition of PTP-1B activity with pervanadate and metformin or knocking down PTP-1B reestablishes IFNα response. Likewise, metformin decreases PTP-1B activity and improves response to IFNα in insulin-resistant obese mice. The use of PTP-1B inhibitors may improve the response to IFNα/ribavirin therapy.     

Histone deacetylase inhibitor belinostat represses survivin expression through reactivation of transforming growth factor beta (TGFbeta) receptor II leading to cancer cell death

Survivin is a cancer-associated gene that functions to promote cell survival, cell division, and angiogenesis and is a marker of poor prognosis. Histone deacetylase inhibitors induce apoptosis and re-expression of epigenetically silenced tumor suppressor genes in cancer cells. In association with increased expression of the tumor suppressor gene transforming growth factor β receptor II (TGFβRII) induced by the histone deacetylase inhibitor belinostat, we observed repressed survivin expression. We investigated the molecular mechanisms involved in survivin down-regulation by belinostat downstream of reactivation of TGFβ signaling. We identified two mechanisms. At early time points, survivin protein half-life was decreased with its proteasomal degradation. We observed that belinostat activated protein kinase A at early time points in a TGFβ signaling-dependent mechanism. After longer times (48 h), survivin mRNA was also decreased by belinostat. We made the novel observation that belinostat mediated cell death through the TGFβ/protein kinase A signaling pathway. Induction of TGFβRII with concomitant survivin repression may represent a significant mechanism in the anticancer effects of this drug. Therefore, patient populations exhibiting high survivin expression with epigenetically silenced TGFβRII might potentially benefit from the use of this histone deacetylase inhibitor.     

Identification of function for CD44 intracytoplasmic domain (CD44-ICD): modulation of matrix metalloproteinase 9 (MMP-9) transcription via novel promoter response element

CD44 is a multifunctional cell receptor that conveys a cancer phenotype, regulates macrophage inflammatory gene expression and vascular gene activation in proatherogenic environments, and is also a marker of many cancer stem cells. CD44 undergoes sequential proteolytic cleavages that produce an intracytoplasmic domain called CD44-ICD. However, the role of CD44-ICD in cell function is unknown. We take a major step toward the elucidation of the CD44-ICD function by using a CD44-ICD-specific antibody, a modification of a ChIP assay to detect small molecules, and extensive computational analysis. We show that CD44-ICD translocates into the nucleus, where it then binds to a novel DNA consensus sequence in the promoter region of the MMP-9 gene to regulate its expression. We also show that the expression of many other genes that contain this novel response element in their promoters is up- or down-regulated by CD44-ICD. Furthermore, hypoxia-inducible factor-1α (Hif1α)-responsive genes also have the CD44-ICD consensus sequence and respond to CD44-ICD induction under normoxic conditions and therefore independent of Hif1α expression. Additionally, CD44-ICD early responsive genes encode for critical enzymes in the glycolytic pathway, revealing how CD44 could be a gatekeeper of the Warburg effect (aerobic glycolysis) in cancer cells and possibly cancer stem cells. The link of CD44 to metabolism is novel and opens a new area of research not previously considered, particularly in the study of obesity and cancer. In summary, our results finally give a function to the CD44-ICD and will accelerate the study of the regulation of many CD44-dependent genes.     

Structure of the Bone Morphogenetic Protein Receptor ALK2 and Implications for Fibrodysplasia Ossificans Progressiva

Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.