HPVE6-USP46 Mediated Cdt2 Stabilization Reduces Set8 Mediated H4K20-Methylation to Induce Gene Expression Changes

E6 from high-risk strains of HPV is well known to transform cells by deregulating p53. We reported that in HPV transformed cell-lines E6 from high-risk HPV can recruit the USP46 deubiquitinase to substrates such as Cdt2 and stabilize the latter, and that USP46 is important for growth of HPV induced tumors in xenografts. Here we show that in cervical cancer biopsies the stabilization of Cdt2 in the HPV-induced cancers leads to the decrease of a CRL4-Cdt2 substrate, the histone H4K20 mono-methyltransferase Set8, and decrease in H4K20me1 or H4K20me3 that can be detected by immunohistochemistry. In HPV-transformed cancer cell lines in vitro, knockdown of E6 decreases Cdt2 and increases Set8. Co-knockdown of Set8 shows that some of the gene expression changes produced by E6 knockdown is due to the increase of Set8. EGFR and EGFR regulated genes were identified in this set of genes. Turning to the mechanism by which E6 stabilizes Cdt2, we find that a purified E6:USP46 complex has significantly more de-ubiquitinase activity in vitro than USP46 alone, demonstrating that E6 can directly interact with USP46 in the absence of other proteins and that it can substitute for the known activators of USP46, UAF1 and WDR20. Deletion mapping of Cdt2 shows that there are three discrete, but redundant, parts of the substrate that are essential for stabilization by E6: USP46. The helix–loop–helix region or the WD40 repeat driven beta-propeller structure of Cdt2 are dispensable for the stabilization implying that interaction with DDB1 (and the rest of the CRL4 complex) or with the substrate of the CRL4-Cdt2 E3 ligase is not necessary for E6:USP46 to interact with and stabilize Cdt2. The identification of 50 amino acid stretches in the 731 amino acid Cdt2 protein as being important for the stabilization by E6 underlines the specificity of the process. In summary, E6 activates the deubiquitinase activity of USP46, stabilizes Cdt2 utilizing multiple sites on Cdt2, and leads to degradation of Set8 and changes in gene-expression in HPV-transformed cells.

ADD3 Deletion in Glioblastoma Predicts Disease Status and Survival

Loss of heterozygosity (LOH) on chromosome 10 frequently occurs in gliomas. Whereas genetic loci with allelic deletion often implicate tumor suppressor genes, a putative tumor suppressor Adducin3 (ADD3) mapped to chromosome 10q25.2 was found to be preferentially downregulated in high-grade gliomas compared with low-grade lesions. In this study, we unveil how the assessment of ADD3 deletion provides clinical significance in glioblastoma (GBM). By deletion mapping, we assessed the frequency of LOH in forty-three glioma specimens using five microsatellite markers spanning chromosome 10q23-10qter. Data were validated in The Cancer Genome Atlas (TCGA) cohort with 203 GBM patients. We found that allelic loss in both D10S173 (ADD3/MXI1 locus) and D10S1137 (MGMT locus) were positively associated with tumor grading and proliferative index (MIB-1). However, LOH events at only the ADD3/MXI1 locus provided prognostic significance with a marked reduction in patient survival and appeared to have diagnostic potential in differentiating high-grade gliomas from low-grade ones. Furthermore, we showed progressive loss of ADD3 in six out of seven patient-paired gliomas with malignant progression, as well as in recurrent GBMs. These findings suggest the significance of ADD3/MXI1 locus as a promising marker that can be used to refine the LOH10q assessment. Data further suggest the role of ADD3 as a novel tumor suppressor, whereby the loss of ADD3 is indicative of a progressive disease that may at least partially account for rapid disease progression in GBM. This study revealed for the first time the downregulation of ADD3 on the genetic level resulting from copy number deletion.

Engineering, Characterization, and Biological Evaluation of an Antibody Targeting the HGF Receptor

The Hepatocyte growth factor (HGF) and its receptor (MET) promote several physiological activities such as tissue regeneration and protection from cell injury of epithelial, endothelial, neuronal and muscle cells. The therapeutic potential of MET activation has been scrutinized in the treatment of acute tissue injury, chronic inflammation, such as renal fibrosis and multiple sclerosis (MS), cardiovascular and neurodegenerative diseases. On the other hand, the HGF-MET signaling pathway may be caught by cancer cells and turned to work for invasion, metastasis, and drug resistance in the tumor microenvironment. Here, we engineered a recombinant antibody (RDO24) and two derived fragments, binding the extracellular domain (ECD) of the MET protein. The antibody binds with high affinity (8 nM) to MET ECD and does not cross-react with the closely related receptors RON nor with Semaphorin 4D. Deletion mapping studies and computational modeling show that RDO24 binds to the structure bent on the Plexin-Semaphorin-Integrin (PSI) domain, implicating the PSI domain in its binding to MET. The intact RDO24 antibody and the bivalent Fab2, but not the monovalent Fab induce MET auto-phosphorylation, mimicking the mechanism of action of HGF that activates the receptor by dimerization. Accordingly, the bivalent recombinant molecules induce HGF biological responses, such as cell migration and wound healing, behaving as MET agonists of therapeutic interest in regenerative medicine. In vivo administration of RDO24 in the murine model of MS, represented by experimental autoimmune encephalomyelitis (EAE), delays the EAE onset, mitigates the early clinical symptoms, and reduces inflammatory infiltrates. Altogether, these results suggest that engineered RDO24 antibody may be beneficial in multiple sclerosis and possibly other types of inflammatory disorders.

The effector FEP3/IRON MAN1 modulates interaction between BRUTUS-LIKE1 and bHLH subgroup IVb and IVc proteins

Plants use the micronutrient iron (Fe) efficiently to balance the requirements for Fe during growth with its potential cytotoxic effects. A cascade of basic helix-loop-helix (bHLH) transcription factors is initiated by bHLH proteins of the subgroups IVb and IVc. This induces more than 50 genes in higher plants that can be grouped in co-expression clusters. Gene co-expression networks contain information on functional protein interactomes. We conducted a targeted yeast two-hybrid screen with pairwise combinations of 23 proteins stemming from previously characterized Fe-deficiency-induced gene co-expression clusters and regulators. We identified novel and described interactions, as well as interaction hubs with multiple interactions within the network. We found that BRUTUS-LIKE E3 ligases (BTSL1, BTSL2) interacted with basic helix-loop-helix (bHLH) transcription factors of the subgroups IVb and IVc including PYE, bHLH104 and ILR3, and with small FE UPTAKE-INDUCING PEPTIDE3/IRON MAN1 (FEP3/IMA1). Through deletion studies and with support of molecular docking, we mapped the interaction sites to three-amino-acid regions in BTSL1 and FEP3/IMA1. The FEP3/IMA1 active residues are present in interacting sites of the bHLH IVc factors. FEP3/IMA1 attenuated interaction of BTSL1 with bHLH proteins in a quantitative yeast three-hybrid assay suggesting that it is an inhibitor. Co-expression of BTSL1 and bHLH IVb and IVc factors uncovered unexpected patterns of subcellular localization. Combining deletion mapping, protein interaction and physiological analysis, we discuss the model that FEP3/IMA1 is a small effector protein inhibiting BTSL1/BTSL2-mediated degradation of bHLH subgroup IVb and IVc proteins.

Snail Upregulates Transcription of FN, LEF, COX2, and COL1A1 in Hepatocellular Carcinoma: A General Model Established for Snail to Transactivate Mesenchymal Genes

SNA is one of the essential EMT transcriptional factors capable of suppressing epithelial maker while upregulating mesenchymal markers. However, the mechanisms for SNA to transactivate mesenchymal markers was not well elucidated. Recently, we demonstrated that SNA collaborates with EGR1 and SP1 to directly upregulate MMP9 and ZEB1. Remarkably, a SNA-binding motif (TCACA) upstream of EGR/SP1 overlapping region on promoters was identified. Herein, we examined whether four other mesenchymal markers, lymphoid enhancer-binding factor (LEF), fibronectin (FN), cyclooxygenase 2 (COX2), and collagen type alpha I (COL1A1) are upregulated by SNA in a similar fashion. Expectedly, SNA is essential for expression of these mesenchymal genes. By deletion mapping and site directed mutagenesis coupled with dual luciferase promoter assay, SNA-binding motif and EGR1/SP1 overlapping region are required for TPA-induced transcription of LEF, FN, COX2 and COL1A1. Consistently, TPA induced binding of SNA and EGR1/SP1 on relevant promoter regions of these mesenchymal genes using ChIP and EMSA. Thus far, we found six of the mesenchymal genes are transcriptionally upregulated by SNA in the same fashion. Moreover, comprehensive screening revealed similar sequence architectures on promoter regions of other SNA-upregulated mesenchymal markers, suggesting that a general model for SNA-upregulated mesenchymal genes can be established.

Snail upregulates FN and LEF transcription negatively feed backed by PPAR-gamma: a general model established for Snail to transactivate mesenchymal genes

Background Snail (SNA) is responsible for epithelial mesenchymal transition, migration and metastasis of hepatocellular carcinoma. SNA represses the transcription of the essential epithelial marker such as E-cadherin and enhances mesenchymal markers including fibronectin and lymphoid enhancer-binding factor. Our previous studies indicated that SNA, in collaboration with EGR1 and SP1, may directly activate transcription of the mesenchymal markers, matrix degradation enzyme matrix metalloproteinases (MMP9) and zinc finger E-box binding homeobox 1 (ZEB1) in HepG2 cell stimulated by the phorbol ester tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Besides, we pinpointed a SNA binding motif (TCACA) upstream of EGR1/SP1 overlapping region on promoters. In this study, we investigated whether LEF and FN are transcriptionally regulated by SNA in a similar fashion. Moreover, a general model for SNA-upregulated mesenchymal markers is proposed.Methods RT/PCR and Western blot were used for analyzing gene expression and shRNA technology for depleting SNA. Dual luciferase assay was used for promoter activation; deletion mapping and mutagenesis were used for confirming the indicated promoter region required for transcription activation. ChIP and EMSA were used for validating the binding of the indicated transcription factor on their putative motifs. Results SNA binding motif and E/S overlapping region are required for TPA-induced transcription of LEF and FN. These were supported by TPA-induced binding of SNA and EGR-1/SP-1 on indicated promoter regions. Moreover, a peroxisome proliferator-activated receptor γ motif upstream of SNA binding motif was found to be a negatively regulatory region in TPA-induced promoter activation of FN, LEF, MMP9 and ZEB1. This was supported by that co-treatment of a PPAR-g inhibitor, GW9662, and mutation of PPAR-g binding motif enhanced TPA-induced promoter activity and expression of the aforementioned genes whereas overexpression of PPAR-g reversed it. Moreover, comprehensive screening of the SNA-upregulated mesenchymal genes revealed similar sequence architecture on the promoter regions of the candidate genes: SNA binding motif (TCACA) coupled with a downstream EGR/SP1 overlapping region and an upstream PPAR-g binding motif. Among them COX2 and COL1A1 were found to potentially exhibit the same transcription mechanisms described above. Conclusions We established a general model for SNA-upregulated mesenchymal gene expressions negatively feed backed by PPAR-g.

Analysis of a Strong Suppressor of Segregation Distorter in Drosophila melanogaster

Segregation Distorter (SD) is a naturally occurring male meiotic drive system in Drosophila melanogaster, characterized by almost exclusive transmission of the SD chromosome owing to dysfunction of sperm receiving the SD+ homolog. Previous studies identified at least three closely linked loci on chromosome 2 required for distortion: Sd, the primary distorting gene; E(SD) (Enhancer of SD), which increases the strength of distortion; and Rsp (Responder), the apparent target of Sd. Strength of distortion is also influenced by linked upward modifiers including M(SD) (Modifier of SD) and St(SD) (Stabilizer of SD), and by various unlinked suppressors. Although Sd is known to encode a mutant RanGAP protein, none of the modifiers have been molecularly identified. This work focuses on the genetic and cytological characterization of a strong X-linked suppressor, Su(SD), capable of restoring Mendelian transmission in SD/SD+ males. Sd and its cohort of positive modifiers appear to act semiquantitatively in opposition to Su(SD) with distortion strength depending primarily on the total number of distorting elements rather than which particular elements are present. Su(SD) can also suppress male sterility observed in certain SD genotypes. To facilitate its eventual molecular identification, Su(SD) was localized by deletion mapping to polytene region 13C7-13E4. These studies highlight the polygenic nature of distortion and its dependence on a constellation of positive and negative modifiers, provide insight into the stability of Mendelian transmission in natural populations even when a drive system arises, and pave the way for molecular characterization of Su(SD) whose identity should reveal new information about the mechanism of distortion.

Bone morphogenetic protein signaling regulates Id1 mediated neural stem cell quiescence in the adult zebrafish brain via a phylogenetically conserved enhancer module

In the telencephalon of adult zebrafish, the inhibitor of DNA binding 1 (id1) gene is expressed in radial glial cells (RGCs), behaving as neural stem cells (NSCs), during constitutive and regenerative neurogenesis. Id1 controls the balance between resting and proliferating states of RGCs by promoting quiescence. Here, we identified a phylogenetically conserved cis-regulatory module (CRM) mediating the specific expression of id1 in RGCs. Systematic deletion mapping and mutation of conserved transcription factor binding sites in stable transgenic zebrafish lines reveal that this CRM operates via conserved smad1/5 and 4 binding motifs (SBMs) under both homeostatic and regenerative conditions. Transcriptome analysis of injured and uninjured telencephala as well as pharmacological inhibition experiments identify a crucial role of bone morphogenetic protein (BMP) signaling for the function of the CRM. Our data highlight that BMP signals control id1 expression and thus NSC proliferation during constitutive and induced neurogenesis.