MYC regulates the core pre-mRNA splicing machinery as an essential step in lymphomagenesis.

Deregulated expression of the MYC transcription factor occurs in most human cancers and correlates with high proliferation, reprogrammed cellular metabolism and poor prognosis. Overexpressed MYC binds to virtually all active promoters within a cell, although with different binding affinities, and modulates the expression of distinct subsets of genes. However, the critical effectors of MYC in tumorigenesis remain largely unknown.

Multiplexed locus-specific analysis of DNA methylation in single cells.

This protocol details a method for measuring the DNA methylation state of multiple target sites in single cells, otherwise known as single-cell restriction analysis of methylation (SCRAM). The basic steps include isolating and lysing single cells, digesting genomic DNA with a methylation-sensitive restriction endonuclease (MSRE) and amplification of multiple targets by two rounds of PCR to determine the methylation status of target sites. The method can reliably and accurately detect the methylation status of multiple target sites in each single cell, and it can be completed in a relatively short time (<2 d) at low cost.

Quantitative assessment of telomerase components in cancer cell lines.

Besides its canonical function of catalyzing the formation of telomeric repeats, many groups have recently reported non-canonical functions of hTERT in particular, and telomerase in general. Regulating transcription is the central basis of non-canonical functions of telomerase. However, unlike reverse transcriptase activity of telomerase that requires only a few molecules of enzymatically active hTERT, non-canonical functions of hTERT or other telomerase components theoretically require several hundred copies.

Understanding of complex protein interactions with respect to anchorage independence.

Anchorage-independent growth of cells in soft agar is one of the hallmark characteristics of cellular transformation and uncontrolled cell growth. It may be considered as one of the most stringent assays for detecting malignant transformation of cells. Here, we describe a retroviral infection of a library of small secretory proteins and the use of the soft agar assay to obtain and study novel interacting protein combinations that cause cell transformation.

Collective cell motility promotes chemotactic prowess and resistance to chemorepulsion.

Collective cell migration is a widespread biological phenomenon, whereby groups of highly coordinated, adherent cells move in a polarized fashion. This migration mode is a hallmark of tissue morphogenesis during development and repair and of solid tumor dissemination. In addition to circulating as solitary cells, lymphoid malignancies can assemble into tissues as multicellular aggregates.

The clinical role of epithelial-mesenchymal transition and stem cell markers in advanced-stage ovarian serous carcinoma effusions.

We recently identified gene signatures that allow classification of ovarian carcinoma into 5 distinct clinically relevant groups. In the present study, we investigated the clinical role of 10 protein products of the discriminating genes, with focus on epithelial-mesenchymal transition and stem cell markers. Expression of E-cadherin, N-cadherin, P-cadherin, Zeb1, HMGA2, Rab25, CD24, NCAM (CD56), Sox11, and vimentin was assessed in 100 advanced-stage (International Federation of Gynecology and Obstetrics stages III-IV) serous ovarian carcinoma effusions using immunohistochemistry.

Arg kinase-binding protein 2 (ArgBP2) interaction with α-actinin and actin stress fibers inhibits cell migration.

Cell migration requires dynamic remodeling of the actomyosin network. We report here that an adapter protein, ArgBP2, is a component of α-actinin containing stress fibers and inhibits migration. ArgBP2 is undetectable in many commonly studied cancer-derived cell lines.

Multiplexed analysis of protein-ligand interactions by fluorescence anisotropy in a microfluidic platform.

Homogeneous assay platforms for measuring protein-ligand interactions are highly valued due to their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive due to the large amounts of reagents required and the need for automation. We implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions.

The structural and functional organization of the podocyte filtration slits is regulated by Tjp1/ZO-1.

Blood filtration in the kidney glomerulus is essential for physiological homeostasis. The filtration apparatus of the kidney glomerulus is composed of three distinct components: the fenestrated endothelial cells, the glomerular basement membrane, and interdigitating foot processes of podocytes that form the slit diaphragm. Recent studies have demonstrated that podocytes play a crucial role in blood filtration and in the pathogenesis of proteinuria and glomerular sclerosis; however, the molecular mechanisms that organize the podocyte filtration barrier are not fully understood.

SPHK1 regulates proliferation and survival responses in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is characterized by unique aggressive behavior and lack of targeted therapies. Among the various molecular subtypes of breast cancer, it was observed that TNBCs express elevated levels of sphingosine kinase 1 (SPHK1) compared to other breast tumor subtypes. High levels of SPHK1 gene expression correlated with poor overall and progression- free survival, as well as poor response to Doxorubicin-based treatment.

ZO-1 and ZO-2 are required for extra-embryonic endoderm integrity, primitive ectoderm survival and normal cavitation in embryoid bodies derived from mouse embryonic stem cells.

The Zonula Occludens proteins ZO-1 and ZO-2 are cell-cell junction-associated adaptor proteins that are essential for the structural and regulatory functions of tight junctions in epithelial cells and their absence leads to early embryonic lethality in mouse models. Here, we use the embryoid body, an in vitro peri-implantation mouse embryogenesis model, to elucidate and dissect the roles ZO-1 and ZO-2 play in epithelial morphogenesis and de novo tight junction assembly. Through the generation of individual or combined ZO-1 and ZO-2 null embryoid bodies, we show that their dual deletion prevents tight junction formation, resulting in the disorganization and compromised barrier function of embryoid body epithelial layers.

Loss of Cdk2 and cyclin A2 impairs cell proliferation and tumorigenesis.

Cell-cycle inhibition has yet to offer a generally effective approach to cancer treatment, but a full evaluation of different combinations of cell-cycle inhibitors has not been evaluated. Cyclin A2, a core component of the cell cycle, is often aberrantly expressed in cancer where it may impact cell proliferation. In this study, we investigated the role of cyclin A2 in tumorigenesis using a conditional genetic knockout mouse model.

DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos.

Methylation of DNA is an essential epigenetic control mechanism in mammals. During embryonic development, cells are directed toward their future lineages, and DNA methylation poses a fundamental epigenetic barrier that guides and restricts differentiation and prevents regression into an undifferentiated state. DNA methylation also plays an important role in sex chromosome dosage compensation, the repression of retrotransposons that threaten genome integrity, the maintenance of genome stability, and the coordinated expression of imprinted genes.

The "histone mimicry" by pathogens.

One of the defining characteristics of human and animal viruses is their ability to suppress host antiviral responses. Viruses express proteins that impair the detection of viral nucleic acids by host pattern-recognition receptors, block signaling pathways that lead to the synthesis of type I interferons and other cytokines, or prevent the activation of virus-induced genes. We have identified a novel mechanism of virus-mediated suppression of antiviral gene expression that relies on the presence of histone-like sequences (histone mimics) in viral proteins.

Noncanonical functions of telomerase: implications in telomerase-targeted cancer therapies.

Telomerase plays a pivotal role in bypassing cellular senescence and maintaining telomere homeostasis, essential properties required for the sustenance and progression of cancer. However, recent investigations have uncovered extratelomeric properties of telomerase that are independent of its role in telomere extension. This review summarizes recent insights to the noncanonical functions of telomerase reverse transcriptase (TERT) catalytic subunit, in particular in cancer progression, and highlights two major signaling mechanisms involved in the cross-talk with TERT-the NF-κB and Wnt/β-catenin pathways.

IFN-gamma AU-rich element removal promotes chronic IFN-gamma expression and autoimmunity in mice.

We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3' untranslated region (3'UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) (-/-) present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del(-/-) mice display increased numbers of pDCs in bone marrow and spleen.

MEN1 tumorigenesis in the pituitary and pancreatic islet requires Cdk4 but not Cdk2.

Recent studies suggest that physiological and tumorigenic proliferation of mammalian cells is controlled by multiple cyclin-dependent kinases (CDKs) largely in tissue-specific manners. We and others previously demonstrated that adult mice deficient for the Cyclin D partner CDK4 (Cdk4(-/-) mice) exhibit hypoplasia in the pituitary and pancreatic islet due to primary postnatal defects in proliferation. Intriguingly, those neuroendocrine tissues affected in Cdk4(-/-) mice are the primary targets of tumorigenesis in the syndrome of multiple endocrine neoplasia type-1 (MEN1).

The Complex Relationship between Liver Cancer and the Cell Cycle: A Story of Multiple Regulations.

The liver acts as a hub for metabolic reactions to keep a homeostatic balance during development and growth. The process of liver cancer development, although poorly understood, is related to different etiologic factors like toxins, alcohol, or viral infection. At the molecular level, liver cancer is characterized by a disruption of cell cycle regulation through many molecular mechanisms.

Basal keratinocytes contribute to all strata of the adult zebrafish epidermis.

The epidermis of terrestrial vertebrates is a stratified epithelium and forms an essential protective barrier. It is continually renewed, with dead corneocytes shed from the surface and replaced from a basal keratinocyte stem cell population. Whilst mouse is the prime model system used for epidermal studies, there is increasing employment of the zebrafish to analyse epidermis development and homeostasis, however the architecture and ontogeny of the epidermis in this system are incompletely described.

p27 is regulated independently of Skp2 in the absence of Cdk2.

Cyclin-dependent kinase 2 (Cdk2) is dispensable for mitotic cell cycle progression and Cdk2 knockout mice are viable due to the compensatory functions of other Cdks. In order to assess the role of Cdk2 under limiting conditions, we used Skp2 knockout mice that exhibit increased levels of Cdk inhibitor, p27(Kip1), which is able to inhibit Cdk2 and Cdk1. Knockdown of Cdk2 abrogated proliferation of Skp2(-/-) mouse embryonic fibroblasts, encouraging us to generate Cdk2(-/-)Skp2(-/-) double knockout mice.

MiR-27 orchestrates the transcriptional regulation of brown adipogenesis.

Objective: Brown adipose tissue (BAT) produces heat using chemical energy of lipids and glucose, a function induced by cold exposure or diet. The brown adipogenesis is tightly controlled in a coordinated interplay between several transcriptional factors. It is not known what enables and coordinates this robust program of concerted cooperation between the transcriptional factors and co-regulators necessary for the brown adipogenesis.

A simple methodology for conversion of mouse monoclonal antibody to human-mouse chimeric form.

Passive immunotherapy has mainly been used as a therapy against cancer and inflammatory conditions. Recent studies have shown that monoclonal antibody-(mAb-) based passive immunotherapy is a promising approach to combat virus infection. Specific mouse mAbs can be routinely generated in large amounts with the use of hybridoma technology but these cannot be used for therapy in human beings due to their immunogenicity.

Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.

The tight control of gene expression at the level of both transcription and post-transcriptional RNA processing is essential for mammalian development. We here investigate the role of protein arginine methyltransferase 5 (PRMT5), a putative splicing regulator and transcriptional cofactor, in mammalian development. We demonstrate that selective deletion of PRMT5 in neural stem/progenitor cells (NPCs) leads to postnatal death in mice.