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*

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*

Enhancers activate transcription in a distance-, orientation-, and position-independent manner, which makes them difficult to be identified. Self-transcribing active regulatory region sequencing (STARR-seq) measures the enhancer activity of millions of DNA fragments in parallel. Here we used STARR-seq to generate a quantitative global map of rice enhancers. Most enhancers were mapped within genes, especially at the 5' untranslated regions (5'UTR) and in coding sequences. Enhancers were also frequently mapped proximal to silent and lowly-expressed genes in transposable element (TE)-rich regions. Analysis of the epigenetic features of enhancers at their endogenous loci revealed that most enhancers do not co-localize with DNase I hypersensitive sites (DHSs) and lack the enhancer mark of histone modification H3K4me1. Clustering analysis of enhancers according to their epigenetic marks revealed that about 40% of identified enhancers carried one or more epigenetic marks. Repressive H3K27me3 was frequently enriched with positive marks, H3K4me3 and/or H3K27ac, which together label enhancers. Intergenic enhancers were also predicted based on the location of DHS regions relative to genes, which overlap poorly with STARR-seq enhancers. In summary, we quantitatively identified enhancers by functional analysis in the genome of rice, an important model plant. This work provides a valuable resource for further mechanistic studies in different biological contexts.
Acetylation ; Base Sequence ; Deoxyribonuclease I ; metabolism ; Enhancer Elements, Genetic ; Epigenesis, Genetic ; Genes, Plant ; Genomics ; methods ; Histone Code ; genetics ; Histones ; metabolism ; Models, Genetic ; Oryza ; genetics ; Promoter Regions, Genetic ; genetics ; Repetitive Sequences, Nucleic Acid ; genetics ; Sequence Analysis, DNA ; Transcription, Genetic

Transposable elements (TEs) have no longer been totally considered as "junk DNA" for quite a time since the continual discoveries of their multifunctional roles in eukaryote genomes. As one of the most important and abundant TEs that still active in human genome, Alu, a SINE family, has demonstrated its indispensable regulatory functions at sequence level, but its spatial roles are still unclear. Technologies based on 3C (chromosome conformation capture) have revealed the mysterious three-dimensional structure of chromatin, and make it possible to study the distal chromatin interaction in the genome. To find the role TE playing in distal regulation in human genome, we compiled the new released Hi-C data, TE annotation, histone marker annotations, and the genome-wide methylation data to operate correlation analysis, and found that the density of Alu elements showed a strong positive correlation with the level of chromatin interactions (hESC: r = 0.9, P < 2.2 × 10(16); IMR90 fibroblasts: r = 0.94, P < 2.2 × 10(16)) and also have a significant positive correlation with some remote functional DNA elements like enhancers and promoters (Enhancer: hESC: r = 0.997, P = 2.3 × 10(-4); IMR90: r = 0.934, P = 2 × 10(-2); Promoter: hESC: r = 0.995, P = 3.8 × 10(-4); IMR90: r = 0.996, P = 3.2 × 10(-4)). Further investigation involving GC content and methylation status showed the GC content of Alu covered sequences shared a similar pattern with that of the overall sequence, suggesting that Alu elements also function as the GC nucleotide and CpG site provider. In all, our results suggest that the Alu elements may act as an alternative parameter to evaluate the Hi-C data, which is confirmed by the correlation analysis of Alu elements and histone markers. Moreover, the GC-rich Alu sequence can bring high GC content and methylation flexibility to the regions with more distal chromatin contact, regulating the transcription of tissue-specific genes.
Alu Elements ; genetics ; Base Composition ; Binding Sites ; Cell Line ; Chromatin ; chemistry ; genetics ; metabolism ; CpG Islands ; DNA ; metabolism ; Databases, Genetic ; Enhancer Elements, Genetic ; genetics ; Genome, Human ; Histones ; metabolism ; Humans ; Methylation

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 generate a gene delivery plasmid carrying the dominant negative form of the protein phosphatase 2A catalytic subunit a (DN-PP2Aca) driven by a hepatocellular carcinoma (HCC) tissue-specific promoter and investigate its ability to inhibit growth of cultured hepatoma cells.

METHODSThe gene delivery plasmid was constructed by PCR-amplifying DN-PP2Aca from wild-type PP2Aca using site-directed mutagenesis and then ligating the sequence-verified amplicon downstream of an alpha-fetoprotein enhancer and phosphoglycerate kinase promoter (AFpg) in the luciferase reporter vector pGL3-Basic. Following transfection into two AFP+ hepatoma cell lines (HepG2 and HepG3) and two AFP- hepatoma cell lines (SK-HEP-1 and L02), the transcriptional activity of the AFpg-driven DN-PP2Aca plasmid was tested using luciferase reporter gene assay and western blotting. The effect on cell growth was tested using MTT assay. Between group differences were assessed by t-test.

RESULTSThe AFpg-driven DN-PP2Aca plasmid showed high transcriptional activity and protein expression in both HepG2 and Hep3B cells. At 72 h after transfection, the proliferation capacities were repressed by 42.65%+/-3.99% (P = 0.0002) and 39.87%+/-3.91% (P = 0.0002) in AFP+ HepG2 and Hep3B cells, respectively (vs. untransfected). In contrast, the plasmid was transcriptionally inactive in and had no effect on proliferation of AFP- cells.

CONCLUSIONThe AFpg-driven DN-PP2Aca plasmid exhibits selective cytotoxicity against AFP+ hepatoma cells, and may represent a useful gene therapy strategy to treat HCC.

Carcinoma, Hepatocellular ; genetics ; metabolism ; Enhancer Elements, Genetic ; Genetic Therapy ; Genetic Vectors ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; metabolism ; Mutation ; Promoter Regions, Genetic ; Protein Phosphatase 2 ; genetics ; alpha-Fetoproteins ; genetics

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

DNA methylation may regulate gene expression by restricting the access of transcription factors. We have previously demonstrated that GATA-1 regulates the transcription of the CCR3 gene by dynamically interacting with both positively and negatively acting GATA elements of high affinity binding in the proximal promoter region including exon 1. Exon 1 has three CpG sites, two of which are positioned at the negatively acting GATA elements. We hypothesized that the methylation of these two CpGs sites might preclude GATA-1 binding to the negatively acting GATA elements and, as a result, increase the availability of GATA-1 to the positively acting GATA element, thereby contributing to an increase in GATA-1-mediated transcription of the gene. To this end, we determined the methylation of the three CpG sites by bisulfate pyrosequencing in peripheral blood eosinophils, cord blood (CB)-derived eosinophils, PBMCs, and cell lines that vary in CCR3 mRNA expression. Our results demonstrated that methylation of CpG sites at the negatively acting GATA elements severely reduced GATA-1 binding and augmented transcription activity in vitro. In agreement, methylation of these CpG sites positively correlated with CCR3 mRNA expression in the primary cells and cell lines examined. Interestingly, methylation patterns of these three CpG sites in CB-derived eosinophils mostly resembled those in peripheral blood eosinophils. These results suggest that methylation of CpG sites at the GATA elements in the regulatory regions fine-tunes CCR3 transcription.
Binding Sites ; Cell Line ; *CpG Islands ; DNA Methylation ; Enhancer Elements, Genetic ; Eosinophils/cytology/*metabolism ; Exons ; Fetal Blood/cytology/metabolism ; GATA1 Transcription Factor/*genetics/metabolism ; Gene Expression Regulation ; Humans ; Promoter Regions, Genetic ; RNA, Messenger/metabolism ; Receptors, CCR3/*genetics/metabolism ; Sequence Analysis, DNA ; *Transcription, Genetic

OBJECTIVETo identify the enhancers of human lung specific X protein (LUNX) and their regulation at the transcription level in vitro.

METHODSThree enhancer fragments (E1:+3770~+3959bp; E2: +6454~+6555bp; E3: +14553~+14652 bp) predicted by bioinformatics software were isolated from the human genomic DNA by PCR amplification. Luciferase assay was performed to detect the activities of the enhancers in transcriptional regulation.

RESULTSPCR products were confirmed by DNA sequencing. The amplified enhancers digested by Kpn I/Xho I and BamH I/Sal I, to generate the sticky-end fragments were inserted into PGL3-promoter in a reporter vector, and 6 luciferase expression vectors were obtained. All the reporter plasmids and pGL3-promoter were transiently transfected into HEK293 cells with an internal control of pSV-β-Galactosidase reporter vector. The enhancer activity of each construct was evaluated by luciferase assay of the cell extracts after transfection for 48 h. The results showed that the 3 fragments, when located upstream, did not increase transcription of reporter gene, but when at the downstream, E1 and E3 increased the transcription by 2.83 and 1.59 folds of that of pGL3-promoter, respectively.

CONCLUSIONLUNX gene sequences from +3770 to +3959 bp and +14553 to +14652 bp possess the capacity to enhance gene transcription.

Base Sequence ; Cloning, Molecular ; Enhancer Elements, Genetic ; Gene Expression Regulation ; Glycoproteins ; genetics ; HEK293 Cells ; Humans ; Molecular Sequence Data ; Phosphoproteins ; genetics ; Transcription, Genetic

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