Control Of Gene Activity Research Articles

The Mu story: how a maverick phage moved the field forward

This article traces the pioneering contributions of phage Mu to our current knowledge of how movable elements move/transpose. Mu provided the first molecular evidence of insertion elements in E. coli, postulated by McClintock to control gene activity in maize in the pre-DNA era. An early Mu-based model successfully explained all the DNA rearrangements associated with transposition, providing a blueprint for navigating the deluge of accumulating reports on transposable element activity. Amplification of the Mu genome via transposition meant that its transposition frequencies were orders of magnitude greater than any rival, so it was only natural that the first in vitro system for transposition was established for Mu. These experiments unraveled the chemistry of the phosphoryl transfer reaction of transposition, and shed light on the nucleoprotein complexes within which they occur. They hastened a similar analysis of other transposons and ushered in the structural era where many transpososomes were crystallized. While it was a lucky break that the mechanism of HIV DNA integration turned out to be similar to that of Mu, it is no accident that current drugs for HIV integrase inhibitors owe their discovery to trailblazing experiments done with Mu. Shining the light on how movable elements restructure genomes, Mu has also given of itself generously to understanding the genome.

Production of human papillomavirus type 33 L1 major capsid protein and virus-like particles from Bacillus subtilis to develop a prophylactic vaccine against cervical cancer

We developed a bacterial expression system to produce human papillomavirus (HPV) type 33 L1 major capsid protein and virus-like particles from a recombinant Bacillus subtilis strain. For the first time, we have isolated self-assembled virus-like particles (VLPs) of HPV type 33 from B. subtilis, a strain generally recognized as safe (GRAS). The gene encoding the major capsid protein L1 of HPV type 33 was amplified from viral DNA isolated from a Korean patient and expressed in B. subtilis; a xylose-induction system was used to control gene activity. HPV33 L1 protein was partially purified by 40% (w/v) sucrose cushion centrifugation and strong cation exchange column chromatography. Eluted samples exhibited immunosignaling in fractions of 0.5–1.0M NaCl. The HPV33 L1 protein was shown to be approximately 56kDa in size by SDS-PAGE and Western blotting; recovery and purity were quantified by indirect immuno-ELISA assay. The final yield and purity were approximately 20.4% and 10.3%, respectively. Transmission electron microscopic analysis of fractions immunoactive by ELISA revealed that the L1 protein formed self-assembled VLPs with a diameter of approximately 20–40nm. Humoral and cellular immune responses provoked by the B. subtilis/HPV33 L1 strain were approximately 100- and 3-fold higher than those of the empty B. subtilis strain as a negative control, respectively. Development of a VLP production and delivery system using B. subtilis will be helpful, in that the vaccine may be convenient production as an antigen delivery system. VLPs thus produced will be safer for human use than those purified from Gram-negative strains such as Escherichia coli. Also, use of B. subtilis as a host may aid in the development of either live or whole cell vaccines administered by antigen delivery system.

MicroRNA Sequence Variation Potentially Contributes to Within-Species Functional Divergence in the NematodeCaenorhabditis briggsae

Mounting evidence points to differences in gene regulation as a major source of phenotypic variation. MicroRNA-mediated post-transcriptional regulation has emerged recently as a key factor controlling gene activity during development. MicroRNA genes are abundant in genomes, acting as managers of gene expression by directing translational repression. Thus, understanding the role of microRNA sequence variation within populations is essential for fully dissecting the origin and maintenance of phenotypic diversity in nature. In this study, we investigate allelic variation at microRNA loci in the nematode Caenorhabditis briggsae, a close relative of C. elegans. Phylogeographic structure in C. briggsae partitions most strains from around the globe into a "temperate" or a "tropical" clade, with a few strains having divergent, geographically restricted genotypes. Remarkably, strains that follow this latitudinal dichotomy also differ in temperature-associated fitness. With this phylogeographic pattern in mind, we examined polymorphisms in 18 miRNAs in a global sample of C. briggsae isolates and tested whether newly isolated strains conform to this phylogeography. Surprisingly, nucleotide diversity is relatively high in this class of gene that generally experiences strong purifying selection. In particular, we find that miRNAs in C. briggsae are substantially more polymorphic than in Arabidopsis thaliana, despite similar background levels of neutral site diversity between the two species. We find that some mutations suggest functional divergence on the basis of requirements for target site recognition and computational prediction of the effects of the polymorphisms on RNA folding. These findings demonstrate the potential for miRNA polymorphisms to contribute to phenotypic variation within a species. Sequences were deposited in GenBank under accession nos. JN251323-JN251744.

ATP-Dependent Chromatin Remodeling Factors and Their Roles in Affecting Nucleosome Fiber Composition

ATP-dependent chromatin remodeling factors of the SNF2 family are key components of the cellular machineries that shape and regulate chromatin structure and function. Members of this group of proteins have broad and heterogeneous functions ranging from controlling gene activity, facilitating DNA damage repair, promoting homologous recombination to maintaining genomic stability. Several chromatin remodeling factors are critical components of nucleosome assembly processes, and recent reports have identified specific functions of distinct chromatin remodeling factors in the assembly of variant histones into chromatin. In this review we will discuss the specific roles of ATP-dependent chromatin remodeling factors in determining nucleosome composition and, thus, chromatin fiber properties.

Effect of native and NH3 plasma-functionalized polymeric membranes on the gene expression profiles of primary hepatocytes

Little is known about how cells respond to different biomaterials at the molecular level. Biomaterials could stimulate specific cellular responses at the molecular level, such as activation of signalling pathways that control gene activity involved in the maintenance, growth and functional regeneration of liver tissue in vitro. This aspect is an important step in liver tissue engineering. Currently, there are no data available concerning the modulation of cellular genomic response by using synthetic membranes in a bioartificial system. For the first time we investigated gene expression profiles of primary hepatocytes cultured on different substrates: collagen sandwich, native and NH(3) plasma-grafted PEEK-WC-PU membranes. Gene expression in cell suspension prepared after cell isolation was used as a control. Generally, microarray data revealed that the expression of the majority of genes remained unchanged compared to the control. Among 31 000 genes, 52 were significantly changed: 20 were upregulated and 32 downregulated. There were similar changes in gene expression of hepatocytes cultured in the membranes and collagen sandwich. However, some genes involved in the cell proliferation and functional metabolic pathways are more expressed in cells cultured on the membranes and especially on the functionalized ones. Both membranes sustained liver functions at the molecular level, demonstrating their suitability for the reconstruction of liver and as a toxicogenomic tool to predict the liver response to novel drugs.

Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity

The human body contains thousands of unique cell types, each with specialized functions. Cell identity is governed in large part by gene transcription programs, which are determined by regulatory elements encoded in DNA. To identify regulatory elements active in seven cell lines representative of diverse human cell types, we used DNase-seq and FAIRE-seq (Formaldehyde Assisted Isolation of Regulatory Elements) to map "open chromatin." Over 870,000 DNaseI or FAIRE sites, which correspond tightly to nucleosome-depleted regions, were identified across the seven cell lines, covering nearly 9% of the genome. The combination of DNaseI and FAIRE is more effective than either assay alone in identifying likely regulatory elements, as judged by coincidence with transcription factor binding locations determined in the same cells. Open chromatin common to all seven cell types tended to be at or near transcription start sites and to be coincident with CTCF binding sites, while open chromatin sites found in only one cell type were typically located away from transcription start sites and contained DNA motifs recognized by regulators of cell-type identity. We show that open chromatin regions bound by CTCF are potent insulators. We identified clusters of open regulatory elements (COREs) that were physically near each other and whose appearance was coordinated among one or more cell types. Gene expression and RNA Pol II binding data support the hypothesis that COREs control gene activity required for the maintenance of cell-type identity. This publicly available atlas of regulatory elements may prove valuable in identifying noncoding DNA sequence variants that are causally linked to human disease.

Combining ability, heterosis and genetic diversity in tropical maize (Zea mays L.) under stress and non-stress conditions

Drought and low soil fertility are considered the most important abiotic stresses limiting maize production in sub-Saharan Africa. Knowledge of the combining ability and diversity of inbred lines with tolerance to the two stresses and for those used as testers would be beneficial in setting breeding strategies for stress and nonstress environments. We used 15 tropical maize inbred lines to (i) evaluate the combining ability for grain yield (GY), (ii) assess the genetic diversity of this set of inbred lines using RFLP, SSR, and AFLP markers, (iii) estimate heterosis and assess the relationship between F1 hybrid performance, genetic diversity and heterosis, and (iv) assess genotype × environment interaction of inbred lines and their hybrids. The F1 diallel hybrids and parental inbreds were evaluated under drought stress, low N stress, and well-watered conditions at six locations in three countries. General combining ability (GCA) effects were highly significant (P < 0.01) for GY across stresses and well-watered environments. Inbred lines CML258, CML339, CML341, and CML343 had the best GCA effects for GY across environments. Additive genetic effects were more important for GY under drought stress and well-watered conditions but not under low N stress, suggesting different gene action in control of GY. Clustering based on genetic distance (GD) calculated using combined marker data grouped lines according to pedigree. Positive correlation was found between midparent heterosis (MPH) and specific combining ability (SCA), GD and GY. Hybrid breeding program targeting stress environments would benefit from the accumulation of favorable alleles for drought tolerance in both parental lines.

DNA compaction: fundamentals and applications

Compaction is the process in which a large DNA molecule undergoes a transition between an elongated conformation and a very compact form. In nature, DNA compaction occurs to package genomic material inside tiny spaces such as viral capsids and cell nuclei. In vitro, several strategies exist to compact DNA. In this review, we first provide a physico-chemical description of this phenomenon, focusing on the modes of compaction, the types of compaction agents and the chemical and physical parameters that control compaction and its reverse process, decompaction. We then describe three main kinds of applications. First, we show how regulated compaction/decompaction can be used to control gene activity in vitro, with a particular emphasis on the use of light to reversibly control gene expression. Second, we describe several approaches where compaction is used as a way to reversibly protect DNA against chemical, biochemical, or mechanical stresses. Third, we show that compact DNA can be used as a nanostructure template to generate nanomaterials with a well-defined size and shape. We conclude by proposing some perspectives for future biochemical and biotechnological applications and enumerate some remaining challenges that we think worth being undertaken.

Behavior of genetic (co)variance components in populations simulated from non-additive genetic models of dominance and overdominance

The aim of this work was to investigate the short-term behavior of the genetic variability of quantitative traits simulated from models with additive and non-additive gene action in control and phenotypic selection populations. Both traits, one with low (h² = 0.10) and the other with high (h² = 0.60) heritability, were controlled by 600 biallelic loci. From a standard genome, it was obtained six genetic models which included the following: only the additive gene effects; complete and positive dominance for 25, 50, 75 and 100% of the loci; and positive overdominance for 50% of the loci. In the models with dominance deviation, the additive allelic effects were also included for 100% of the loci. Genetic variability was quantified from generation to generation using the genetic variance components. In the absence of selection, genotypic and additive genetic variances were higher. In the models with non-additive gene action, a small magnitude covariance component raised between the additive and dominance genetic effects whose correlation tended to be positive on the control population and negative under selection. Dominance variance increased as the number of loci with dominance deviation or the value of the deviation increased, implying on the increase in genotypic and additive genetic variances among the successive models.

Deciphering the cancer imprintome

Genetic events alone cannot explain the entire process of carcinogenesis. It is estimated that there are more epigenetic alterations in cancer than DNA mutations, and disiphering driver and secondary events is essential to understand early processes of tumorigenesis. Epigenetic modifications control gene activity, governing whether a gene is transcribed or silent. In cancer, global patterns of two epigenetic marks, histone modifications and DNA methylation, are known to be extensively deregulated. Tumour cells are also characterized by loss-of-imprinting, a key epigenetic developmental mechanism. Genomic imprinting is the parent-of-origin, monoallelic expression of genes and is controlled by differentially DNA-methylated regions and allelic-histone modifications. With specific emphasis on imprinted loci this review will discuss alterations in DNA methylation and histone modifications in cancer. The recent advances in technology that might facilitate the identification and characterization of the epigenetic profiles of cancer will also be described.

The Expanding Mi-2/NuRD Complexes: A Schematic Glance

This mini-review will schematically update the progress of the expanding Mi-2/Nucleosome Remodeling Deacetylase (NuRD) complexes in cancer and in normal development such as stemness, with a focus on mammals and the increasingly popular and powerful model organism Caenorhabditis elegans. The Mi-2/NuRD complexes control gene activity during the development of complex organisms. Every Mi-2/NuRD complex contains many different core polypeptides, which form distinct multifunctional complexes with specific context-dependent regulators. The Mi-2/NuRD complexes have unique ATP-dependent chromatin remodeling, histone deacetylase, demethylase activities and higher order chromatin organization. They can regulate the accessibility of transcription factors or repair proteins to DNA. In this review, we summarize our current knowleges in the composition, interaction and function of the subunits within the Mi-2/NuRD complex, the methodology used for the identification of Mi-2/NuRD complexes, as well as the clinical and therapeutic implications targeting the Mi-2/NuRD subunits.

Active promoters and insulators are marked by the centrosomal protein 190

For the compact Drosophila genome, several factors mediating insulator function, such as su(Hw) and dCTCF, have been identified. Recent analyses showed that both these insulator-binding factors are functionally dependent on the same cofactor, CP190. Here we analysed genome-wide binding of CP190 and dCTCF. CP190 binding was detected at CTCF, su(Hw) and GAF sites and unexpectedly at the transcriptional start sites of actively transcribed genes. Both insulator and transcription start site CP190-binding elements are strictly marked by a depletion of histone H3 and, therefore, a loss of nucleosome occupancy. In addition, CP190/dCTCF double occupancy was seen at the borders of many H3K27me3 'islands'. As before, these sites were also depleted of H3. Loss of either dCTCF or CP190 causes an increase of H3 and H3K27 trimethylation at these sites. Thus, for both types of cis-regulatory elements, domain borders and promoters, the chromatin structure is dependent on CP190.

DNA demethylation: a lesson from the garden

Gene silencing by DNA methylation is well documented and known to be essential for various biological phenomena in many organisms. In contrast, the processes that convert the silent state of a gene whose DNA is methylated and predicted to form facultative heterochromatin to the actively transcribed state remain elusive. In Arabidopsis, recent studies have shown that the DNA glycosylases DEMETER (DME) and REPRESSOR OF SILENCING1 (ROS1) participate in DNA demethylation. DME is necessary for genomic imprinting in the endosperm, while ROS1 is involved in pruning DNA methylation patterns in transposons and genic regions of vegetative tissues. These findings provide us with molecular clues for understanding the underlying mechanisms of DNA demethylation and gene activation. In this review, we will consider and discuss the processes of controlling gene activation through DNA demethylation, which are predicted to include the recognition of target sequences, DNA demethylation, the transformation of the chromatin to the active state, and transcription. Many of these processes remain poorly understood at this stage.

A “FLP-Out” System for Controlled Gene Expression in Caenorhabditis elegans

We present a two-part system for conditional FLP-out of FRT-flanked sequences in Caenorhabditis elegans to control gene activity in a spatially and/or temporally regulated manner. Using reporters, we assess the system for efficacy and demonstrate its use as a cell lineage marking tool. In addition, we construct and test a dominant-negative form of hlh-12, a gene that encodes a basic helix-loop-helix (bHLH) transcription factor required for proper distal tip cell (DTC) migration. We show that this allele can be conditionally expressed from a heat-inducible FLP recombinase and can interfere with DTC migration. Using the same DTC assay, we conditionally express an hlh-12 RNAi-hairpin and induce the DTC migration defect. Finally, we introduce a set of traditional and Gateway-compatible vectors to facilitate construction of plasmids for this technology using any promoter, reporter, and gene/hairpin of interest.

The relationship of 5HTT (SLC6A4) methylation and genotype on mRNA expression and liability to major depression and alcohol dependence in subjects from the Iowa Adoption Studies

Serotonin Transporter (5HTT or SLC6A4) mRNA transcription is regulated by both genetic and epigenetic mechanisms. Unfortunately, despite intense scrutiny, the exact identity and contribution of each of these regulatory mechanisms, and their relationship to behavioral illness remain unknown. This lack of knowledge is critical because alterations in SLC6A4 function are posited to be central to a wide variety of CNS disorders. In order to address this shortcoming, we quantified 5HTTLPR genotype, SLC6A4 mRNA production and CpG methylation using biomaterial from 192 lymphoblast cell lines derived from subjects who participated in the latest wave of the Iowa Adoption Studies. We then analyzed the resulting data with respect to clinical characteristics. We confirmed prior findings that the short (s) 5HTTLPR allele is associated with lower amounts of mRNA transcription, but there was no significant effect of the "Long G" allele on mRNA transcription. We also found that CpG methylation was higher (P < 0.0008) and mRNA production (P < 0.0001) was lower in females as compared to males. Those subjects with a lifetime history of Alcohol Dependence had higher levels of SLC6A4 mRNA. There was a trend for an association of increased overall methylation with lifetime history of major depression. Finally, we confirm our prior findings that the exact levels of 5HTT mRNA expression are dependent on how it is measured. We conclude that both genetic variation and epigenetic modifications contribute to the regulation of SLC6A4 function and that more in-depth studies of the molecular mechanisms controlling gene activity and the relationship of these mechanisms to behavioral illness are indicated.

Developmentally Regulated Activation of a SINE B2 Repeat as a Domain Boundary in Organogenesis

The temporal and spatial regulation of gene expression in mammalian development is linked to the establishment of functional chromatin domains. Here, we report that tissue-specific transcription of a retrotransposon repeat in the murine growth hormone locus is required for gene activation. This repeat serves as a boundary to block the influence of repressive chromatin modifications. The repeat element is able to generate short, overlapping Pol II-and Pol III-driven transcripts, both of which are necessary and sufficient to enable a restructuring of the regulated locus into nuclear compartments. These data suggest that transcription of interspersed repetitive sequences may represent a developmental strategy for the establishment of functionally distinct domains within the mammalian genome to control gene activation.

Idefix insulator activity can be modulated by nearby regulatory elements

Insulators play important roles in controlling gene activity and maintaining regulatory independence between neighbouring genes. In this article, we show that the enhancer-blocking activity of the insulator present within the LTR retrotransposon Idefix can be abolished if two copies of the region containing the insulator—specifically, the long terminal repeat (LTR)—are fused to the retrotransposon's 5′ untranslated region (5′ UTR). The presence of this combination of two [LTR-5′ UTR] modules is a prerequisite for the loss of enhancer-blocking activity. We further show that the 5′ UTR causes flanking genomic sequences to be displaced to the nuclear periphery, which is not observed when two insulators are present by themselves. This study thus provides a functional link between insulators and independent genomic modules, which may cooperate to allow the specific regulation of defined genomic loci via nuclear repositioning. It further illustrates the complexity of genomic regulation within a chromatic environment with multiple functional elements.

Regulating gene expression with light-activated oligonucleotides

Since the development of light-responsive amino acids, the activity of numerous biomolecules has been photomodulated in biochemical, biophysical, and cellular assays. Biological problems of even greater complexity motivate the development of quantitative methods for controlling gene activity with high spatial and temporal resolution, using light as an external trigger. Photoresponsive DNA and RNA oligonucleotides would optimally serve this purpose, but have proven difficult to expand from proofs-of-concept to in vivo experiments. Until recently, the development of this technology was limited by the synthesis of oligonucleotides whose function could be significantly modulated with near-UV light. New synthetic protocols and strategies for both up- and down-regulating gene activity finally make it possible to address biological considerations. In the near future, we can expect photoresponsive DNA and RNA molecules that are relatively non-toxic, nuclease-resistant, and maintain their specificity and activity in vivo. Quantitative, laser-initiated methods for controlling DNA and RNA function will illuminate new areas in cell and developmental biology.

The evolution of genomes and language

The evolution of genomes and language

Pushing the Envelope

Cell biologists once dismissed lamins as the backside of the nuclear membrane, but within the last 10 years, the proteins have hit the big time. Scientists have identified many new functions for lamins: The proteins help repair DNA, control gene activity, and spur precursor fat cells to grow up. Faulty lamins cause at least 12 diseases, and researchers have been discovering new mutations every year. Although lamin-related diseases remain untreatable, recent findings suggest that certain cancer drugs might alleviate symptoms in one type of illness. New work suggests that lamin defects might also underlie aging.