Expression from cell type-specific enhancer-modified retroviral vectors after transduction: influence of marker gene stability

The enhanced green fluorescent protein (EGFP) is increasingly used as a reporter gene in viral vectors for a number of applications. To establish a system to study the activity of cis-acting cellular regulatory sequences, we deleted the viral enhancer in EGFP-carrying retroviral vectors and replaced it with cell type-specific elements. In this study, we use this system to demonstrate the activity of the human CD2 lymphoid-specific and the Tie2 endothelial cell type-specific enhancers in cell lines and in primary cells transduced by retroviral vectors. Furthermore, we compare findings obtained with EGFP as the reporter gene to those obtained replacing EGFP with d2EGFP, an unstable variant of EGFP characterized by a much shorter half-life compared to EGFP, and by reduced accumulation in the cells. d2EGFP-carrying vectors were generated at titers which were not different from those generated by the corresponding vectors carrying EGFP. Moreover, the activity of a Moloney murine leukemia virus enhancer could be readily detected following transduction of target cells with either EGFP- or d2EGFP-carrying vectors. However, the activity of the relatively weak CD2 and Tie2 enhancers was exclusively detected using EGFP as the reporter gene.These findings indicate that enhancer replacement is a feasible and promising approach to address the function of cell type-specific regulatory elements in retroviral vectors carrying the EGFP gene.     

A new technique for selective identification and mapping of enhancers within long genomic sequences

We report a new experimental method of direct selection, identification, and mapping of potential enhancer sequences within extended stretches of genomic DNA. The method allows simultaneous cloning of a quantity of sequences instead of tedious screening of the separate ones, thus providing a robust and high-throughput approach to the mapping of enhancers. The selection procedure is based on the ability of such sequences to activate a minimal promoter that drives expression of a selective gene. To this end a mixture of short DNA fragments derived from the segment of interest was cloned in a retroviral vector containing the neomycin phosphotransferase II gene under control of a cytomegalovirus (CMV) minimal promoter. The pool of retroviruses obtained was used to infect HeLa cells and then to select neomycin-resistant colonies containing constructs with enhancer-like sequences. The pool of the genomic fragments was rescued by PCR and cloned, forming a library of the potential enhancers. Fifteen enhancer-like fragments were selected from 1-Mb human genome locus, and enhancer activity of 13 of them was verified in a transient transfection reporter gene assay. The sequences selected were found to be predominantly located near 5' regions of genes or within gene introns.     

Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures

Enhancers are important regulators of gene expression in eukaryotes. Enhancers function independently of their distance and orientation to the promoters of target genes. Thus, enhancers have been difficult to identify. Only a few enhancers, especially distant intergenic enhancers, have been identified in plants. We developed an enhancer prediction system based exclusively on the DNase I hypersensitive sites (DHSs) in the Arabidopsis thaliana genome. A set of 10,044 DHSs located in intergenic regions, which are away from any gene promoters, were predicted to be putative enhancers. We examined the functions of 14 predicted enhancers using the β-glucuronidase gene reporter. Ten of the 14 (71%) candidates were validated by the reporter assay. We also designed 10 constructs using intergenic sequences that are not associated with DHSs, and none of these constructs showed enhancer activities in reporter assays. In addition, the tissue specificity of the putative enhancers can be precisely predicted based on DNase I hypersensitivity data sets developed from different plant tissues. These results suggest that the open chromatin signature-based enhancer prediction system developed in Arabidopsis may serve as a universal system for enhancer identification in plants.     

Modeling the Evolutionary Architectures of Transcribed Human Enhancer Sequences Reveals Distinct Origins,Functions, and Associations with Human Trait Variation

Despite the importance of gene regulatory enhancers in human biology and evolution, we lack a comprehensive model of enhancer evolution and function. This substantially limits our understanding of the genetic basis of species divergence and our ability to interpret the effects of noncoding variants on human traits.To explore enhancer sequence evolution and its relationship to regulatory function, we traced the evolutionary origins of transcribed human enhancer sequences with activity across diverse tissues and cellular contexts from the FANTOM5 consortium. The transcribed enhancers are enriched for sequences of a single evolutionary age (“simple” evolutionary architectures) compared with enhancers that are composites of sequences of multiple evolutionary ages (“complex” evolutionary architectures), likely indicating constraint against genomic rearrangements. Complex enhancers are older, more pleiotropic, and more active across species than simple enhancers. Genetic variants within complex enhancers are also less likely to associate with human traits and biochemical activity. Transposable-element-derived sequences (TEDS) have made diverse contributions to enhancers of both architectures; the majority of TEDS are found in enhancers with simple architectures, while a minority have remodeled older sequences to create complex architectures. Finally, we compare the evolutionary architectures of transcribed enhancers with histone-mark-defined enhancers.Our results reveal that most human transcribed enhancers are ancient sequences of a single age, and thus the evolution of most human enhancers was not driven by increases in evolutionary complexity over time. Our analyses further suggest that considering enhancer evolutionary histories provides context that can aid interpretation of the effects of variants on enhancer function. Based on these results, we propose a framework for analyzing enhancer evolutionary architecture.     

Comparative cis-regulatory analyses identify new elements of the mouse Hoxc8 early enhancer

The Hoxc8 early enhancer is a 200 bp region that controls the early phase of Hoxc8 expression during mouse embryonic development. This enhancer defines the domain of Hoxc8 expression in the neural tube and mesoderm of the posterior regions of the developing embryo. Five distinct cis-acting elements, A-E, were previously shown to govern early phase Hoxc8 expression. Significant divergence between mammalian and fish Hoxc8 early enhancer sequences and activities suggested additional cis-acting elements. Here we describe four additional cis-acting elements (F-I) within the 200 bp Hoxc8 early enhancer region identified by comparative regulatory analysis and transgene-mutation studies. These elements affect posterior neural tube and mesoderm expression of the reporter gene, either singly or in combination. Surprisingly, these new elements are missing from the zebrafish and Fugu Hoxc8 early enhancer sequences. Considering that fish enhancers direct robust reporter expression in transgenic mouse embryos, it is tempting to postulate that fish and mammalian Hoxc8 early enhancers utilize different sets of elements to direct Hoxc8 early expression. These observations reveal a remarkable plasticity in the Hoxc8 early enhancer, suggesting different modes of initiation and establishment of Hoxc8 expression in different species. We postulate that extensive restructuring and remodeling of Hox cis-regulatory regions occurring in different taxa lead to relatively different Hox expression patterns, which in turn may act as a driving force in generating diverse axial morphologies.     

Aberrant cryptic responsiveness of the pCAT 3- and pGL3-promoter reporter vectors

Transfection analyses are an informative method to assess the activity of specific promoter or enhancer elements in mammalian cells. Commercially available reporter vectors can be extremely useful investigative tools for such studies. This study reports that the pCAT 3- and pGL3-promoter vectors display cryptic responsiveness to androgens when they contain a DNA insert, while the empty vector, a commonly used negative control, is nonresponsive. Our studies initially aimed to characterize novel androgen-responsive DNA sequences in human genomic DNA through transactivational analyses. An isolated DNA fragment, designated ARC-3, contained three putative androgen response element "half-sites" and was androgen-responsive when cloned into the pCAT3-promoter vector. While we originally believed this to be a novel enhancer element, subsequent analyses of this clone revealed that this vector displays cryptic activity in the presence of an androgen. This was confirmed by cloning several unrelated DNA fragments that did not contain any known classic response elements into the pCAT3-promoter vector, all of which were found to be responsive. The empty vector (negative control) was again nonresponsive. The ARC-3 DNA fragment was also weakly responsive to stimulation when cloned into the pGL3-promotor vector, which is identical to the pCAT3-promoter vector, with the exception of an intron located 5' of the chloramphenicol acetyltransferase gene, and the reporter genes. This work demonstrates that both the pCAT3- and pGL3-promoter vectors are inappropriate to assess androgen-responsive enhancers and emphasizes the importance of the careful selection of reporter vectors and controls when conducting transactivational analysis.     

Comparison of CMV, RSV, SV40 viral and Vlambda1 cellular promoters in B and T lymphoid and non-lymphoid cell lines.

Determining the activity of viral and cellular regulatory elements in B or T lymphoid cell lines would facilitate appropriate utilization of the regulatory sequences for gene transfer- and expression-dependent applications. We have compared the activity of the CMV, RSV and SV40 viral promoter/enhancers as well as the Vlambda1 cellular promoter, in three B cell lines (REH, SMS-SB, C3P), three T cell lines (CEM, Jurkat, ST-F10), and two non-lymphoid cell lines (K-562, HeLa) using the luciferase reporter gene. In B cell lines, the activity of the CMV promoter/enhancer construct was the highest ranging from 10- to 113-fold greater than that of SV40. In contrast, in T cell lines the RSV promoter/enhancer activity was 11-65-fold higher than that of SV40. The Vlambda1 promoter activity was close to that of SV40 promoter/enhancer in most of the cell lines tested. We conclude that CMV and RSV promoter/enhancers contain stronger regulatory elements than do the SV40 and Vlambda1 for expression of genes in lymphoid cell lines.     

Identification and experimental validation of splicing regulatory elements in Drosophila melanogaster reveals functionally conserved splicing enhancers in metazoans

RNA sequence elements involved in the regulation of pre-mRNA splicing have previously been identified in vertebrate genomes by computational methods. Here, we apply such approaches to predict splicing regulatory elements in Drosophila melanogaster and compare them with elements previously found in the human, mouse, and pufferfish genomes. We identified 99 putative exonic splicing enhancers (ESEs) and 231 putative intronic splicing enhancers (ISEs) enriched near weak 5' and 3' splice sites of constitutively spliced introns, distinguishing between those found near short and long introns. We found that a significant proportion (58%) of fly enhancer sequences were previously reported in at least one of the vertebrates. Furthermore, 20% of putative fly ESEs were previously identified as ESEs in human, mouse, and pufferfish; while only two fly ISEs, CTCTCT and TTATAA, were identified as ISEs in all three vertebrate species. Several putative enhancer sequences are similar to characterized binding-site motifs for Drosophila and mammalian splicing regulators. To provide additional evidence for the function of putative ISEs, we separately identified 298 intronic hexamers significantly enriched within sequences phylogenetically conserved among 15 insect species. We found that 73 putative ISEs were among those enriched in conserved regions of the D. melanogaster genome. The functions of nine enhancer sequences were verified in a heterologous splicing reporter, demonstrating that these sequences are sufficient to enhance splicing in vivo. Taken together, these data identify a set of predicted positive-acting splicing regulatory motifs in the Drosophila genome and reveal regulatory sequences that are present in distant metazoan genomes.     

Sequence-Specific and/or Stereospecific Constraints of the U3 Enhancer Elements of MCF 247-W Are Important for Pathogenicity

The oncogenic potential of many nonacute retroviruses is dependent on the duplication of the enhancer sequences present in the unique 3′ (U3) region of the long terminal repeat (LTR). In a molecular clone (MCF 247-W) of the murine leukemia virus MCF 247, a leukemogenic mink cell focus-inducing (MCF) virus, the U3 enhancer sequences are tandemly repeated in the LTR. We mutated the enhancer region of MCF 247-W to test the hypothesis that the duplicated enhancer sequences of this virus have a sequence-specific and/or a stereospecific role in enhancer function required for transformation. In one virus, we inserted 14 nucleotide bp into the novel sequence generated at the junction of the two enhancers to generate an MCF virus with an interrupted enhancer region. In the second virus, only one copy of the enhancer sequences was present. This second virus also lacked the junction sequence present between the two enhancers of MCF 247-W. Both viruses were less leukemogenic and had a longer mean latency period than MCF 247-W. These data indicate that the sequence generated at the junction of the two enhancers and/or the stereospecific arrangement of the two enhancer elements are required for the full oncogenic potential of MCF 247-W. We analyzed proviral LTRs within the c-myc locus in tumor DNAs from mice injected with the MCF virus with the interrupted enhancer region. Some of the proviral LTRs integrated upstream of c-myc contain enhancer regions that are larger than those of the injected virus. These results are consistent with the suggestion that the virus with an interrupted enhancer changes in vivo to perform its role in the transformation of T cells.     

Characterization of a cell type-specific enhancer found in the human papilloma virus type 18 genome.

We have previously isolated long-range acting enhancer elements from the HeLa genome by functional selection. In this report, the structural and functional characteristics of one (GA1) of the enhancers are reported. By cloning various restriction fragments and by deletion mutagenesis, the activity of GA1 was located in a 230-bp region. The nucleotide sequence of GA1 and genomic Southern blot analysis indicated that GA1 is derived from human papilloma virus (HPV) 18 DNA that had been integrated into the HeLa genome. The enhancer is located in the non-coding region of the HPV 18 genome. The HPV 18 enhancer consists of two functional domains, both of which have full enhancer activity in HeLa cells. The enhancer does not contain enhancer core sequences but contains several blocks of potential Z-DNA sequence. Like SV40 and polyoma virus enhancers, the activity of the HPV 18 enhancer was repressed by adenovirus E1a products. The HPV 18 enhancer shows a narrow cell type specificity: it is active in some cervical carcinoma cell lines, but inactive in all non-cervical cells except for one neuroblastoma cell line. These results suggest that the HPV 18 enhancer plays an important role in regulation of the viral genes.