Minna U. Kaikkonen

Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA

Mammalian genomes are populated with thousands of transcriptional enhancers that orchestrate cell-type-specific gene expression programs, but how those enhancers are exploited to institute alternative, signal-dependent transcriptional responses remains poorly understood. Here we present evidence that cell-lineage-specific factors, such as FoxA1, can simultaneously facilitate and restrict key regulated transcription factors, exemplified by the androgen receptor (AR), to act on structurally and functionally distinct classes of enhancer. Consequently, FoxA1 downregulation, an unfavourable prognostic sign in certain advanced prostate tumours, triggers dramatic reprogramming of the hormonal response by causing a massive switch in AR binding to a distinct cohort of pre-established enhancers. These enhancers are functional, as evidenced by the production of enhancer-templated non-coding RNA (eRNA) based on global nuclear run-on sequencing (GRO-seq) analysis, with a unique class apparently requiring no nucleosome remodelling to induce specific enhancer–promoter looping and gene activation. GRO-seq data also suggest that liganded AR induces both transcription initiation and elongation. Together, these findings reveal a large repository of active enhancers that can be dynamically tuned to elicit alternative gene expression programs, which may underlie many sequential gene expression events in development, cell differentiation and disease progression.

Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription

Rev-Erb-α and Rev-Erb-β are nuclear receptors that regulate the expression of genes involved in the control of circadian rhythm, metabolism and inflammatory responses. Rev-Erbs function as transcriptional repressors by recruiting nuclear receptor co-repressor (NCoR)–HDAC3 complexes to Rev-Erb response elements in enhancers and promoters of target genes, but the molecular basis for cell-specific programs of repression is not known. Here we present evidence that in mouse macrophages Rev-Erbs regulate target gene expression by inhibiting the functions of distal enhancers that are selected by macrophage-lineage-determining factors, thereby establishing a macrophage-specific program of repression. Remarkably, the repressive functions of Rev-Erbs are associated with their ability to inhibit the transcription of enhancer-derived RNAs (eRNAs). Furthermore, targeted degradation of eRNAs at two enhancers subject to negative regulation by Rev-Erbs resulted in reduced expression of nearby messenger RNAs, suggesting a direct role of these eRNAs in enhancer function. By precisely defining eRNA start sites using a modified form of global run-on sequencing that quantifies nascent 5′ ends, we show that transfer of full enhancer activity to a target promoter requires both the sequences mediating transcription-factor binding and the specific sequences encoding the eRNA transcript. These studies provide evidence for a direct role of eRNAs in contributing to enhancer functions and suggest that Rev-Erbs act to suppress gene expression at a distance by repressing eRNA transcription.

Non-coding RNAs as regulators of gene expression and epigenetics

Genome-wide studies have revealed that mammalian genomes are pervasively transcribed. This has led to the identification and isolation of novel classes of non-coding RNAs (ncRNAs) that influence gene expression by a variety of mechanisms. Here we review the characteristics and functions of regulatory ncRNAs in chromatin remodelling and at multiple levels of transcriptional and post-transcriptional regulation. We also describe the potential roles of ncRNAs in vascular biology and in mediating epigenetic modifications that might play roles in cardiovascular disease susceptibility. The emerging recognition of the diverse functions of ncRNAs in regulation of gene expression suggests that they may represent new targets for therapeutic intervention.

Effect of natural genetic variation on enhancer selection and function

The mechanisms by which genetic variation affects transcription regulation and phenotypes at the nucleotide level are incompletely understood. Here we use natural genetic variation as an in vivo mutagenesis screen to assess the genome-wide effects of sequence variation on lineage-determining and signal-specific transcription factor binding, epigenomics and transcriptional outcomes in primary macrophages from different mouse strains. We find substantial genetic evidence to support the concept that lineage-determining transcription factors define epigenetic and transcriptomic states by selecting enhancer-like regions in the genome in a collaborative fashion and facilitating binding of signal-dependent factors. This hierarchical model of transcription factor function suggests that limited sets of genomic data for lineage-determining transcription factors and informative histone modifications can be used for the prioritization of disease-associated regulatory variants.

Avidin-biotin technology in targeted therapy

Importance of the field: The goal of drug targeting is to increase the concentration of the drug in the vicinity of the cells responsible for disease without affecting healthy cells. Many approaches in cancer treatment are limited because of their broad range of unwanted side effects on healthy cells. Targeting can reduce side effects and increase efficacy of drugs in the patient. Areas covered in this review: Avidin, originally isolated from chicken eggs, and its bacterial analogue, streptavidin, from Streptomyces avidinii, have extremely high affinity for biotin. This unique feature is the basis of avidin-biotin technology. This article reviews the current status of avidin-biotin systems and their use for pretargeted drug delivery and vector targeting. What the reader will gain: The reader will gain an understanding of the following approaches using the avidin-biotin system: i) targeting antibodies and therapeutic molecules are administered separately leading to a reduction of drug dose in normal tissues compared with conventional (radio)immunotherapies; ii) introducing avidin gene into specific tissues by local gene transfer, which subsequently can sequester and concentrate considerable amounts of therapeutic ligands; and iii) enabling transductional targeting of gene therapy vectors. Take home message: Avidin and biotin technology has proved to be an extremely versatile tool with broad applications, such as pretargeting, delivering avidin gene into cells enabling targeting of biotinylated compounds and targeting of viral vectors.

Control of Proinflammatory Gene Programs by Regulated Trimethylation and Demethylation of Histone H4K20

Regulation of genes that initiate and amplify inflammatory programs of gene expression is achieved by signal-dependent exchange of coregulator complexes that function to read, write, and erase specific histone modifications linked to transcriptional activation or repression. Here, we provide evidence for the role of trimethylated histone H4 lysine 20 (H4K20me3) as a repression checkpoint that restricts expression of toll-like receptor 4 (TLR4) target genes in macrophages. H4K20me3 is deposited at the promoters of a subset of these genes by the SMYD5 histone methyltransferase through its association with NCoR corepressor complexes. Signal-dependent erasure of H4K20me3 is required for effective gene activation and is achieved by NF-κB-dependent delivery of the histone demethylase PHF2. Liver X receptors antagonize TLR4-dependent gene activation by maintaining NCoR/SMYD5-mediated repression. These findings reveal a histone H4K20 trimethylation/demethylation strategy that integrates positive and negative signaling inputs that control immunity and homeostasis.

Mechanisms establishing TLR4-responsive activation states of inflammatory response genes.

Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Comprehensive genome-wide analysis of the epigenetic and transcription status of the TLR4-induced transcriptional program in macrophages suggests that Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early- (I/E) and late-response genes results from a sequential process in which signal-independent factors initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by encoding a distinct set of signal-dependent transcription factor elements, including TATA boxes, which lead to preferential binding of TBP and basal enrichment for RNA polymerase II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases the overall rates of both transcriptional initiation and the efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. Collectively, these findings reveal broadly utilized mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses.

In Vitro Targeting of Avidin-Expressing Glioma Cells with Biotinylated Persistent Luminescence Nanoparticles

Far red emitting persistent luminescence nanoparticles (PLNP) were synthesized and functionalized with biotin to study their targeting ability toward biotin-binding proteins. First, the interaction of biotin-decorated PLNP with streptavidin, immobilized on a plate, was shown to be highly dependent on the presence of a PEG spacer between the surface of the nanoparticles and the biotin ligand. Second, interaction between biotin-PEG-PLNP and free neutravidin in solution was confirmed by fluorescence microscopy. Finally, in vitro binding study on BT4C cells expressing lodavin fusion protein, bearing the extracellular avidin moiety, showed that such biotin-covered PLNP could successfully be targeted to malignant glioma cells through a specific biotin-avidin interaction. The influence of nanoparticle core diameter, incubation time, and PLNP concentration on the efficiency of targeting is discussed.

Truncated vesicular stomatitis virus G protein improves baculovirus transduction efficiency in vitro and in vivo

Pseudotyping of viral vectors has been widely used to enhance viral transduction efficiency. One of the most popular pseudotyping proteins has been the G-protein of the vesicular stomatitis virus, VSV-G. In the present study, we show that the 21-amino-acid ectodomain with transmembrane and cytoplasmic tail domains of VSV-G (VSV-GED) augments baculovirus-mediated gene delivery in vertebrate cells by aiding viral entry. The VSV-GED pseudotyped virus replicated efficiently in insect cells yielding high titers. Five out of six studied cell lines showed improved transduction, as measured by a number of transduced cells or transgene expression level. Nearly 15-fold increase in the transduction efficiency was detected in rat malignant glioma cells as compared to the control virus. In the rat brain, transgene expression could be detected in the walls of lateral ventricles and in subarachnoid membranes. Increased transduction efficiency was also observed in the rabbit muscle. Our results suggest that VSV-GED enhances baculoviral gene transfer by augmenting gp64-mediated endosomal release. Moreover, no cytotoxicity was associated with improved gene transfer efficiency. Thus, VSV-GED pseudotyping provides a simple means to enhance baculovirus-mediated gene transfer in vitro and in vivo.

Non-invasive Imaging in Gene Therapy.

Several methods are available for non-invasive imaging of gene delivery and transgene expression, including magnetic resonance imaging (MRI), single photon emission tomography (SPECT)/positron emission tomography (PET), and fluorescence and bioluminescence imaging. However, these imaging modalities differ greatly in terms of their sensitivity, cost, and ability to measure the signal. Whereas MRI can produce a resolution of approximately 50 μm, optical imaging achieves only 3-5 mm but outperforms MRI in terms of the cost of the imaging device. Similarly, SPECT and PET give a resolution of only 1-2 mm but provide for relatively easy quantitation of the signal and need only nanograms of probe, compared with the microgram or milligram levels required for MRI and optical imaging. To develop safer and more efficient gene delivery vectors, it is essential to perform rigorous in vivo experiments, to image particle biodistribution and transduction patterns, and to quantify the transgene expression profile. Differences between modalities have a significant effect on the resultant imaging resolution for gene therapy. This review describes the methodologies in use and highlights recent key approaches using the latest imaging modalities in gene therapy. Future trends in gene therapy imaging are also discussed.