Multigenic Regulation Research Articles

Development of a temperature-responsive yeast cell factory using engineered Gal4 as a protein switch.

Conflict between cell growth and product accumulation is frequently encountered in biosynthesis of secondary metabolites. Herein, a temperature-dependent dynamic control strategy was developed by modifying the GAL regulation system to facilitate two-stage fermentation in yeast. A temperature-sensitive Gal4 mutant Gal4M9 was created by directed evolution, and used as a protein switch in ΔGAL80 yeast. After EGFP-reported validation of its temperature-responsive induction capability, the sensitivity and stringency of this system in multi-gene pathway regulation was tested, using lycopene as an example product. When Gal4M9 was used to control the expression of PGAL -driven pathway genes, growth and production was successfully decoupled upon temperature shift during fermentation, accumulating 44% higher biomass and 177% more lycopene than the control strain with wild-type Gal4. This is the first example of adopting temperature as an input signal for metabolic pathway regulation in yeast cell factories.

A p53 enhancer region regulates target genes through chromatin conformations in cis and in trans

We examined how a p53 enhancer transmits regulatory information in vivo. Using genetic ablation together with digital chromosome conformation capture and fluorescent in situ hybridization, we found that a Drosophila p53 enhancer region (referred to as the p53 response element [p53RE]) physically contacts targets in cis and across the centromere to control stress-responsive transcription at these sites. Furthermore, when placed at ectopic genomic positions, fragments spanning this element re-established chromatin contacts and partially restored target gene regulation to mutants lacking the native p53RE. Therefore, a defined p53 enhancer region is sufficient for long-range chromatin interactions that enable multigenic regulation.

The Size of the Plasmacytoid Dendritic Cell Compartment Is a Multigenic Trait Dominated by a Locus on Mouse Chromosome 7

Plasmacytoid dendritic cells (pDC) compose one of the many distinct dendritic cell subsets. The primary function of pDC is to potently produce type 1 IFNs upon stimulation, which is highly relevant in antiviral responses. Consequently, the ability to manipulate the size of the pDC compartment in vivo may increase the capacity to clear viral infections. In an attempt to identify genetic loci affecting the size of the pDC compartment, defined by both the proportion and absolute number of pDC, we undertook an unbiased genetic approach. Linkage analysis using inbred mouse strains identified a locus on chromosome 7 (Pdcc1) significantly linked to both the proportion and the absolute number of pDC in the spleen. Moreover, loci on either chromosome 11 (Pdcc2) or 9 (Pdcc3) modified the effect of Pdcc1 on chromosome 7 for the proportion and absolute number of pDC, respectively. Further analysis using mice congenic for chromosome 7 confirmed Pdcc1, demonstrating that variation within this genetic interval can regulate the size of the pDC compartment. Finally, mixed bone marrow chimera experiments showed that both the proportion and the absolute number of pDC are regulated by cell-intrinsic hematopoietic factors. Our findings highlight the multigenic regulation of the size of the pDC compartment and will facilitate the identification of genes linked to this trait.

How chromatin remodelling allows shuffling of immunoglobulin heavy chain genes

Cellular identity is determined by the switching on and off of lineage-specific genes. This dynamic process is regulated by a highly co-ordinated series of chromatin remodelling mechanisms that control DNA accessibility to facilitate transcription, replication and recombination. The identity of an individual B-lymphocyte is defined by the expression of a unique antibody protein, composed of two identical immunoglobulin heavy and two identical light chain polypeptides, which recognize a single foreign antigen with high specificity. However, the mammalian adaptive immune system requires an enormous variety of antibody-expressing B cells to combat the millions of foreign antigens it may encounter. This diversity is generated primarily at the multigene immunoglobulin loci by V(D)J recombination, a specialised form of DNA recombination in which numerous variable (V), diversity (D) and joining (J) genes are cut and pasted together in a strict order to allow shuffling of immunoglobulin genes. The mouse immunoglobulin heavy chain (Igh) locus is the largest known multigene locus. It spans approximately 3 Mb and comprises more than 200 genes. Its size and complexity pose an enormous logistic challenge to the chromatin remodelling machinery, but recent major advances in our understanding of how the 200 genes are shuffled have begun to reveal an exquisitely co-ordinated set of chromatin remodelling mechanisms which exploit every aspect of nuclear dynamics, and provide a global view of multigene regulation. This review will explore the numerous processes implicated in opening up and positioning of the locus to enable shuffling of the Igh locus genes, including non-coding RNA transcription, histone modifications, transcription factors, nuclear relocation and locus contraction.

Activity-dependent Neuroprotective Protein Constitutes a Novel Element in the SWI/SNF Chromatin Remodeling Complex

Complete deficiency in activity-dependent neuroprotective protein (ADNP), a heterochromatin 1-binding protein, results in dramatic changes in gene expression, neural tube closure defects, and death at gestation day 9 in mice. To further understand the cellular roles played by ADNP, the HEK293 human embryonic kidney cell line that allows efficient transfection with recombinant DNA was used as a model for the identification of ADNP-interacting proteins. Recombinant green fluorescent protein (GFP)-ADNP was localized to cell nuclei. When nuclear extracts were subjected to immunoprecipitation with specific GFP antibodies followed by polyacrylamide gel electrophoresis, several minor protein bands were observed in addition to GFP-ADNP. In-gel protein digests followed by mass spectrometry identified BRG1, BAF250a, and BAF170, all components of the SWI/SNF (mating type switching/sucrose nonfermenting) chromatin remodeling complex, as proteins that co-immunoprecipitate with ADNP. These results were verified utilizing BRG1 antibodies. ADNP short hairpin RNA down-regulation resulted in microtubule reorganization and changes in cell morphology including reduction in cell process formation and cell number. These morphological changes are closely associated with the SWI/SNF complex multifunctionality. Taken together, the current study uncovers a molecular basis for the essential function of the ADNP gene and protein.

Engineered Tet repressors with recognition specificity for the tetO-4C5G operator variant

We created a new DNA recognition specificity for tetracycline repressor (TetR) binding to the tet operator variant tetO-4C5G containing four bp exchanges compared to tetO. TetR variants created by doped oligonucleotide mutagenesis of residues in the DNA recognition helix yielded several mutants binding to tetO-4C5G. These variants contained exchanges of the amino acids at positions 36, 37, 39 and 42. The two amino acid exchanges in TetR E37A P39K are sufficient for tetO-4C5G specific binding. The E37A mutation increases the affinity of TetR for tetO variants and seems to be essential for binding to modified operator sequences. The Lys39 residue is in a position to directly contact the fourth and fifth bps of tetO thereby creating specificity for tetO-4C5G. Combinations of these mutations with others that lead to a reverse phenotype or altered inducer specificity yielded new TetR mutants with the respective combined activities. Single chain TetR variants were constructed that contain DNA reading heads with two different operator binding specificities. Specific binding of this TetR mutant to the respective mixed tetO-wt/4C5G variants containing one wild type and one double exchange operator half site was only accomplished at a low expression level of TetR variant, while cross-talk with other operator variants were observed at an elevated expression level. This observation emphasizes the importance of the transcription factor expression level for in vivo DNA binding specificity. These new TetR variants can be useful for multigene regulation systems.

Inheritance of Day-neutrality in Octoploid Species of Fragaria

The inheritance of day-neutrality in octoploid Fragaria L. was investigated in crosses between day-neutral (DN) × short day (SD) and DN × DN types using F. ×ananassa Duchesne in Lamarck cultivars and elite selections of F. virginiana Miller and F. chiloensis (L.) Miller. Genotypes were considered as DN if they flowered under both the SDs of spring before 30 May (<14 hours) and the long days of summer after 24 July (>15 hours). Wide ranges in the percentage of DN progeny were found among the families regardless of species background (30% to 87% in DN × SD and 22% to 93% in DN × DN crosses). None of the families fit the segregation ratios expected if DN was regulated by recessive alleles at one locus, and only about half of the families fit the segregation ratios expected if a single dominant allele regulated DN. Several two-gene models fit the segregation data better than the single locus ones, but none of the genetic models tested fit the DN segregation ratios at the ends of the distribution range. The wide range observed in the percentage of DN progeny across all the families is most consistent with a polygenic model. Several other kinds of observations supported the multigenic regulation of DN: 1) Different DN parents crossed to the same SD genotype often produced different percentages of DN progeny, 2) Some of the day-neutrality sources were more powerful than others in producing of DNs, and 3) None of the DN parents produced 100% DN progeny, which would be expected if there were homozygous dominant DN individuals. Specific combining abilities for DN and flowering strength were significant, while general combining abilities for these traits were not. Our results suggest that parental combinations can be selected that will generate very high proportions of DN progeny that bloom for long periods of time.

Functional consequences of IGFBP excess?lessons from transgenic mice

The functions of insulin-like growth factor-binding proteins (IGFBPs) have been studied extensively in vitro, revealing IGF-dependent and also IGF-independent effects on cell growth, differentiation, and survival. In contrast, the biological relevance of IGFBPs in vivo is only partially understood. In the past decade, mouse models lacking or overexpressing specific IGFBPs have been generated by transgenic technology. Phenotypic analysis revealed features that are common for most IGFBPs (growth inhibition), but also effects that appear to be specific for some but not all IGFBPs, such as disturbed glucose homeostasis (IGFBP-1 and -3) or impaired fertility (IGFBP-1, -5, and -6). Future systematic comparison of IGFBP functions in transgenic mice will be facilitated by targeted insertion of IGFBP expression vectors and by standardized phenotype assessment. Furthermore, analysis of IGFBP expression in growth-selected mouse lines or pedigrees segregating for growth phenotypes will be important to understand the roles of IGFBPs in multigenic growth regulation.

Study on high-rate and low-rate metastatic large-cell lung cancer cell lines via differential display reverse transcript PCR

To investigate the differentially expressed gene profile between high-rate and low-rate metastatic large-cell lung cancer cell lines via differential display reverse transcript PCR (DDRT-PCR). DDRT-PCR was performed in two large-cell lung cancer cell lines with different metastatic ability. The differentially displayed cDNA were cloned and sequenced and compared with the sequences in the GenBank. Fifty differentially displayed cDNAs were acquired, including 18 function-known genes, 16 function-unknown genes, 16 genomic DNA fragments and 4 ESTs. The cell lines have different gene expression profiles, and metastasis is affected by multi-gene expression and regulation. Some genes which contribute to the metastasis of lung cancer could be found by DDRT-PCR, and this study gives some new clues to cancer diagnosis and therapy.

Neural Model of the Genetic Network

Many cell control processes consist of networks of interacting elements that affect the state of each other over time. Such an arrangement resembles the principles of artificial neural networks, in which the state of a particular node depends on the combination of the states of other neurons. The lambda bacteriophage lysis/lysogeny decision circuit can be represented by such a network. It is used here as a model for testing the validity of a neural approach to the analysis of genetic networks. The model considers multigenic regulation including positive and negative feedback. It is used to simulate the dynamics of the lambda phage regulatory system; the results are compared with experimental observation. The comparison proves that the neural network model describes behavior of the system in full agreement with experiments; moreover, it predicts its function in experimentally inaccessible situations and explains the experimental observations. The application of the principles of neural networks to the cell control system leads to conclusions about the stability and redundancy of genetic networks and the cell functionality. Reverse engineering of the biochemical pathways from proteomics and DNA micro array data using the suggested neural network model is discussed.

Neural network model of gene expression.

Many natural processes consist of networks of interacting elements that, over time, affect each other's state. Their dynamics depend on the pattern of connections and the updating rules for each element. Genomic regulatory networks are networks of this sort. In this paper we use artificial neural networks as a model of the dynamics of gene expression. The significance of the regulatory effect of one gene product on the expression of other genes of the system is defined by a weight matrix. The model considers multigenic regulation including positive and/or negative feedback. The process of gene expression is described by a single network and by two linked networks where transcription and translation are modeled independently. Each of these processes is described by different network controlled by different weight matrices. Methods for computing the parameters of the model from experimental data are discussed. Results computed by means of the model are compared with experimental observations. Generalization to a 'black box' concept, where the molecular processes occurring in the cell are considered as signal processing units forming a global regulatory network, is discussed.-Vohradský, J. Neural network model of gene expression.

Regulation of adjacent yeast genes

In the genomes of prokaryotes many cases are known where a single regulatory system controls two or more functionally related genes1. Although important differences exist between the regulatory systems of prokaryotes and eukaryotes, it has been suggested that multi-gene regulation also exists in eukaryotic genomes. We explore this notion through the analysis of gene-expression data because observed changes in gene-expression levels quantify the effect of the regulatory mechanisms. The yeast Saccharomyces cerevisiae is our model organism because the whole genome has been sequenced, all open reading frames have been identified, and much expression data is available.

Modeling the complexity of genetic networks: Understanding multigenic and pleiotropic regulation

AbstractMolecular genetics presents an increasingly complex picture of the genome and biological function. Evidence is mounting for distributed function, redundancy, and combinatorial coding in the regulation of genes. Satisfactory explanation will require the concept of a parallel processing signaling network. Here we provide an introduction to Boolean networks and their relevance to present‐day experimental research. Boolean network models exhibit global complex behavior, self‐organization, stability, redundancy and periodicity, properties that deeply characterize biological systems. While the life sciences must inevitably face the issue of complexity, we may well look to cybernetics for a modeling language such as Boolean networks which can manageably describe parallel processing biological systems and provide a framework for the growing accumulation of data. We finally discuss experimental strategies and database systems that will enable mapping of genetic networks. The synthesis of these approaches holds an immense potential for new discoveries on the intimate nature of genetic networks, bringing us closer to an understanding of complex molecular physiological processes like brain development, and intractable medical problems of immediate importance, such as neurodegenerative disorders, cancer, and a variety of genetic diseases.

Genes responsible for quantitative regulation of antibody production.

Evidence is first presented demonstrating that the individual capacity for antibody responsiveness has an inheritable component. The main evidence consists of spontaneous mutations causing either immunodeficiency or predisposition to autoimmunity in human and animals, and the involvement of some obvious candidate genes (in particular those belonging to the highly polymorphic histocompatibility and Igh loci), extensively analyzed in many mouse strain combinations. One finding constantly emerging from these studies is that single gene expression depends on environmental effects, lowering the genotype/phenotype correlation, but also on multigenic interactions appearing as background effects. The models available for the analysis of this multigenic regulation are discussed with special emphasis on the high and low antibody responder mice produced for this purpose by bidirectional selective breeding. The major advantage of this model is that the interline difference is huge and multispecifically expressed. The second part of the review presents the recent results on positional mapping of genes with immunomodulatory effects in this model and in one appropriate recombinant congenic strain series. This in vivo genetic dissection of antibody responsiveness discriminated the involvement of candidate genes and suggested that unsuspected genes might be identified by means of this wide open search.

Multigenic regulation of the primary immune response to ovalbumin in mice

At least four genes regulate the primary immune response to ovalbumin in mice. The ability to be sensitized to transfer delayed type hypersensitivity to ovalbumin is controlled by two genes. One gene,OVA-β, is linked to theH-2 complex and maps to the left ofI-E. The linkage of the other gene,OVA-Bg1, has not been determined, but it segregates independently of theLy M locus, of the heavy chain allotype genes and of certain genes controlling coat color. At least two genes regulate the ability to respond with a primary antiovalbumin antibody response. One gene,OVA-α, is linked to theH-2 complex and maps to the right ofI-E. Discordance of the minimum dose of antigen needed to elicit delayed type hypersensitivity response and antibody suggests that non-H-2 gene(s) regulating the primary antibody response are different fromOVA-Bg1.