The Experimental Research And Bioinformatics Analysis Of Effects Of A Short Sequence Enhancer And Alu Repeats On Gene Expression

Objective: Cis-regulatory elements are regions of DNA that regulate the expression of genes located on the same DNA strand. Though these elements are important for gene expression regulation, the functions of cis elements remain largely unknown. The studies of the mechanisms of cis-regulatory elements on gene expression regulation have important value for understanding the biological behavior of mammals including differentiation, epigenetics, tumorigenesis and aging.Enhancers are cis elements that activate gene expression and play an significant role in regulating gene expression. Gene expression regulation is a central issue of contemporary biology and relates to various biological phenomena, such as differentiation, tumorigenesis and aging. To clarify these biological phenomena, the mechanisms of gene expression regulation must be elucidated. Though the mechanisms for gene expression regulation are complicated, they are often studied by simplified systems. To explore the mechanisms of gene activation regulation, we have established one such simplified system. In this system, we examined the enhancer activity of short sequence DNA and its derived sequences using a GFP expression system. We found that the insertion of particular DNA sequences upstream of Alu elements can eliminate the inhibition of GFP gene expression induced by Alu repeats. In Our previous works, 14 piece of Alu repeats were inserted downstream of GFP gene in p EGFP-C1 to construct C1-Alu×14. Through C1-Alu×14 being transiently transfected into He La cells, we found that Alu×14 can significantly repress expression of GFP. When C1-AA×2-Alu×14 constructed by AA inserted between GFP and Alu×14 was transiently transfected into He La cells, the inhibitory effect of Alu×14 on GFP gene expression was partially released and AA sequence showed certain enhancer activity. In this paper, we paid attention to mechanisms of AA sequences and its derived sequences activating GFP expression.Alu elements are present in more than one million copies which altogether account for 10% of the whole human genome, Most scholars think that SINE（Alu） have important biological functions because they mandatorily exist during evolution process. However, the biological fuctions of most Alu repeats are unknown. Several lines of evidence demonstrated that Alus belonging to silencer suppressed gene expression. Alu RNAs acted as transcriptional co-repressors in the nucleus and as the most abundant fraction of the antisense transcriptome, these have the potential to downregulate sense expression both in cis as well as trans. Our previous studies have shown that Alu elements inhibited GFP reporter gene expression via triggering chromatin tight wrapping. Alu elements belong to silencer, but they are abundant in housekeeping genes and high expressed chromosomes, which puzzles biologists for a long time. To study the biological function of Alu repeats, we performed experimental research and bioinformatics analysis of Alu repeats elements.Methods: 1 The synthesis of primers and DNA fragments.Entrust the DNA synthesis company in Beijing to synthetize the primers with appropriate enzyme sites and DNA sequences. Attached table in this paper listed the sequences and names of primers and DNA template used in this study. 2 The construction of expression vector. 2.1 The construction of expression vector related to AA and its derived sequences.Primers with two appropriate enzyme loci（Ecor I/Xba I or Kpn I/Nhe I） were designed. DNA sequences with 4 restriction enzyme loci（Ecor I/Xba I and Kpn I/Nhe I） were amplified with template AA and its derived sequences using PCR. The PCR products with four enzyme loci and C1- Alu14 plasmid were digested by restriction enzymes Ecor I and Kpn I respectively. Both the small fragments and big frgments were with cohesive ends. Then we connected the small fragments of AA and its derived sequences and the big fragments of C1-Alu14 with T4 DNA ligase respectively. Using transformation, extraction of plasmid, etc, expression vectors of AA and its derived sequences were successfully constructed. Subsequently to get small fragments and big fragments of AA and its derived sequences of expression vectors with one piece of AA or its derived sequences, expression vectors one AA or its derived sequences were digested by restriction enzymes Hindâ…¢/Nheâ… and Hindâ…¢/Xbaâ…  respectively. Using T4 DNA ligase, small fragments and big fragments were connected and expression vectors with two piece of AA or its derived sequences were successfully constructed. 2.2 Construction of expression vectors related to Alu.Alu sequences derived from Alu on chromosome Xq13.1 RP11-29107 by PCR amplication. Lac Z sequences derived from E.coli. 4TMI（5′-GTGAAATAAATGCTTTTTTTGT） was a sequence with high enhancer activity, which were mentioned in our previous paper. The promoter CMV derived from plasmid pc DNA3.1+（1-882bp）, which contained intact sequence of the promoter CMV. The method for construction of plasmid was the same as above.All the plasmids for transfection were identified by enzyme digestion and gene sequencing. 3 Cell transfectionCell transfection contained transient transfection and stable transfection. The cells in logarithmic growth phase were transfected with LipofectamineTM2000 reagent according to the user manuals. By positive cell screening with G418 reagent, stably tranfected cells were obtained. 4 Observation for FluorescenceAfter cell transfection, firstly the GFP-positive cells were counted under fluorescence microscope in UV radiation, secondly the total cell number in the same field were counted under white light. Every sample was counted at least 500. Then calculate positive percentage of the GFP-positive cells. The formula was as follow:Positive percentage of GFP-positive cells=（the number of GFP-positive cells/ the total cell number in the same field）×100% 5 RT-PCRTotal RNA was extracted from transfected cells and DNA in total RNA was digested by DNase I. Reverse transcription was performed using Revert Aid reverse transcriptase（Thermo Scientific） and a 9-nt random primer. 6 Polyacrylamide gel electrophoresis（PAGE）To determine whether AA sequence can form secondary structure by atypical base-pairing, the conformation of 22 nt oligonucleotide was detected by PAGE. Conformation of seven piece of 22 nt oligonucleotide was determined by PAGE and silver staining. The sequence containing affluent T bases was used as marker for PAGE. After PAGE under different temperature, different ionic strength, denaturing PAGE and non-denaturing PAGE condition and silver staining, the ability of these oligonucleotides to form secondary structure was speculated by electrophoretic mobility shift assay. Thereby the relationship between gene spatial conformation and gene biological function was speculated. 7 Flow cytometric analysisThe transfected cells were determined by Flow Cytometry. Flow cytometric analysis were performed by No.4 Hospital of Hebei Medical University. 8 Bioinformatic analysis.Gene sequences of five different animals（people, dogs, cats, rats and mice） alpha globin gene cluster and its flanking sequence were obtained from the databases as follows: Homo sapiens（assembly GRCh38）（http://www.ncbi.nlm.nih.gov/genome/?term=human）;Canis lupus familiaris（assembly Can Fam3.1）（http://www.ncbi.nlm.nih.gov/ genome/?term=dog）; Felis catus（assembly Felis_catus-6.2）（http:// www.ncbi.nlm.nih.gov/genome/?term=cat）; Rattus norvegicus（assembly Rnor₆.0）（http://www.ncbi.nlm.nih.gov/genome/?term=rat）; Mus musculus（assembly GRCm38.p2）（http://www.ncbi.nlm.nih.gov/genome /?term=mouse）. Repeat Masker server（http://www.repeatmasker.org） was used to find repetitive sequences within α globin gene clusters and their flanking sequences.The mutation of human alpha globin gene cluster and its flanking sequence would cause haemoglobinopathy. The mutation data（deletion mutation） of human α globin gene cluster and its flanking sequence are from Hb Var database（http://globin.bx.psu.edu/hbvar/menu.html）.Results:1 Construction and identification of expression vectorsAll the expression vectors used for transfection were identified to be correct by enzyme digestion and gene sequencing.2 the effect of AA and its derived sequences on GFP report geneThrough GFP-positive percentage obtained from GFP-positive cells count after cell transfection, we found that sequence 22 R, 4TMI, AA could activate GFP report gene expression, However, sequence AA, TT, 7pie A and 7Pie T not, The results had statistical significance. The other 15 sequence derived from AA sequence all had enhancer activity, and most activity of the derived sequences was lower than that of AA sequence, the results had statistical significance.3 AA sequence activate GFP report gene expression at the transcriptional levelThe ability of AA sequence to activate GFP report gene expression was higher than that of 7Pie A sequence.To clarify the machnism of AA sequence activate GFP report gene expression, we determined the expression levels of RNA by RT-PCR. In order to increase the experimental accuracy, we used three pairs of primers to amplify GFP gene of different gene location. Our study found that AA sequence could induce more GFP m RNA than 7Pie A and Alu14 sequence. This result was the same as that of protein determination. Accordingly we could come to a conclusion that AA sequence activate GFP gene m RNA expression level. Both increased gene transcription and slowed RNA degradation can contribute to an increased intracellular RNA content, that is to say GFP gene m RNA expression increase can occur at transcritional level or m RNA degradation level. To figure out the answer to this quenstion, firstly we used actinomycin D to suppress the production of m RNA. Secondly m RNA content was determined by RT-PCR. Under the same experimental conditions, the degradation rate should be identical. The RT-PCR experiment found that GFP m RNA content of cells transfected with AA plasmids was higher than than of cells transfected with 7pie A plasmids. These results showed that the reason for AA sequence induced more GFP protein was that GFP gene transcription increased. That is to say, AA sequence activate GFP report gene expression at the transcriptional level.4 PAGE distinguish DNA oligonucleotide conformation.Using 20% denaturing PAGE at room temperature, we found that the electrophoretic mobility of the seven oligonucleotides, from fast to slow, as 7pie A>AA>22R>4T>4TMI>TT>7pie T. The number of A bases in 7pie A, AA, 22 R, 4T, 4TMI, TT, 7pie T sequences is 14, 12, 8, 7, 6, 2 and 0, respectively. We found that the number of A bases was proportional to the electrophoretic mobility. These findings demonstrated that the electrophoretic mobility of sequences containing more A bases was faster than that of sequences containing more T bases.Additionally, we found that the electrophoretic mobility of the T-rich sequences was less affected by ionic strength and low temperature, therefore, we used the T-rich sequences as a marker of PAGE. In 1×TBE and at a low temperature, the electrophoretic mobility of the 4T sequence was the fastest, and it was faster than that of the 15 nt marker; the electrophoretic velocities of 22 R, 7pie A, AA and 4TMI were similar to that of the 15 nt marker; and the electrophoretic mobility of 7pie T and TT were the slowest. These results show that the 4T sequence forms a secondary structure; and that the AA, 22 R, 7pie A and 4TMI sequences form a partial secondary structure; and that the TT sequence form a weak secondary structure and 7pie T sequence can not form a secondary structure.5 The combined sense and antisense Alus in the same vector activated GFP reporter geneThe expression vectors, C1-4TMI-Alu × 14-CMV-Alu × 2（4TMI-Alu-sense-sense）, C1-4TMI-Alu × 14-CMV-Alu × 2as（4TMI-Alu-sense-antisense）, C1-4TMI-Alu × 14-CMV-Lac Z2F2 R × 2（4TMI-Alu sense-control）, C1-4TMI-Lac Z3F3 R × 14-CMV-lac Z3F3 R × 2（4TMI-Lac Z-sense-sense）, C1-4TMI-Lac Z3F3 R × 14-CMV-lac Z3F3 R × 2as（4TMI-Lac Z-sense-antisense） and C1-4TMI-Lac Z3F3R×14-CMV-Lac Z2F2R× 2（4TMI-Lac Z-sense-control） were transfected into He La cells. GFP expression was determined using a flow cytometer after stability screening. GFP labeled amount of 4TMI-Alu-sense-antisense is 4.0 times than that of 4TMI-Alu-sense-sense, and is 2.9 times than that of 4TMI-Alu sense-control. All expression vectors containing Alus induced higher GFP labeled amount than the expression vectors containing Lac Z fragments. 4TMI-Alu-sense-antisense induced the hightest GFP labeled amount, suggesting that combined sense and antisense Alu elements activated GFP reporter gene.6 The proper combined sense and antisense Alus activating GFP reporter gene is cell type dependentThe expression vectors were transfected into BHK21 cells and He La cells. GFP expression was determined using a flow cytometer after stability screening. All expression vectors did not activate GFP reporter gene in BHKL21 cells while some expression vectors activated GFP reporter gene in He La cells. These results indicate that combined sense and antisense Alus activating gene expression is cell type denpendent.7 The combined sense and antisense Alu activting GFP reporter gene needs enhancer existenceThe inserted sequences of expression vectors had different copys of enhancer. When the expression vectors were transfected into He La cells. GFP expression was determined using a flow cytometer after cell stable transfection. The results showed that the labeled amount increase with enhancer number increasing, which indicated that combined sense and antisense Alus activating gene expression needs enhancer existence.8 The combined sense and antisense Alu activating GFP reporter gene needs enough copy of AluOur experiment showed that when there were enhancers upstream of 5’ tandem Alu and upstream of 3’ tandem Alu, Alu-sense-antisense expression vectors increased GFP gene expression with Alu copy number increasing of 3’ tandem Alu, which indicated that the combined sense and antisense Alu activating GFP reporter gene needed enough copy of Alu.9 The combined sense and antisense Alu activating GFP reporter gene needs stable transfectionWe compared the effects of transient transfection and stable transfection on GFP gene expression. The highest GFP labeled amount induced by Alu-sense-sense and Alu-sense-antisense expression vectors occours at 48 h after transient transfection. There is no significant difference of GFP labeled amount between the two kinds of expression vectors. However, Alu-sense-antisense expression vector started to induce more GFP gene activation at 24 days after stable transfection than that of Alu-sense-sense. The results above showed that the combined sense and antisense Alu activating GFP reporter gene needed stable transfection.10 The repetitive sequence distribution of α globin gene clusters in different speciesWe analyzed SINE content with 100 kb upstream and downstream of α globin gene clusters in human and other 4 animals and found that the arrangement and distribution of repetitive sequences in α globin gene clusters are similar though different species use different repetitive sequences. There are significent high SINEs content in α globin gene clusters compared with in β globin gene clusters by Bioinformatics analysis. The results illustrated that SINE sequences accumulate within α globin gene clusters and over long distances. SINE content with 100 kb upstream and downstream of α globin gene clusters was higher than the average value of distribution of SINE in the whole genom. The results above showed that SINE in α globin gene of the five species may play the same role in gene regulation process.11 Bioinformatics analysis of deletion of large gene fragments which cause human hemoglobin diseaseBioinformatics analysis of gene deletion of α globin gene clusters in human showed that many types of gene deletion could cause human hemoglobin disease. However, there was no case with only large deletion of downstream sequences of HBA1 gene but keeping HBA1 gene. We analyzed the repetitive sequence variability within downstream of HBA genes in normal individuals from 1000 genome plan（http://browser.1000 genomes.org） and did not find large fragment deletion within downstream of HBA genes. We came to the conclusion that the deletion of Alu sequences located downstream of HBA1 gene would lead to abnormal activation of HBA1 and HBA2 genes that contain promoters and enhancers and lead to heterozygous death.12 According to the result of experiment research and bioinformatics analysis, the model of activating gene induced by repeats is put forward in this article: repeats along with other non-coding sequences make genes in the critical state of transcribed and non-transcribed. Repeats play the role of inhibiting gene expression, which is a gene silencer. Once genes transcribed, RNAs produced from repetitive sequences of gene itself, can add to an RNA networks to activate gene in a positive feedback way, namely the transcripts act as the input to activate the output.Conlusion:1 AA sequence, which has enhancer activity, can activate genes and increase transcription of genes.2 AA sequence forms an unstable stem-loop via atypical complementation to play a role of enhancer activity.3 Alu elements activated GFP reporter gene only when simultaneously satisfying the following conditions:（1） The combination of the sense and antisense Alus;（2） Cell type dependence:in the He La cells Alu can activate genes but in BHK21 cells not;（3） The enhancer exsitence in upstream of Alus;（4） Enough Alu copy number;（5） Stable transfection. Alu elements silence gene, but once transcribed, The combined sense and antisense Alu elements have the ability to activate gene.4 The model of activating gene induced by repeats is put forward: genes in genome are in the critical state of transcribed and non-transcribed, once genes transcribed, repeats in the transcripts have the ability of activating genes.