Upstream DNA Research Articles

Epigenetic regulation of pyruvate carboxylase gene expression in the postpartum liver

Hepatic gluconeogenesis is essential for maintenance of whole body glucose homeostasis and glucose supply for mammary lactose synthesis in the dairy cow. Upregulation of the gluconeogenic enzyme pyruvate carboxylase (PC) during the transition period is vital in the adaptation to the greater glucose demands associated with peripartum lactogenesis. The objective of this study was to determine if PC transcription in hepatocytes is regulated by DNA methylation and if treatment with a nonsteroidal anti-inflammatory drug (NSAID) alters methylation of an upstream DNA sequence defined as promoter 1. Dairy cows were left untreated (n=20), or treated with a NSAID during the first 5 d postcalving (n=20). Liver was biopsied at d 7 precalving and d 7, 14, and 28 postcalving. Total PC and transcript specific gene expression was quantified using quantitative PCR and DNA methylation of promoter 1 was quantified using bisulfite Sanger sequencing. Expression of PC changed over the transition period, with increased expression postcalving occurring concurrently with increased circulating concentration of nonesterified fatty acids. The DNA methylation percentage was variable at all sites quantified and ranged from 21 to 54% across the 15 CpG dinucleotides within promoter 1. The DNA methylation at wk 1 postcalving, however, was not correlated with gene expression of promoter 1-regulated transcripts and we did not detect an effect of NSAID treatment on DNA methylation or PC gene expression. Our results do not support a role for DNA methylation in regulating promoter 1-driven gene expression of PC at wk 1 postcalving. Further research is required to determine the mechanisms regulating increased PC expression over the transition period.

Structures of E. coli σS-transcription initiation complexes provide new insights into polymerase mechanism

In bacteria, multiple σ factors compete to associate with the RNA polymerase (RNAP) core enzyme to form a holoenzyme that is required for promoter recognition. During transcription initiation RNAP remains associated with the upstream promoter DNA via sequence-specific interactions between the σ factor and the promoter DNA while moving downstream for RNA synthesis. As RNA polymerase repetitively adds nucleotides to the 3'-end of the RNA, a pyrophosphate ion is generated after each nucleotide incorporation. It is currently unknown how the release of pyrophosphate affects transcription. Here we report the crystal structures of E coli transcription initiation complexes (TICs) containing the stress-responsive σ(S) factor, a de novo synthesized RNA oligonucleotide, and a complete transcription bubble (σ(S)-TIC) at about 3.9-Å resolution. The structures show the 3D topology of the σ(S) factor and how it recognizes the promoter DNA, including likely specific interactions with the template-strand residues of the -10 element. In addition, σ(S)-TIC structures display a highly stressed pretranslocated initiation complex that traps a pyrophosphate at the active site that remains closed. The position of the pyrophosphate and the unusual phosphodiester linkage between the two terminal RNA residues suggest an unfinished nucleotide-addition reaction that is likely at equilibrium between nucleotide addition and pyrophosphorolysis. Although these σ(S)-TIC crystals are enzymatically active, they are slow in nucleotide addition, as suggested by an NTP soaking experiment. Pyrophosphate release completes the nucleotide addition reaction and is associated with extensive conformational changes around the secondary channel but causes neither active site opening nor transcript translocation.

UpCoT: an integrated pipeline tool for clustering upstream DNA sequences of orthologous genes in prokaryotic genomes.

UpCoT is a pipeline tool developed by automating the series of steps involved in prediction of cis-regulatory elements. UpCoT generates orthologs for each gene in target genome using bi-directional best blast hit against the reference genomes, then identifies potential orthologous transcriptional units using intergenic distance. Finally it generates the FASTA files containing upstream sequences of orthologous transcriptional units of each gene in target genome. The inputs of UpCoT are protein sequence files (*.faa), genome sequence files (*.fna) and gene co-ordinate files (*.ptt) for target and reference genomes. The clustered-upstream DNA sequences can be used by motif prediction tool, such as MEME, Bio-prospector, Gibbs motif sampler, MDscan for prediction of conserved DNA elements. We tested the performance of UpCoT by selecting the genome of Synechocystis sp PCC 6803 as the target and 13 different cyanobacterial genomes as reference. The clustered upstream sequences generated by UpCoT of groES, ycf24 and nirA were used for cis-regulatory element prediction. The results were consistent with the experimentally identified cis-regulatory elements. Therefore, UpCoT is a reliable and automated pipeline package for prediction of orthologs, orthologous transcriptional units, and orthologous upstream sequences of a selected prokaryotic genome. UpCoT can be downloaded from http://jssplab.uohyd.ac.in/upcot/.

Kinetics and Mechanism of Formation and Stabilization of the RNA Polymerase-Promoter Open Compex

Specific binding of E. coli RNA polymerase holoenzyme (RNAP; α2ββ’σ70) to promoter DNA sets in motion a series of conformational changes that advance the initial closed complex (RPc), open 12-14 bp including the −10 region and the transcription start site, and at some promoters then stabilize the initial open complex dramatically. Our research focuses on the kinetics and mechanism of these conformational changes at different promoters, using Forster resonance energy transfer (FRET) and protein induced fluorescence enhancement (PIFE) together with fast footprinting and filter binding assays to determine the kinetics of open complex formation and dissociation, and to obtain structural and thermodynamic information about key intermediates. FRET and PIFE results to date demonstrate the importance of wrapping of upstream promoter DNA on RNAP for efficient bending of the downstream duplex into the active site cleft to form the advanced closed complex which is opened by RNAP in the rate limiting step. The initial open complex is found to be unstable, and is subsequently stabilized at some but not all promoters by conformational changes and interactions of in-cleft and downstream mobile elements of RNAP, directed by the discriminator sequence of the promoter. Dissociation kinetic studies with PIFE, FRET and filter binding assays are being used to probe the mechanism of these conformational changes in open complex stabilization. This project is funded by NIH support(GM103061).

Structure of transcribing mammalian RNA polymerase II

RNA polymerase (Pol) II produces messenger RNA during transcription of protein-coding genes in all eukaryotic cells. The Pol II structure is known at high resolution from X-ray crystallography for two yeast species. Structural studies of mammalian Pol II, however, remain limited to low-resolution electron microscopy analysis of human Pol II and its complexes with various proteins. Here we report the 3.4 Å resolution cryo-electron microscopy structure of mammalian Pol II in the form of a transcribing complex comprising DNA template and RNA transcript. We use bovine Pol II, which is identical to the human enzyme except for seven amino-acid residues. The obtained atomic model closely resembles its yeast counterpart, but also reveals unknown features. Binding of nucleic acids to the polymerase involves 'induced fit' of the mobile Pol II clamp and active centre region. DNA downstream of the transcription bubble contacts a conserved 'TPSA motif' in the jaw domain of the Pol II subunit RPB5, an interaction that is apparently already established during transcription initiation. Upstream DNA emanates from the active centre cleft at an angle of approximately 105° with respect to downstream DNA. This position of upstream DNA allows for binding of the general transcription elongation factor DSIF (SPT4-SPT5) that we localize over the active centre cleft in a conserved position on the clamp domain of Pol II. Our results define the structure of mammalian Pol II in its functional state, indicate that previous crystallographic analysis of yeast Pol II is relevant for understanding gene transcription in all eukaryotes, and provide a starting point for a mechanistic analysis of human transcription.

Duplications upstream and downstream of SHOX identified as novel causes of Leri-Weill dyschondrosteosis or idiopathic short stature.

Leri-Weill dyschondrosteosis is a pseudoautosomal dominantly-inherited skeletal dysplasia ascribed to haploinsufficiency of the SHOX gene caused by deletions, point mutations, or partial duplications of the gene, or to heterozygous deletions upstream or downstream of the intact SHOX gene involving conserved non-coding cis-regulatory DNA elements that show enhancer activity. Recently, two SHOX conserved non-coding element duplications, one upstream and one downstream, were reported in patients referred with idiopathic short stature. To further evaluate the role of these duplications in SHOX-related disorders, we describe seven patients (five with Leri-Weill dyschondrosteosis and two with short stature) all of whom have duplications of part of the upstream or downstream conserved non-coding element regions, identified by multiplex ligation-dependent probe amplification. In addition, we show data from 32 patients with an apparently identical downstream duplication that includes a proposed putative regulatory element (identified by multiplex ligation-dependent probe amplification or array comparative genome hybridization), which results in a variable phenotype from normal to mild Leri-Weill dyschondrosteosis. These additional data provide further evidence that duplications of upstream and downstream long range cis-regulatory DNA elements can result in a SHOX-related phenotype.

Engineering an efficient and tight D-amino acid-inducible gene expression system in Rhodosporidium/Rhodotorula species.

BackgroundRhodosporidium and Rhodotorula are two genera of oleaginous red yeast with great potential for industrial biotechnology. To date, there is no effective method for inducible expression of proteins and RNAs in these hosts.ResultsWe have developed a luciferase gene reporter assay based on a new codon-optimized LUC2 reporter gene (RtLUC2), which is flanked with CAR2 homology arms and can be integrated into the CAR2 locus in the nuclear genome at >90 % efficiency. We characterized the upstream DNA sequence of a d-amino acid oxidase gene (DAO1) from R. toruloides ATCC 10657 by nested deletions. By comparing the upstream DNA sequences of several putative DAO1 homologs of Basidiomycetous fungi, we identified a conserved DNA motif with a consensus sequence of AGGXXGXAGX11GAXGAXGG within a 0.2 kb region from the mRNA translation initiation site. Deletion of this motif led to strong mRNA transcription under non-inducing conditions. Interestingly, DAO1 promoter activity was enhanced about fivefold when the 108 bp intron 1 was included in the reporter construct. We identified a conserved CT-rich motif in the intron with a consensus sequence of TYTCCCYCTCCYCCCCACWYCCGA, deletion or point mutations of which drastically reduced promoter strength under both inducing and non-inducing conditions. Additionally, we created a selection marker-free DAO1-null mutant (∆dao1e) which displayed greatly improved inducible gene expression, particularly when both glucose and nitrogen were present in high levels. To avoid adding unwanted peptide to proteins to be expressed, we converted the original translation initiation codon to ATC and re-created a translation initiation codon at the start of exon 2. This promoter, named PDAO1-in1m1, showed very similar luciferase activity to the wild-type promoter upon induction with d-alanine. The inducible system was tunable by adjusting the levels of inducers, carbon source and nitrogen source.ConclusionThe intron 1-containing DAO1 promoters coupled with a DAO1 null mutant makes an efficient and tight d-amino acid-inducible gene expression system in Rhodosporidium and Rhodotorula genera. The system will be a valuable tool for metabolic engineering and enzyme expression in these yeast hosts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0357-7) contains supplementary material, which is available to authorized users.

Restoration of Physiologically Responsive Low-Density Lipoprotein Receptor-Mediated Endocytosis in Genetically Deficient Induced Pluripotent Stem Cells.

Acquiring sufficient amounts of high-quality cells remains an impediment to cell-based therapies. Induced pluripotent stem cells (iPSC) may be an unparalleled source, but autologous iPSC likely retain deficiencies requiring correction. We present a strategy for restoring physiological function in genetically deficient iPSC utilizing the low-density lipoprotein receptor (LDLR) deficiency Familial Hypercholesterolemia (FH) as our model. FH fibroblasts were reprogrammed into iPSC using synthetic modified mRNA. FH-iPSC exhibited pluripotency and differentiated toward a hepatic lineage. To restore LDLR endocytosis, FH-iPSC were transfected with a 31 kb plasmid (pEHZ-LDLR-LDLR) containing a wild-type LDLR (FH-iPSC-LDLR) controlled by 10 kb of upstream genomic DNA as well as Epstein-Barr sequences (EBNA1 and oriP) for episomal retention and replication. After six months of selective culture, pEHZ-LDLR-LDLR was recovered from FH-iPSC-LDLR and transfected into Ldlr-deficient CHO-a7 cells, which then exhibited feedback-controlled LDLR-mediated endocytosis. To quantify endocytosis, FH-iPSC ± LDLR were differentiated into mesenchymal cells (MC), pretreated with excess free sterols, Lovastatin, or ethanol (control), and exposed to DiI-LDL. FH-MC-LDLR demonstrated a physiological response, with virtually no DiI-LDL internalization with excess sterols and an ~2-fold increase in DiI-LDL internalization by Lovastatin compared to FH-MC. These findings demonstrate the feasibility of functionalizing genetically deficient iPSC using episomal plasmids to deliver physiologically responsive transgenes.

Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1.

Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent remodeling reactions is largely absent. The single subunit remodeler Mot1 (modifier of transcription 1) dissociates TATA box-binding protein (TBP):DNA complexes, offering a useful system to address the structural mechanisms of Swi2/Snf2 ATPases. Here, we report the crystal structure of the N-terminal domain of Mot1 in complex with TBP, DNA, and the transcription regulator negative cofactor 2 (NC2). Our data show that Mot1 reduces DNA:NC2 interactions and unbends DNA as compared to the TBP:DNA:NC2 state, suggesting that Mot1 primes TBP:NC2 displacement in an ATP-independent manner. Electron microscopy and cross-linking data suggest that the Swi2/Snf2 domain of Mot1 associates with the upstream DNA and the histone fold of NC2, thereby revealing parallels to some nucleosome remodelers. This study provides a structural framework for how a Swi2/Snf2 ATPase interacts with its substrate DNA:protein complex.

ARID1A Deficiency Impairs the DNA Damage Checkpoint and Sensitizes Cells to PARP Inhibitors.

ARID1A, SWI/SNF chromatin remodeling complex subunit, is a recently identified tumor suppressor that is mutated in a broad spectrum of human cancers. Thus, it is of fundamental clinical importance to understand its molecular functions and determine whether ARID1A deficiency can be exploited therapeutically. In this article, we report a key function of ARID1A in regulating the DNA damage checkpoint. ARID1A is recruited to DNA double-strand breaks (DSB) via its interaction with the upstream DNA damage checkpoint kinase ATR. At the molecular level, ARID1A facilitates efficient processing of DSB to single-strand ends and sustains DNA damage signaling. Importantly, ARID1A deficiency sensitizes cancer cells to PARP inhibitors in vitro and in vivo, providing a potential therapeutic strategy for patients with ARID1A-mutant tumors. ARID1A has been identified as one of the most frequently mutated genes across human cancers. Our data suggest that clinical utility of PARP inhibitors might be extended beyond patients with BRCA mutations to a larger group of patients with ARID1A-mutant tumors, which may exhibit therapeutic vulnerability to PARP inhibitors.

Bacteriophage Xp10 anti-termination factor p7 induces forward translocation by host RNA polymerase.

Regulation of transcription elongation is based on response of RNA polymerase (RNAP) to various pause signals and is modulated by various accessory factors. Here we report that a 7 kDa protein p7 encoded by bacteriophage Xp10 acts as an elongation processivity factor of RNAP of host bacterium Xanthomonas oryzae, a major rice pathogen. Our data suggest that p7 stabilizes the upstream DNA duplex of the elongation complex thus disfavouring backtracking and promoting forward translocated states of the elongation complex. The p7-induced ‘pushing’ of RNAP and modification of RNAP contacts with the upstream edge of the transcription bubble lead to read-through of various types of pauses and termination signals and generally increase transcription processivity and elongation rate, contributing for transcription of an extremely long late genes operon of Xp10. Forward translocation was observed earlier upon the binding of unrelated bacterial elongation factor NusG, suggesting that this may be a general pathway of regulation of transcription elongation.

MicroRNA-1298 is regulated by DNA methylation and affects vascular smooth muscle cell function by targeting connexin 43.

Growing evidence links microRNA to the process of peripheral vascular disease. Recently, we have found that microRNA-1298(miR-1298) is one of the most significantly down-regulated microRNAs in human arteries with arteriosclerosis obliterans (ASO) of the lower extremities. However, little is known regarding its role in the process of ASO. The present study aimed to investigate the expression, regulatory mechanisms, and functions of miR-1298 in the process of ASO. Using quantitative reverse-transcription PCR and in situ hybridization assays, miR-1298 was observed predominantly expressed in the vascular smooth muscle cells (VSMCs) and was significantly down-regulated in ASO compared with normal arteries. Pyrosequencing analysis revealed that the miR-1298 DNA upstream of CpG sites were hypermethylated in ASO compared with normal arteries. Next, the luciferase reporter assay revealed that miR-1298 down-regulation is related with upstream DNA CpG site hypermethylation. Introducing a miR-1298 mimic into cultured VSMCs significantly attenuated cell proliferation and migration. Connexin 43 (Cx43) was validated to be a functional target of miR-1298 that was involved in the miR-1298-mediated cellular effects. Finally, lentivirus-mediated delivery of miR-1298 and its target Cx43 into a rat carotid balloon injury model indicated that re-overexpression of miR-1298 significantly decreased neointimal formation by targeting connexin 43. Our data demonstrate a specific role of the upstream DNA methylation/miR-1298/Cx43 pathway in regulating VSMC function and suggest that modulation of miR-1298 levels may offer a novel therapeutic approach for ASO.

Okazaki fragment maturation involves α-segment error editing by the mammalian FEN1/MutSα functional complex.

During nuclear DNA replication, proofreading-deficient DNA polymerase α (Pol α) initiates Okazaki fragment synthesis with lower fidelity than bulk replication by proofreading-proficient Pol δ or Pol ε. Here, we provide evidence that the exonuclease activity of mammalian flap endonuclease (FEN1) excises Pol α replication errors in a MutSα-dependent, MutLα-independent mismatch repair process we call Pol α-segment error editing (AEE). We show that MSH2 interacts with FEN1 and facilitates its nuclease activity to remove mismatches near the 5' ends of DNA substrates. Mouse cells and mice encoding FEN1 mutations display AEE deficiency, a strong mutator phenotype, enhanced cellular transformation, and increased cancer susceptibility. The results identify a novel role for FEN1 in a specialized mismatch repair pathway and a new cancer etiological mechanism.

Genetics of the serrated pathway to colorectal cancer.

Genetics of the serrated pathway to colorectal cancer.

Angiotensin II Upregulates CAPON Expression via ERK‐MAPK‐CREB Pathway in the Paraventricular Nucleus of Rats with Chronic Heart Failure

Elevated levels of angiotensin II (Ang II) and its receptor (AT1) in brain are responsible for increase sympatho‐excitation during heart failure (HF).Previously, we have demonstrated that higher levels of CAPON (nitric oxide synthase 1‐adaptor protein) down regulate neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN) of rats with HF. The significant increase in CAPON transcript (15 folds) in the PVN of HF rats was ameliorated by Losartan treatment (10 mg/kg/day for three weeks), suggesting the involvement of AngII/AT1R signaling in transcriptional up‐regulation of CAPON. In vitro studies using NG108 cells showed that Ang II dependent increases in CAPON expression was abrogated in presence of the mitogen‐activated protein kinase/ERK kinase 1/2 inhibitor U0126. The in silico analysis of the ~7.5 kb upstream DNA sequences from the translational start site of CAPON genes identified two likely promoter elements (Promoter 2.0 Prediction Server) containing the sequences responsive (TES Software) to transcriptional factor CREB binding. Concurrently, there was activation of ERK and CREB transcriptional factors in the PVN of rats with HF as evident by increased ERK‐P/ERK (1.43±0.1 Sham. vs. 1.98±0.1 HF) and CREB‐P/CREB (1.07±0.03 Sham. vs. 1.56±0.05 HF) ratios. The siRNA‐mediated silencing of ERK and CREB in NG108 cells ameliorate Ang II mediated upregulation of CAPON further supporting the involvement of MAP kinase in facilitating CAPON up‐regulation. Taken together, our results demonstrate the involvement of the ERK1/2 pathway in selective up‐regulation of CAPON expression, possibly via activation of CREB.

Architecture of the RNA polymerase II-Mediator core initiation complex.

The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.

Single-Molecule FRET for Dynamic Structural Biology: DNA Polymerase i Structure and Mechanism with Angstrom Precision

A key question in the function of DNA-processing enzymes is their mechanism for rapid location and binding of target sites in DNA. DNA polymerase I (Pol) is involved in high-fidelity DNA replication and repair, and must recognize gapped and flapped DNA substrates. Due to their transient and dynamic nature, the structures of these complexes and the recognition mechanism by which they are formed are largely unknown.Combining single-molecule Forster Resonance Energy Transfer (smFRET) with dye-position modelling, we can obtain absolute distance measurements with angstrom precision. We have used 120 such distances in a rigid-body docking approach to determine the structure of the single-nucleotide-gapped DNA-Pol binary complex, and that of the gapped-DNA substrate alone.In the complex structure, the DNA substrate exhibits a 120-degree bend. The upstream DNA is channeled into the Pol active site, and its position is in excellent agreement (RMSD < 3 A) with the position of short DNA fragments seen in crystal structures of Pol. The downstream DNA is partially melted and held in close proximity to F771, a residue important for strand displacement.The structure of the gapped-DNA substrate alone adopts a 20-degree bend and a twist. Our coarse-grained simulations show short excursions of the substrate to partially kinked conformations not accessible to duplex DNA. Together, these data are consistent with a two-step model whereby Pol recognizes and binds a partially kinked conformation of gapped DNA, which then further rearranges to the fully bent state seen in the complex structure.Finally, using our method of internalization by electroporation, we have observed single-molecule FRET signals corresponding to unbound and Pol-bound gapped DNA in live bacteria, confirming the physiological relevance of the proposed substrate and complex structures and the recognition mechanism.

저산소와 NO 조건에서 Mycobacterium smegmatis의 adenylyl cyclase 유전자 발현

In Mycobacterium tuberculosis H37Rv 16 adenylyl cyclase (AC) genes have been identified, while 10 AC genes have been found in non-pathogenic Mycobacterium smegmatis. Expression of 6 AC genes (MSMEG_0218, MSMEG_3243, MSMEG_3780, MSMEG_4279, MSMEG_4477, and MSMEG_6154) among 10 AC genes in M. smegmatis was increased when M. smegmatis was subjected to hypoxic growth conditions. On the other hand, only MSMEG_3780 and MSMEG_4279 were slightly induced in the presence of NO. The cAMP levels in cells and culture media were 450and 9.8-fold increased, respectively, when M. smegmatis was grown under hypoxic conditions relative to those grown aerobically. Intracellular levels of cAMP were increased 5.8-fold on the exposure to NO. The DevSR two-component system is known to be involved in the induction of many genes whose expression is induced under hypoxic conditions and in the presence of NO. Expression of 6 hypoxically induced AC genes was observed to be induced in a devR deletion mutant grown under hypoxic conditions, indicating that the induction of the 6 AC genes under hypoxic conditions is independent of the DevSR two-component system. In order to identify a trans-acting regulatory element that is pertinent in the hypoxic induction of MSMEG_3780, ligand-fishing analysis was performed using the upstream DNA of MSMEG_3780 and MSMEG_5136 protein was identified.

Characterization of DNA Methylation in Short Tandem Repeats in Human Genome

Background: DNA methylation plays a critical role in the DNA regulation, which occurs mainly at the C5 position of CpG dinucleotides, and can be affected by environmental pollutants. Short Tandem Repeats (STRs) are repeating sequences of 2-6 base pairs of DNA in human genome. DNA methylation in certain STRs has been shown to contribute to the development of various diseases, including cancer. However, the characterization of DNA methylation in STRs throughout the human genome remains largely unknown. Aims: To characterize STRs cross the human genome using the Illumina HumanMethylation450 BeadChip (450K array), and estimate the correlation of DNA methylation with STRs. Methods: To locate the STRs associated with 450K probes, we adopted the 450K array to examine DNA methylation level throughout the genome using data from 80 healthy pesticide applicators, and retrieved STRs in the 1050 bps DNA region which included both 500 bps upstream and downstream DNA sequences of each probe. For each probe, we obtained the STRs factors, including counts of CG and GC, STRs repeats size, copy number, and distance to the probe. Based on their repeat size (2 to 6), these probes were divided into 5 groups, and tested with Wilcoxon Rank-Sum Test to examine the methylation level of each group against the rest. For each group, we utilized linear regression analyses to determine the association of DNA methylation level with these STRs factors. Results: We found 29,528 probes with STRs, and 27,446 of them were kept for analysis. We observed significantly differences in methylation level of each repeat size group against the rest (P-values were 2.2E-16, 2.2E-16, 2.2E-16, 3.803E-05, and 2.2E-16, respectively) and DNA methylation level was associated with the CG and GC counts, distance between STRs and probes, copy number and the repeat size. Conclusion: The CG and GC counts, copy number, repeat size and the distance to probes of STRs may determine their DNA methylation level.

Anti-viral gene induction is absent upon secondary challenge with double-stranded RNA in the Pacific oyster, Crassostrea gigas

Oyster farming is one of the most important aquaculture industries in the world. However, its productivity is increasingly limited by viral disease and we do not yet have management practices, such as protective vaccination, that can control these disease outbreaks. Hence, in the current study we investigated the expression of known anti-viral genes in oysters (Crassostrea gigas) in response to primary and secondary encounter with a virus associated molecular pattern (dsRNA), and tested whether a common form of epigenetic gene regulation (DNA methylation) was associated with the expression of these anti-viral genes. Injection of dsRNA into the adductor muscle resulted in the rapid and transient expression of virus recognition receptors (TLR & MDA5), whereas several anti-viral signalling (IRF & SOC-1) and effector (PKR & viperin) genes were still up-regulated at one week post primary challenge (p < 0.05). This primary encounter with dsRNA appeared to deplete the immune system because anti-viral gene induction was absent in the gills when oysters were given a second injection of dsRNA at 1-week post-primary injection. The expression of DNA methylation genes (DNMT1, DNMT3b, TDG, TET2) and DNA methylation profiles up-stream of specific anti-viral genes (STING, SOC-1 & Viperin) did not change in response to dsRNA injection (p > 0.05). These results collectively suggest that C. gigas does not have an enhanced anti-viral gene response (immune-priming) to secondary dsRNA challenge and that the sustained up-regulation of anti-viral signalling and effector genes following primary challenge is unlikely to be associated with upstream DNA methylation levels.