Special Issue "Gene Expression and Regulation"

Guest Editor
Dr. Mary O'Connell
Medical Research Council, Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Scotland, UK
Website: http://www.hgu.mrc.ac.uk/people/m.oconnell.html
E-Mail: M.OConnell@hgu.mrc.ac.uk
Interests: RNA editing; RNA Protein interaction; RNA interference

Dear Colleagues,

Gene expression and how it is regulated is a fundamental question in biology and therefore its complexity is not surprising. Initially the major focus of research was on transcription factors as the major modulators of gene expression. Then enhancers and enhancer binding proteins were found to be critical for gene expression. In recent years epigenetic changes such as DNA methylation and histone methylation have added layers of complexity as they also regulate gene expression. Long range DNA looping can also influence gene expression as it brings promoters and enhancers proximal to genes. With the availability of high throughput sequencing profiling of gene expression in different tissues and also in different diseases has become a very active area of research. What has become a major challenge is to draw a coherent picture from a large volume of data, of what are key regulators of gene expression. This special issue will cover original research papers on this very broad topic. The submission of reviews is also welcomed.

Dr. Mary O'Connell
Guest Editor

Submission

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• transcription factors
• enhancers
• enhancer binding proteins
• DNA methylation
• histone methylation
• DNA looping
• expression profiling
• miRNA and post-translational gene silencing
• asymmetrical distribution of genetic factors

Type of Paper: Article
Title: A Novel Method on Selection Marker-Independent Isolation of Transgene-High-Expressing Mammalian Cells
Authors: Masahiro Sato ¹, Eri Akasaka ², Issei Saitoh ², Masato Ohtsuka ³, Shingo Nakamura ⁴, Takayuki Sakurai ⁵ and Satoshi Watanabe ⁶
Affiliations: ¹ Section of Gene Expression Regulation, Frontier Science Research Center, Japan
² Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan
³ Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa, Japan
⁴ Department of Surgery, National Defense Medical College, Saitama, Japan
⁵ Department of Organ Regeneration, Graduate School of Medicine, Shinshu University, Nagano, Japan
⁶ Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki, Japan; E-Mail: masasato@ms.kagoshima-u.ac.jp
Abstract: Almost all of the experiments for transfection of mammalian cells employ drug resistance gene for drug selection of transfected cells. However, these drug resistant cells are often heterogeneous with respect to transgene expression. Therefore, characterization of each isolated clone is prerequisite to obtain a transgene-high-expressing clone. In this study, we developed a novel method for selective isolation of transgene-high-expressing clones without such characterization. An expression vector pCEIEnd that enables simultaneous expression of two types of protein [EGFP (as gene of interest to be expressed) and endo β-galactosidase (EndoGalC)] upon transfection was constructed. EndoGalC isolated from chrostoridium can digest β-Gal epitope present on the cell surface of almost all mammalian cells except for human and old-world ape. BS-I-B4 lectin (IB4) specifically binds to this epitope. Porcine embryonic fibroblasts (PEFs) were first transfected with pCEIEnd and 4-5 days later the surviving cells (5 X 105) were treated with 1 mg/ml of IB4 conjugated with a strong toxin saporin (SAP) (IB4-SAP) for 2 h at 37C. These treated cells were then incubated in normal medium for 7-10 days. During these periods, β-Gal epitope-expressing cells, including non-transfectants and cells expressing EndoGalC weakly, would have been killed by SAP, because when SAP is incorporated inside the cell via binding of IB4 to the β-Gal epitope it causes cell death. On the other hand, cells expressing EndoGalC strongly would survive, because they can not bind to IB4, due to absence of β-Gal epitope on their surface. The surviving colonies after treatment with IB4-SAP were negative for staining with IB4 and also expressed EGFP strongly. This method is applicable to cells that express β-Gal epitope, and also useful for isolation of transgene-high-expressing cells that exhibit multi-drug resistance.

Type of Paper: Review
Title: Understanding the Dynamics of Gene Regulatory Systems in Disease; Polymorphisms, Synergy and Epigenetics.
Authors: Philip Cowie, Ruth Ross and Alasdair MacKenzie
Affiliations: Gene Regulatory Systems Lab, School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, AB39 3UW, UK; E-Mail; alasdair.mackenzie @abdn.ac.uk
Abstract: Modern genetic analysis has shown that most polymorphisms associated with human disease are non-coding. Much of the functional information contained in the non-coding genome consists of cis-regulatory sequences (CRSs) that are required to respond to signal transduction cues that direct cell specific gene expression. It has been hypothesised that many diseases may be due to polymorphisms within CRS that alter their responses to signal transduction cues. However, identification of CRSs, and the effects of allelic variation on their ability to respond to signal transduction cues, is still at an early stage. In the current review we describe the use of comparative genomics that allows for the identification of CRSs. In addition we describe techniques that allow for the analysis of the effects of allelic variation and epigenetic modification on CRS responses to signal transduction cues. Using specific examples we show that the interactions driving these elements are highly complex and the effects of disease associated polymorphisms often subtle. It is clear that gaining an understanding of the functions of CRSs, and how they are affected by SNPs and epigenetic modification, is essential to understanding the genetic basis of human disease and provides novel directions for the development of personalised medicine.

Type of Paper: Review
Title: Control of Myofibroblast Gene Expression in Cardiopulmonary Fibrosis
Authors: Arthur R. Strauch and Seethalakshmi Hariharan
Affiliation: Department of Physiology & Cell Biology, the Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University College of Medicine, Columbus, OH 43210, USA; E-Mail: strauch.1@osu.edu
Abstract: Myofibroblasts (MFBs) are contractile, smooth muscle-like cells that normally mediate tissue repair and remodeling during wound healing but also accumulate in a dysfunctional state during chronic fibrotic diseases of the heart and lung. While MFBs may differentiate from tissue fibroblasts as well as circulating bone marrow fibrocytes, these cells also may arise from the epithelial and vascular endothelial beds as part of a developmental process referred to as mesenchymal transition. TGFβ1 is the wound healing-associated agonist for both MFB-specific gene expression and epithelial- and endothelial-mesenchymal transition (EMT). Via its receptorregulated Smad 2/3/4 proteins, TGFβ1 stimulates transcription of genes encoding the prototypical MFB-specific markers smooth muscle α-actin (SMαA) and type I collagen (COL1α2) as well as activates pro-survival Akt kinase. Subsequent inhibition of GSK3β kinase by Akt prevents β-catenin degradation and allows activation of β-catenin-dependent genes such as cyclin D and matrix metalloproteinases needed for mesenchymal cell proliferation and migration. While β-catenin signaling can explain mesenchymal cell hyperplasia during EMT, there must be concurrent mechanisms for activating the SMαA and COL1α2 promoters to allow their maturation into contractile, force-transducing MFBs. This aspect of cellular control will be reviewed with an emphasis on dynamic interplay between serum response factor (SRF), TGFβ1-activated Smads, and the single strand-specific DNA- and mRNA-binding proteins, Purα, Purβ, and YB-1, in governing expression of the SMαA and type I collagen genes in normal fibroblasts and injury-activated MFBs.

Type of Paper: Review
Title: Control of Myofibroblast Gene Expression in Cardiopulmonary Fibrosis
Authors: Arthur R. Strauch and Seethalakshmi Hariharan
Affiliation: Department of Physiology & Cell Biology, the Dorothy M. Davis Heart & Lung Research Institute, The Ohio State University College of Medicine, Columbus, OH 43210, USA; E-Mail: strauch.1@osu.edu
Abstract: Myofibroblasts (MFBs) are contractile, smooth muscle-like cells that normally mediate tissue repair and remodeling during wound healing but also accumulate in a dysfunctional state during chronic fibrotic diseases of the heart and lung. While MFBs may differentiate from tissue fibroblasts as well as circulating bone marrow fibrocytes, these cells also may arise from the epithelial and vascular endothelial beds as part of a developmental process referred to as mesenchymal transition. TGFβ1 is the wound healing-associated agonist for both MFB-specific gene expression and epithelial- and endothelial-mesenchymal transition (EMT). Via its receptorregulated Smad 2/3/4 proteins, TGFβ1 stimulates transcription of genes encoding the prototypical MFB-specific markers smooth muscle α-actin (SMαA) and type I collagen (COL1α2) as well as activates pro-survival Akt kinase. Subsequent inhibition of GSK3β kinase by Akt prevents β-catenin degradation and allows activation of β-catenin-dependent genes such as cyclin D and matrix metalloproteinases needed for mesenchymal cell proliferation and migration. While β-catenin signaling can explain mesenchymal cell hyperplasia during EMT, there must be concurrent mechanisms for activating the SMαA and COL1α2 promoters to allow their maturation into contractile, force-transducing MFBs. This aspect of cellular control will be reviewed with an emphasis on dynamic interplay between serum response factor (SRF), TGFβ1-activated Smads, and the single strand-specific DNA- and mRNA-binding proteins, Purα, Purβ, and YB-1, in governing expression of the SMαA and type I collagen genes in normal fibroblasts and injury-activated MFBs.

Type of Paper: Review
Title: MicroRNA Mediated Regulation of Gene Expression
Authors: Aida Martinez-Sanchez and Chris L Murphy
Affiliation: Kennedy Institute, University of Oxford, UK; E-Mail: chris.murphy@kennedy.ox.ac.uk
Abstract: MicroRNAs (miRNAs) are small non-coding RNA molecules of 21-23 nucleotides that control gene expression at the post-transcriptional level and have been shown to play a vital role in a wide variety of biological processes. Moreover, dysregulated expression of miRNAs is found in many pathologies, including cancer and cardiac disease. Understanding the mechanism of action and defining functional mRNA targets of a specific miRNA is essential to unravel its biological function and to develop therapeutic opportunities. This review summarizes the current understanding of the mechanistic aspects of miRNA-induced gene repression and focuses on the different approaches for miRNA target identification that have been proposed in recent years.

Type of Paper: Article
Title: Ankyrins and the Genomic Sex dichotomy of the Heart Rhythm
Authors: Sanda Iacobas, Neil Thomas and Dumitru A Iacobas
Affiliation: DP Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx-New York, NY, USA; E-Mail: dumitru.iacobas@einstein.yu.edu
Abstract: We performed a differential topological analysis of the heart rhythm determinant (HRD) genomic fabric in atria and ventricles of adult male and female mice to identify the mechanisms underlying the sex dichotomy in the heart function and disease. Fabric genes were selected to form the most interconnected and stably expressed gene web responsible for generating, maintaining and modulating the heart rhythm and their expression level, control and intercoordination determined in each chamber, for both sexes. Remarkably, although composed by the same genes (adrenergic receptors, ankyrins, ion channels and transporters, connexins, cadherins, plakophilins and other components of the intercalated discs), atrial and ventricular HRD fabrics presented profound topological differences between the sexes. Transcriptomic data were then used to deconvolute the networks by which the sex hormones influence the fabric organizational principles. The analysis revealed major sex differences in the ankyrin-based pathways targeting ion channels. Finally, individual genes, gene pairs and linear pathways that are pivotal for the sexual dichotomy were identified and their contribution to the HRD fabric quantified (partially illustrated in Figures below).

Type of Paper: Review
Title: PRDM Proteins Molecular Mechanism in Signal Transduction and Transcriptional Regulation
Authors: Erika di Zazzo and Bruno Moncharmont
Affiliation: Department of Medicine and Health sciences, Università degli studi del Molise – Via Francesco De Sanctis snc, 86100 Campobasso, Italy; E-Mail: moncharmont@unimol.it
Abstract: PRDM family proteins are characterized by the presence of the PR domain and a variable number of zinc finger repeats. Experimental evidence showed that the PRDM proteins play an important role in gene expression regulation, modifying the chromatin structure either directly, through the intrinsic methyltransferase activity or indirectly through the recruitment of chromatin remodeling complexes. PRDM proteins have a dual action: they mediate the effect induced by different cell signals, such as steroid hormones, and control the expression of growth factors. PRDM proteins have therefore a pivotal role in the transduction of signals controlling cell proliferation or differentiation and, consequently, neoplastic transformation. In the present review we describe the pathways in which PRDM proteins are involved and the molecular mechanisms of transcriptional regulation.

Type of Paper: Article
Title: MiRNA Scavenging: a Versatile Tool for Blocking microRNA Activity
Authors: Paride Pelucchi ¹, Valeria Tria ¹, Valentina Martino ¹, Davood Sabour ², Giovanni Bertalot ³, Mira Palizban ¹, Martin Götte ⁴, Ileana Zucchi ¹ and Rolland A. Reinbold ^1,2
Affiliations: ¹ Institute of Biomedical Technologies, National Research Council, 20090 Segrate-Milan, Italy
² Max Planck Institute for Molecular Biomedicine, Cell and Developmental Biology, Muenster, Germany 48149
³ Servizio di Anatomia Patologia, Presidio Ospedaliero di Manerbio, Azienda Ospedaliera di Desenzano d/G, 25025 Manerbio, Italy
⁴ University of Münster Medical Center, Gynecology and Obstetrics, Münster, Germany 48149
E-Mails: rreinbold@mpi-muenster.mpg.de (R.A.R); ileana.zucchi@itb.cnr.it (I.Z.)
Abstract: MicroRNAs (miRNAs) are a class of small RNAs (18-22nt) that post transcriptionally regulate gene expression by binding to complementary sequences on target mRNAs, resulting in translational repression or target degradation and gene silencing. As aberrant expression of miRNAs is implicated in important diseases including cancer, miRNA-based therapies are under intensive investigation. We optimized strategies to stably or conditionally generate miRNA inhibitors for continuous block of miRNA activity that allow for probing miRNA function in long-term cell culture experiments, cancer xenografts, 3D tissue models and for in vivo studies with transgenic organisms.

Type of Paper: Article
Title: Small RNA world: Isolation and Cloning of Small RNA for DNA Deep-Sequencing
Authors: Paride Pelucchi ¹, Giovanni Bertalot ², Valentina Martino ¹, Valeria Tria ¹, Davood Sabour ³, Mira Palizban ¹, Martin Götte ⁴, Rolland A. Reinbold ^1,3 and Ileana Zucchi ¹
Affiliations: ¹ Institute of Biomedical Technologies, National Research Council, 20090 Segrate-Milan, Italy
² Servizio di Anatomia Patologia, Presidio Ospedaliero di Manerbio, Azienda Ospedaliera di Desenzano d/G, 25025 Manerbio, Italy
³ Max Planck Institute for Molecular Biomedicine, Cell and Developmental Biology, Münster, Germany 48149
⁴ University of Münster Medical Center, Gynecology and Obstetrics, Münster, Germany 48149
E-Mails: rreinbold@mpi-muenster.mpg.de (R.A.R.); ileana.zucchi@itb.cnr.it
Abstract: In recent years, small non-coding RNAs (ncRNAs) have emerged as pivotal players in controlling cellular and developmental processes by their capacity to modulate protein expression (microRNA, small interfering RNA) or direct epigenetic chromatin modification (piwi-interacting RNA). Moreover, clinical evidence supports their involvement in several pathologies including cancer. As a large part of the small ncRNA world is still unknown, the identification of novel ncRNAs is paramount for understanding their potential as targets or agents in clinical applications. We developed protocols to generate comprehensive libraries of small ncRNAs for deep-sequencing starting from limiting number of cells, which allow for both novel and sensitive ncRNA identification and differential expression screenings.

Type of Paper: Review
Title: The Role of DNA Methylation in Common Skeletal Disorders
Authors: Jesús Delgado-Calle and José A. Riancho
Affiliation: Department of Internal Medicine. H.U. Marqués de Valdecilla-IFIMAV-University of Cantabria. Santander, Spain; E-Mail: jose.riancho@unican.es
Abstract: Bone is a complex connective tissue formed by an extracellular calcified matrix. This mineralized matrix iis constantly being formed and resorbed throughout life, allowing the bone to adapt to daily requirements and maintain skeletal properties and composition. The imbalance between bone formation and bone resorption leads to changes in the bone mass. This is the case of osteoporosis and osteoarthritis, two common skeletal disorders showing opposite changes in bone mineral density. While osteoporosis is characterized by a decreased bone mass, and, consequently, with higher susceptibly to bone fractures, bone mass tend to be higher in osteoarthritis patients. It is known that these diseases are influenced by heritable factors. However, the DNA polymorphisms identified so far in GWAS explain less than the 10% of the genetic risk, suggesting that other factors may be involved in the development of these diseases. This could be the case of epigenetic marks. This review summarizes current knowledge about the influence of epigenetic marks on bone homeostasis, paying special attention to the role of DNA methylation in the onset and development of osteoporosis and osteoarthritis.

Type of Paper: Review
Title: The Chemokine Receptor CCR5: from Genetic to Epigenetic Transcriptional Regulation.
Authors: Rutger J. Wierda ¹ and Peter J. van den Elsen ^1,2
Affiliations: ¹ Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, Albinusdreef 2, 2333 ZA Leiden, The Netherlands
² Department of Pathology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands; E-Mail: P.J.van_den_Elsen@lumc.nl
Abstract: The chemokine receptor CCR5 regulates trafficking of immune cells of the lymphoid or the myeloid lineage (such as monocytes, macrophages and immature dendritic cells) and microglia. Because of this, there is an increasing recognition of the important role of CCR5 in the pathology of (neuro-) inflammatory diseases, as atherosclerosis and multiple sclerosis. Expression of CCR5 is under the control of a complexly organized promoter region upstream of the gene. The transcription factor cAMP-responsive element binding protein 1 (CREB 1) transactivates the CCR5 P1 promoter. The cell-specific expression of CCR5 however is realized by using many ‘non-classical’ forms of epigenetic regulation. Here we discuss the transcriptional regulation of CCR5 with a focus on the epigenetic peculiarities of CCR5 transcription.

Type of Paper: Review
Title: Does Mitochondrial Activity Influence Gene Expression and Could the Distribution of Mitochondria in the Vertebrate Egg Mark Subsequent Boundaries between Left and Right?
Author: Ursula Mittwoch 
Affiliation: Department of Genetics, Evolution and Environment, Darwin Building, Gower Street, London, WC1 6BT, UK; E-Mail: u.mittwoch@ucl.ac.uk
Abstract: The basic bilateral asymmetry of vertebrates is consistently broken in the development of different organs, and in addition, a significant proportion of congenital malformations of paired structures are preferentially situated on either the left or right side. These facts make it unlikely that all discrepancies between genotype and phenotype can be explained exclusively in terms of intra-nuclear events. The likelihood that mitochondrial activity is also involved in gene expression is supported by recent findings on mitochondrial activity in mammalian oocytes and embryos. The maturation of the oocyte is accompanied by a redistribution of mitochondria, whose localization must be strictly controlled if the embryo is to be viable. This sequence of events opens the possibility of cell divisions giving rise to blastomeres with different numbers of mitochondria, and hence slight differences in phenotype, which might be regarded as the beginning of the regulation of gene expression in the embryo. One could further speculate whether the many mitochondria, carefully distributed in the cytoplasm of the egg, and accompanied by bursts of ATP production, might also hold the long-kept secret regarding the origin of the left/right asymmetry. Evidence for its early developmental origin is increasing.

Type of Paper: Article
Title: A Novel Approach for High-Throughput Screening of Mammary Gland Normal and Cancer Stem Cells
Authors: Cinzia Cocola ¹, Eleonora Piscitelli ¹, Davood Sabour ², Brian D. Gray ³, Martin Götte ⁴, Stefano Molgora ¹, Hans R. Schöler ², Mira Palizban ¹, Rolland A. Reinbold ^1,2,*, Gaia Cecilia Luvoni ⁵, Valeria Grieco ⁶, and Ileana Zucchi ^1,*
Affiliations: ¹ Institute of Biomedical Technologies, National Research Council, Segrate-Milan 20090, Italy; E-Mail: rreinbold@mpi-muenster.mpg.de
² Max Planck Institute for Molecular Biomedicine, Cell and Developmental Biology, Muenster 48149, Germany
³ Molecular Targeting Technologies, Inc. West Chester, PA 19380, USA
⁴ University of Münster Medical Center, Gynecology and Obstetrics, Münster 48149, Germany
⁵ Department of Veterinary Clinical Sciences, Obstetrics and Gynecology, Università degli Studi di Milano, Italy
⁶ Department of Veterinary Science and Public Health, General Anatomy and Pathology, Università degli Studi di Milano, Italy
Abstract: High-throughput screening of mammary gland cancer stem cells such as for drug testing and discovery has proven elusive due to the inability to culture these cells in adherent conditions. Current methods for culturing are based on three-dimensional non-adherent conditions that promote generation of spheres, conditions in which target cells are not physically accessible to chemical, genetic or pharmacological agents in long-term cultures. We have therefore developed a two-dimensional adherent culture approach that allows for long-term maintenance, direct visualization, screening, isolation and expression profiling of canine mammary gland normal and cancer stem cells.