As a key regulatory element of gene differential expression, enhancer plays a crucial role in craniomaxillofacial development through regulating the spatiotemporal expression of target genes to promote tissue-specific differentiation. With the development of CRISPR and chromosome conformation capture technique, the function of enhancer and its regulatory mechanism has been explored in depth. This paper gave a systematic review on the mechanism of enhancer regulating target gene expression and the role of enhancer in oral craniofacial development and malformation.
Animals ; Enhancer Elements, Genetic ; Mammals/genetics*

To establish a transgenic cell model based on anti-oxidative response element (ARE) and green fluorescence protein(GFP) reporter gene, the TK minimal promoter was amplified by PCR and cloned into pEGFP-N1 for constructing reporter vector pTK-GFP/Neo. Four synthetic oligonucleotide ARE motifs were annealed and purified and then were inserted into pTK-GFP/Neo one by one to construct the eukaryotic reporter vector p4ARE-TK-GFP/Neo. Two reconstruct eukaryotic reporter vectors were transfected into HepG2 cells mediated by lipofectamine. The positive clones were obtained by the screen of G418. The cell model was tested with PDTC and tBHQ, well known inducers of phase II enzymes, by determining GFP activity. The results showed that the expression level of GFP was significantly increased by PDTC and tBHQ, and a transgenic cell model based on ARE was established successfully.
Antioxidants ; metabolism ; Base Sequence ; Enhancer Elements, Genetic ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hep G2 Cells ; Humans ; Molecular Sequence Data ; Response Elements ; genetics ; Thymidine Kinase ; biosynthesis ; genetics ; Transfection ; Transgenes

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as 180degrees and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.
DNA End-Joining Repair ; DNA Transposable Elements ; DNA* ; Enhancer Elements, Genetic ; Hand ; Homologous Recombination ; Retroelements

In mammalian cells, transcribed enhancers (TrEns) play important roles in the initiation of gene expression and maintenance of gene expression levels in a spatiotemporal manner. One of the most challenging questions is how the genomic characteristics of enhancers relate to enhancer activities. To date, only a limited number of enhancer sequence characteristics have been investigated, leaving space for exploring the enhancers' DNA code in a more systematic way. To address this problem, we developed a novel computational framework, Transcribed Enhancer Landscape Search (TELS), aimed at identifying predictive cell type/tissue-specific motif signatures of TrEns. As a case study, we used TELS to compile a comprehensive catalog of motif signatures for all known TrEns identified by the FANTOM5 consortium across 112 human primary cells and tissues. Our results confirm that combinations of different short motifs characterize in an optimized manner cell type/tissue-specific TrEns. Our study is the first to report combinations of motifs that maximize classification performance of TrEns exclusively transcribed in one cell type/tissue from TrEns exclusively transcribed in different cell types/tissues. Moreover, we also report 31 motif signatures predictive of enhancers' broad activity. TELS codes and material are publicly available at http://www.cbrc.kaust.edu.sa/TELS.
Enhancer Elements, Genetic ; Genomics ; methods ; Humans ; Nucleotide Motifs ; Sequence Analysis, DNA ; methods ; Transcription, Genetic

OBJECTIVE: To explore the effect of rare synonymous variants of the ATP7B gene on the splicing of its precursor mRNA. METHODS: A total of 248 rare synonymous variants with allelic frequency of <0.005 were retrieved from the ExAc database. Human Splicing Finder (HSF) was used to predict their effect on the splicing of precursor mRNA. And ESE Finder 3.0 was used to predict the effect of such variants on the binding ability of SR protein family. Rare synonymous variants affecting the binding of two or more SR proteins were selected and verified with an in vitro mini gene splicing report system. RESULTS: HSF analysis indicated that 136 of the 248 rare synonymous variants may destroy the exonic splicing enhancer (ESE) motif. Analysis using ESE Finder 3.0 indicated that 19 of them may affect the binding of two or more SR proteins at the same time. In vitro mini gene experiment confirmed that the c.1620C>T (p.L540L) and c.3888C>T (p.A1296A) variants could lead to abnormal splicing of the corresponding exons, resulting in complete skipping of exon 4 and 25% increase in the skipping of exon 18, respectively. CONCLUSION Synonymous variants may affect the splicing of precursor mRNA in various ways, particularly the destruction of ESE motif. This study confirmed that the c.1620C>T (p.L540L) and c.3888C>T (p.A1296A) variants can affect the mRNA splicing of the ATP7B gene, resulting in skipping of corresponding exons, which may provide a basis for genetic diagnosis and consultation of carriers.
Alternative Splicing ; Copper-Transporting ATPases/genetics* ; Enhancer Elements, Genetic ; Exons ; Gene Frequency ; Humans ; RNA, Messenger/genetics*

BACKGROUNDAlthough DNA vaccine is considered as the next generation of vaccine, most DNA vaccine candidates are still suffering from the relatively weak immunogenicity despite the increased dosage of plasmid DNA administered. In order to enhance the immune responses elicited by a codon-optimized HIV gag DNA vaccine, a modified plasmid vector pDRVI1.0 and a booster immunization with replicating Tiantan vaccinia (RTV) strain expressing the same gene were employed.

METHODSVector pDRVI1.0 was constructed through inserting the 72-bp element from the SV40 enhancer, which was reported promoting nuclear transport of plasmid DNA, to the upstream of cytomegalovirus enhancer/promoter region of the plasmid vector pVR1012. Gene expression levels from expression plasmids based on pDRVI1.0 and pVR1012 were tested. Humoral and cellular immune responses induced by DNA vaccine alone or DNA prime-RTV boost regimen were determined in mice.

RESULTSIt was shown that the 72-bp element significantly enhanced the gene expression level in non-dividing cells. gag-specific humoral and cellular immune responses induced by DNA vaccination were both significantly improved, while the Th1/Th2 balance was not obviously affected by the 72-bp element. RTV boosting further significantly enhanced DNA vaccine-primed antibody and T cell responses in a Th1-biased manner.

CONCLUSIONSThe 72-bp SV40 enhancer element should be included in the DNA vaccine vector and RTV strain is a very efficient live vector for boosting immunization.

AIDS Vaccines ; immunology ; Amino Acid Sequence ; Animals ; Blotting, Western ; CD8-Positive T-Lymphocytes ; immunology ; Enhancer Elements, Genetic ; Female ; Gene Products, gag ; immunology ; HIV Antibodies ; blood ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Plasmids ; Simian virus 40 ; genetics ; Vaccination ; Vaccines, DNA ; immunology ; Vaccinia ; immunology

By altering the electrostatic charge of histones or providing binding sites to protein recognition molecules, Chromatin marks have been proposed to regulate gene expression, a property that has motivated researchers to link these marks to cis-regulatory elements. With the help of next generation sequencing technologies, we can now correlate one specific chromatin mark with regulatory elements (e.g. enhancers or promoters) and also build tools, such as hidden Markov models, to gain insight into mark combinations. However, hidden Markov models have limitation for their character of generative models and assume that a current observation depends only on a current hidden state in the chain. Here, we employed two graphical probabilistic models, namely the linear conditional random field model and multivariate hidden Markov model, to mark gene regions with different states based on recurrent and spatially coherent character of these eight marks. Both models revealed chromatin states that may correspond to enhancers and promoters, transcribed regions, transcriptional elongation, and low-signal regions. We also found that the linear conditional random field model was more effective than the hidden Markov model in recognizing regulatory elements, such as promoter-, enhancer-, and transcriptional elongation-associated regions, which gives us a better choice.
Binding Sites ; Chromatin ; genetics ; Enhancer Elements, Genetic ; Epigenomics ; Histones ; genetics ; Humans ; Markov Chains ; Models, Genetic ; Models, Statistical ; Promoter Regions, Genetic ; Regulatory Elements, Transcriptional

OBJECTIVETo screen enhancer-like sequences from vaccinia virus genome, to construct an expression vector harboring prokaryotic enhancer-like sequence and study the effect of interferon gene expression.

METHODSEnhancer-like element from vaccinia virus genome was obtained by using the chloramphenicol acetyl-transferase cat gene as reporter gene. An expression vector harboring prokaryotic enhancer-like sequence VV1 from vaccinia virus was constructed. Interferon was expressed and assayed.

RESULTSEighteen enhancing sequences were found. From them two enhancer-like sequences with distance and orientation independence property were screened and named VV1 and VV16 respectively. Quantification test showed that the direct and reverse orientation of VV1 could increase the activity of beta-galactosidase with 10.9 and 3.8 times and those of VV16 could increase by 9.0 times and 4.1 times respectively. The enhancing activity of the element was on transcription level. An expression vector harboring prokaryotic enhancer-like sequence VV1 was constructed. Using this vector the antiviral activity of interferon alpha-2b was increased by 2.6 times in comparison with the original expression plasmid.

CONCLUSIONTwo enhancer-like sequences were screened from vaccinia virus genome. Interferon gene was highly expressed by using an expression vector harboring enhancer-like sequences.

Enhancer Elements, Genetic ; Genetic Vectors ; genetics ; Interferon-alpha ; biosynthesis ; genetics ; pharmacology ; Plasmids ; Recombinant Proteins ; Vaccinia virus ; genetics

OBJECTIVETo learn the effect of hepatitis B virus (HBV) sequence nt250-453 on the HBV X promoter.

METHODSA plasmid pNRE-XP which contains the NRE and the HBV X promoter was constructed to co-transfect HepG2 cell line with plasmid RL-TK. The firefly luciferase activity and mRNA expression of the firefly luciferase gene were both detected. Then, nt250-453 of HBV was removed from LJ196, which contained HBV full genes. The mutated plasmid LJ196 and plasmid LJ96 which provided core protein and the viral DNA polymerase were used to co-transfect HepG2 cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the X gene mRNA level.

RESULTSThe activity of firefly luciferase and the expression of firefly luciferase gene mRNA were both down-regulated in the presence of the NRE, while the HBV X gene mRNA expression increased as it was removed from the HBV genes.

CONCLUSIONThis study demonstrates that nt250-453 of HBV acts as a novel negative regulatory element which could suppress the HBV X promoter activity.

Enhancer Elements, Genetic ; Gene Expression ; Gene Expression Regulation, Viral ; Hepatitis B virus ; genetics ; Promoter Regions, Genetic ; Trans-Activators ; genetics

Chromosome Mapping ; Enhancer Elements, Genetic ; Erythropoietin ; genetics ; Exercise ; Humans ; Military Personnel ; Physical Endurance ; Polymorphism, Single Nucleotide ; Young Adult