Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes. The ability to translate genetic messages into functional proteins is an absolute necessity for life forms on our planet, and is achieved by the largest known enzyme, the ribosome. The mammalian ribosome is a 4 MDa complex of 4 catalytic RNA molecules and more than 80 proteins that uses amino-acid charged transfer RNAs (tRNAs) to decode the genome via the intermediary of the messenger RNAs (mRNAs) . The effSince a mammalian cell contains up to 10 million ribosomes, e.g. , 8, theiIn human and mouse, the 47S rRNA is encoded on some 200 haploid gene copies that are organized in direct repeats on the short arms of five chromosomes , 14, 15.in vivo. Perhaps most importantly, the data reveal the existence of an Enhancer Boundary Complex formed by CTCF and Cohesin and three or four phased nucleosomes lying immediately adjacent to the Spacer Promoter-Enhancer repeat unit and an arrested RPI elongation complex. We find that this boundary complex is the only significant site of active histone modifications in the whole 45kbp rDNA repeat. Strikingly, the Enhancer Boundary Complex not only clearly delimits the functional rRNA gene unit, but is stably maintained after inactivation of RPI transcription and the replacement of UBF by nucleosomal chromatin. Our data potentially places the Enhancer Boundary Complex as the key entry point for the remodelling complexes that activate rDNA transcription and defines the poised state of rDNA chromatin.Transcription of the rDNA is stimulated in response to nutrients, growth factors and a wide range of cellular stresses, and both Rrn3 and UBF are direct targets of growth regulatory pathways , 18–20. In order to better understand the factors that determine rRNA gene activity, we first needed to establish high resolution, low noise interaction maps for the components of the RPI transcription machinery. We achieved this using a combination of Chromatin Immuno-Precipitation, massively parallel DNA sequencing (ChIP-Seq) and normalization to sequence coverage obtained on control (input) DNA, see in vitro data showing that Rrn3 is released sometime after initiation -uridine (PerkinElmer) to the culture medium and incubation for a further 3h. RNA was extracted using Trizol (Life Technologies) according to the manufacturer’s protocol, analyzed by gel electrophoresis and fluoroimaging  and RNA species quantitated by scintillation counting as previously described [As previously described , cells were initially plated in 6 cm petri dishes (0.8x10out 4-HT . CultureDS-PAGE)  loading escribed , 73.Rrn3fl/fl-, and Ubffl/fl/ER-Cre+/+/p53-/- and matched Rrn3+/+-, and Ubf+/+/ER-Cre+/+/p53-/- MEFs were treated with 4-HT as described above, and 48h later each culture was replated in duplicate at dilutions of 10 000, 50 000, 100 000 cells per 60 mm petri. 144h post 4-HT treatment, petri dishes were fixed for 5 mins with 4% paraformaldehyde/PBS and stained with 0.05% crystal violet in distilled water (filtered) for 30 mins. Dishes were then rinsed 3 times with water and left inverted to dry. Crystal violet staining was quantified by adding 1ml of methanol to each dish to solubilize the dye, the methanol recovered and its optical density at 540nm determined after appropriate dilution.Rabbit polyclonal antibodies against mouse UBF, RPI large subunit (RPA194/Polr1A), TTF1, TAF1B (TAF68) and RRN3 were generated in the laboratory. The UBF, RPI and TTF1 antibodies have been previously described . Rabbit 6 cells in IP Buffer  overnight at 4°C. The antibody slurry was prepared with 50 μl A-, 50 μl G-Dynabeads and 60 μg.ml-1 antibody per IP. Immunoprecipitated chromatin was treated with RNaseA and the DNA isolated using 2% Na SDS and 2mg.ml-1 Proteinase-K. Two or more biological replicates were analyzed for each antibody.Cells were fixed with 1% formaldehyde for 8 min at room temperature. Nuclei were isolated using Lysis Buffer , transferred to Sonication Buffer  and sonicated  for 30 cycles of 30 sec on / 30 sec off at high intensity. Each immunoprecipitation (IP) was carried out on the equivalent of 50 x 10ChIP DNA samples were quality controlled by qPCR before being sent for library preparation and 50 base single-end sequencing on an Illumina HiSeq 2000 (McGill University and Génome Québec Innovation Centre). For qPCR analysis, reactions (20 μl) were performed in triplicate using 2.5 μl of sample DNA, 20 pmol of each primer, and 10 μl of Quantitect SYBR Green PCR Master Mix (QIAGEN). Forty reaction cycles of 10 s at 95°C and 30 s at 58°C were carried out on a Multiplex 3005 Plus (Stratagene/Agilent). The amplicon coordinates relative to the 47S rRNA initiation site (BK000964v3) were as follows: IGS3, 42646–42903; SpPr, 43089–43253; Tsp, 43267–43421; 47SPr, 45133–40; 47S, 159–320; ETS, 3078–3221; 28S, 10215–10411; T1–3, 13412–13607. Data was analyzed using the MxPro software (Agilent). The relative occupancy of each factor was determined by comparison with a standard curve of amplification efficiency for each amplicon using a range of input DNA amounts and generated in parallel with each qPCR run.DNase-Seq analysis of MEFs was carried out following the published protocol , the onlJ = smoothed base coverage, j = aligned coverage and n = base position).The data analysis pipeline was developed to take best advantage of the sequencing depth achievable across the rDNA. We noted that sequence coverage was strongly, but reproducibly biased by the underlying DNA sequences regardless of sample type and devised a simple approach to correct for this bias. Briefly, the raw sequence data from experimental and input DNA samples was checked for quality using FastQC version 0.11.4 . The data was then trimmed using Trimmomatic version 0.33  and the Jnorm was calculated for each base position using AWK in Ubuntu as:JChIP = smoothed sample DNA coverage, and Jinput = smoothed input DNA coverage.The data was then converted to reads per million (RPM) and the sample DNA coverage normalized to the input DNA coverage. The normalized sample data The resulting normalized BED file was converted to BEDgraph format and visualized using IGV .www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-5839.The ChIP-Seq and DNase-Seq data have been deposited in the ArrayExpress database at EMBL-EBI  membrane and nucleosome ladders detected by DNA hybridization using the same probes as for the psoralen analysis.After trypsinizing and washing with PBS the cells were resuspended in buffer A  plus 0.25 M sucrose, 1 mM EDTA, 0.1 mM EGTA, 0.5 mM spermine, 0.15 mM spermidine, 0.1 mM PMSF and protease inhibitors. Triton X100 was added to final concentration 0.25% and the cells homogenized in a Dounce homogenizer using 10 piston strokes and centrifuged 5 min at 2000 rpm. The pellet was washed once by resuspending in buffer A plus 0.34M glucose and then centrifuged for 5 min at 2000 rpm. It was then resuspended in 1 ml buffer A without sucrose, recentrifuged and resuspended in buffer A containing 60 units MNase per 10Rrn3wt/wt-, Rrn3fl/fl-, and UBFfl/fl/ER-Cre+/+/p53-/- MEFs were grown on poly-lysine coated coverslips and treated with 4-HT to induce full inactivation of floxed genes (72h time point), and gene inactivation monitored in parallel by genotyping and measurement of protein levels. 1mM 5-ethynyl uridine  was added to the culture medium and cultures incubated for 1h at 37°C. Cells were then washed with PBS, fixed in 4% paraformaldehyde (PFA) in PBS for 15 minutes and permeabilized with 0.5% Triton in PBS for 5 minutes. EU incorporation was revealed following the manufacturers protocol . Cells were then incubated with the indicated primary antibodies for 1h in PBS plus 5% BSA or Maxblock (Active Motif) and subsequently with AlexaFluor 568/647 conjugated anti-rabbit or -mouse secondary antibodies (Molecular Probes / Thermo Fisher) and counterstained with DAPI. After mounting in 50% glycerol/50% 0.2 M Na-glycine, 0.3 M NaCl, image stacks were acquired using a Leica SP5 II scanning confocal microscope and formatted with Volocity software (Perkin Elmer Improvision).All animal care and animal experiments were conducted in accordance with the guidelines provided by the Canadian Council for Animal Protection, under the surveillance and authority of the institutional animal protection committees of Laval University and the CHU de Québec. The specific studies described were performed under protocol #2011–054, 2014–100, and 2014–101 examined and accepted by the “Comité de protection des animaux du CHU de Québec”. This ensured that all aspects of the work were carried out following strict guidelines to ensure careful, consistent and ethical handling of mice.S1 FigThe normalized map profile of UBF binding across the full rDNA repeat unit and the percent G+C content of the rDNA calculated over 50bp non-overlapping sliding windows . A scale(PDF)Click here for additional data file.S2 FigRrn3-Tif1a+/- mice either before or after (BL6) backcrossing to the C57BL/6 background. B) Example images of E3.5 embryos obtained from the matings. C) Double heterozygous Ubf+/-/Rrn3-Tif1a+/- mice are both viable and generated with the expected Mendelian frequency in matings between Ubf+/-and Rrn3-Tif1a+/- mice. It was previously found that homozygous deletion of the mouse Fibrillarin (Fbl), RPI second largest subunit (Rpa135/Rpo1-2/Polr1b) or Upstream Binding Factor (Ubf/Ubtf) genes all cause developmental arrest during the cleavage divisions and well before the blastula stage [Rrn3-Tif1a-/- mouse embryos were reported not to arrest development until E9.5 at which point they clearly displayed axis formation, tissue differentiation and the beginnings of organogenesis [Rrn3-Tif1a conditional cell lines carrying the Tif1aflox allele created by Yuan et al., we also generated mice carrying the same Rrn3-Tif1a-null allele studied by these authors. When progeny from the Rrn3-TIF1A+/- mice were analyzed we found that null embryos in fact arrested during the cleavage divisions as un-compacted morulae. The same result was obtained after extensive backcrossing to C57BL/6, the mouse strain used in the original publication. Since our Rrn3-Tif1a-/+ mouse lines were extensively backcrossed to remove any transgenes used in recombining the Rrn3-Tif1aflox allele, we presently have no explanation for the discrepancy with the previous study. We concluded that, despite previous data to the contrary, Rrn3-TIF1a, like UBF and RPI, is essential in mouse soon after the normal onset of rRNA gene activity. This strongly argues that the mouse Rrn3 is indeed an essential and non-redundant part of the RPI transcription machinery. It further suggests that the fertilised oocyte does not contain a significant amount of maternal Rrn3 message or protein or, as has been suggested for UBF [A) Genotyping of embryos and live pups from matings of la stage , 95. Thila stage , 96. In ogenesis . By E9.5ogenesis , 47. Thu for UBF , that th(PDF)Click here for additional data file.S3 FigRrn3fl/fl-, and UBFfl/fl/ER-Cre+/+/p53-/- (flox/flox) and matched Rrn3+/+-, and UBF+/+/ER-Cre+/+/p53-/- (wt/wt) MEFs. Cultures were standardly treated with 4-HT, replated 48h later and crystal violet staining of resulting cell colonies determined at 144h post 4-HT treatment. The assay showed that around 4% of Rrn3fl/fl cells, but less than 1% of Ubffl/fl cells, were able to form colonies and hence retained a functional Rrn3 or Ubf after the 4-HT treatment.A) and D) show maps of the wild type (wt), conditional (flox) and deleted (Δ) Rrn3 and UBF gene alleles. B) and E) show typical time courses (Hours post 4-HT) of Rrn3 and UBF gene deletion determined by PCR genotyping (Genotype), in parallel with corresponding protein levels for each factor. C) The anti-Rrn3 antibody generated in our laboratory and used for ChIP analyses revealed a single endogenous Rrn3 polypeptide that corresponded in mobility with the known Rrn3 species (Genbank XP_156394 and NP_001034610) expressed by transient transfection (Exogenous). F) Time courses of relative 47S rRNA synthesis rates in conditional and wild type Rrn3 and UBF MEFs post 4-HT treatment as determined by metabolic labeling . G) Colo(PDF)Click here for additional data file.S4 FigThe normalized ChIP-Seq profiles for TTF1 before and after (72h post 4-HT) Rrn3 or UBF gene deletion and compared to the same data for RPI. The data are similar to those in Figs (PDF)Click here for additional data file.S5 FigA) The mapped position of the hybridization probes relative to the start of the 47S rRNA are indicated above a diagram of the rDNA repeat. B) The profiles of increasing MNase cleavage of chromatin from UBF conditional MEFs before or after (72h post 4-HT) inactivation of the gene, and C) from Rrn3 wild type and conditional (floxed) MEFs after 72h of treatment with 4-HT. The total DNA cleavage ladders were revealed by ethidium bromide (EtBr) staining, and the cleavage ladders within the IGS and the 47S gene body were revealed by hybridization with the corresponding probes shown in A). The positions of mono- (1), di- (2), etc nucleosomes are indicated.(PDF)Click here for additional data file.S6 FigThe RPM profiles for H3K9ac, H3K36me3 and H3K27ac realigned from public data (ENCODE GSE32218) over the Promoter and Enhancer region of the mouse rDNA are shown in comparison with the CTCF and H3K4me3 enrichment profiles established in the present study.(PDF)Click here for additional data file.S7 FigRrn3fl/fl- and Rrn3+/+/ER-Cre+/+/p53-/- and Ubffl/fl- and Ubf+/+/ER-Cre+/+/p53-/- MEFs. The data show the results for Histone H3, H3-K4me3, CTCF and either Rrn3 or UBF, the factor targeted by deletion.A) Positions of QPCR amplicons relative to rDNA sequence motifs. B) and C) ChIP-QPCR analyses at 3 days post 4HT treatment respectively for (PDF)Click here for additional data file.


It is increasingly clear that DNA viruses exploit cellular epigenetic processes to control their life cycles during infection. This review will address epigenetic regulation in members of the polyomaviruses, adenoviruses, human papillomaviruses, hepatitis B, and herpes viruses. For each type of virus, what is known about the roles of DNA methylation, histone modifications, nucleosome positioning, and regulatory RNA in epigenetic regulation of the virus infection will be discussed. The mechanisms used by certain viruses to dysregulate the host cell through manipulation of epigenetic processes and the role of cellular cofactors such as BRD4 that are known to be involved in epigenetic regulation of host cell pathways will also be covered. Specifically, this review will focus on the role of epigenetic regulation in maintaining viral episomes through the generation of chromatin, temporally controlling transcription from viral genes during the course of an infection, regulating latency and the switch to a lytic infection, and global dysregulation of cellular function. The words of the Nobel Laureate Bob Dylan “Because something is happening here, but ya don’t know what it is, do you Mister Jones?” could very well apply to our understanding of the role that epigenetics plays in regulating viral life cycles. While there is lots of evidence that epigenetics plays a critical role in the regulation of virus infections, in many cases the details of the role have not been completely elucidated. While there are likely to be many factors contributing to our relative lack of understanding of the details of viral epigenetic regulation, one of the major contributors is the inherent complexity of epigenetic regulation. There are presently five well-characterized processes that are thought to make up epigenetic regulation including: DNA methylation, nucleosome positioning, histone variants, histone modifications, and regulatory RNA . NeedlesTwo relatively recent technological advances have substantially contributed to our understanding of epigenetic regulation. The first, chromatin immunoprecipitation (ChIP) ,2,3 madeDNA methylation is generally thought to be associated with repression of transcription . NucleosDNA viruses, which exist as nuclear episomes , are very likely to exploit epigenetic mechanisms to regulate biological activities during their life cycle for a number of reasons. First, these viruses typically exist as chromatin either throughout their life cycle or at least when present in the nucleus of a cell. Second, the viruses usually utilize a number of cellular regulatory factors, co-factors, and enzymes to accomplish gene expression and its regulation. Finally and importantly, these viruses require epigenetic or similar processes to allow the different viral genome states necessary to complete an infection to coexist. Many DNA viruses are known to coordinately perform divergent biological processes within the same infected nucleus of a cell. For example, at later times in a typical infection by a DNA virus one might expect to find viral genomes replicating, transcribing one or more distinct genetic units, and also undergoing encapsidation to form new virus particles. Each one of these distinct biological processes would be expected to be tightly controlled relative to the other processes and yet would all be based upon the same DNA sequence of the virus. Differences in one or more epigenetic process listed above would allow for the coexistence of the different functional forms of the viral genome and maintain the necessary tight control. The viruses may also epigenetically dysregulate host cell biology in order to enhance their own biological processes. For example, they may stimulate the synthesis of factors involved in DNA replication or transcription or alternatively inhibit pathways of immune surveillance.This review will focus on DNA viruses which exist as episomes for at least part of their life cycle and will update a prior publication  with datThe polyomaviruses are a family of small (<10 kb) double-strand closed-circular DNA tumor viruses ,15. BaseInterestingly, epigenetic regulation of SV40 was first identified in the late 1970s, although the changes in nucleosome positioning around the SV40 promoter ,23,24,25In order to address these two issues we have taken a three-stage approach. In the first stage, we have determined the global location of nucleosomes in SV40 chromatin isolated at different times in infection and from virus particles using micrococcal nuclease to prepare sequencing libraries and next generation sequencing (NGS). This recently published work  will be SV40 chromatin was analyzed for the location of all nucleosomes on the SV40 genome using micrococcal nuclease to generate nucleosome-sized fragments of DNA. Because of the large number of reads that can be obtained from SV40 chromatin, the data was analyzed without having to bin the reads, which allowed for mapping to the genome with essentially single base resolution. Based upon this analysis of global nucleosome positioning, a model was proposed in which nucleosomes play a critical role in regulating transcription during the SV40 life cycle. In chromatin from virus particles, nucleosomes were observed to be present at nt 5223 and nt 363 effectively covering the major early and late start sites for transcription. In contrast, in chromatin from minichromosomes isolated at 30 min and 48 h post-infection, nucleosomes were found at nt 5119 and nt 212, exposing the early and late start sites. The inhibition of transcription by the nucleosomes blocking the early and late transcription start sites was further confirmed by analyzing the SV40 mutant cs1085. This mutant lacks T-antigen binding Site I and as a consequence does not downregulate early transcription. If the nucleosome located at nt 5223 was involved in blocking early transcription one would expect to see that, in the mutant, the nucleosome would be absent. Interestingly, both the nucleosome at nt 5223 and the one located at nt 363 were substantially reduced on the SV40 chromatin in the mutant. While this was consistent with the expectation for the early transcription it was unexpected that the late start site would also be affected . This reIn addition to the changes in the promoter/regulatory region of SV40, changes in the nucleosome location around the 5′ end of the early splice site, nt 4885, and near the termination of transcription, nt 2800–2900 were also observed in this study. The significance of these changes were not further explored in the publication . NucleosIn the second stage of our studies we are mapping onto the SV40 genome the location of nucleosomes present in SV40 minichromosomes and virions, which contain each of the histone modifications described above. From this analysis, we expect to answer a number of questions related to nucleosome organization and regulation. First, we will determine whether there are preferred locations on the genome for nucleosomes containing each of the specific tail modifications. If, as expected, preferred locations are found for one or more of the histone modifications, we will then characterize the relationships between each preferred location, the underlying DNA sequences, and the location of all nucleosomes as determined in our recent publication . This wiThe third stage of these studies is based upon the strategy shown in None of the other polyomaviruses—such as John Cunningham (JC) virus, BK virus, or Merkel cell virus—have been studied to the same extent as SV40 with respect to epigenetic regulation. Since the viruses are similar to SV40 they are likely to use epigenetics in the same way to regulate infection and the various functional forms of the viral minichromosome. There has been recent progress in investigating the epigenetics of these viruses with respect to miRNAs, histone modification, and the epigenetic reader, BRD4, primarily in explaining how these viruses may dysregulate infected cells. Many if not all polyomaviruses contain a miRNA in the late message strand which can be cleaved to yield a miRNA which inhibits expression of the early genes (reviewed in ). This mA role for global dysregulation of histone modification in cancers following transformation by Merkel cell polyomavirus has also been recently described . A stronBRD4 is thought to serve as a scaffold for activating transcription by binding to acetylated lysines present on histones in chromatin regions available for transcription and also binding to transcription factors necessary to initiate transcription. The function of BRD4 in the reactivation of JC virus in glial cells was investigated using either a BRD4 inhibitor or by mutating lysines targeted for acetylation by BRD4 on the transcription factor NF-κB p65 . Since bThe adenoviruses are a family of double-strand linear DNA viruses approximately 35 KB in length . Within The recent adenovirus publications focusing on epigenetics have addressed epigenetic effects on the host cell as a consequence of infection ,45,46,47The global effects on cellular transcription and histone modifications was also analyzed using the adenovirus early regulatory protein E1A wild-type and mutant viruses in the context of infection . In thisOur understanding of the epigenetic role of adenovirus protein VII was recently extended in a publication combining biochemistry, cell biology, and mass spectrometry . ProteinThe human papillomaviruses (HPV) are a family of closed-circular double-stranded DNA viruses with an approximately 8000 bp genome . BecauseBecause HPV is a very important human pathogen causing a number of different types of cancer there is much more known about the role of epigenetics in the transformation of normal cells and in cancers. This role has been recently reviewed and will not be discussed in detail . Not surDespite the difficulties associated with studying epigenetic regulation in HPV lytic infections there has been some notable recent work ,56,57,58Hepatitis B virus (HBV), a significant human pathogen, exists as a 3.2 kb single strand of DNA in the virus particle and as a covalently closed circular DNA (cccDNA) in the infected nucleus of a cell . Not surUsing a modified ChIP-Seq procedure with enhanced sensitivity, the location of nucleosomes containing specific histone modifications and RNA PII were mapped to the HBV genome in cccDNA chromatin . These sSimilarly, it seems very likely that there will be further studies on the role of the HBV protein HBx which appears to function as an epigenetic regulator to dysregulate a number of cellular genes as well as regulate the virus (reviewed in ). HBx apHerpesviridae are a family of relatively large double-strand DNA viruses approximately 160 kb in size . BecauseThere are two aspects of the life cycle of the typical herpesvirus which are particularly relevant to epigenetics . The firDepending upon the cellular environment, infection by herpesviruses can result in either a lytic infection, in which, the infected cell is ultimately killed, or a latent infection in which the virus exists as an episome (minichromosome) stably in the nucleus of the infected cell. To exist as a stable episome, the virus must be able to coordinate its own replication with that of the cell and to segregate itself along with the chromosomes of the cell following replication. This aspect of regulation is generally controlled by a viral protein, which interacts with the cellular chromosomes to tether the newly replicated virus for segregation along with the chromosome. Each of the viruses has its own multifunctional protein to accomplish this. Finally, there must be a way for the virus present in a latent infection to reactivate to generate a lytic infection usually as a result of a change in the cellular environment. Epigenetics is thought to contribute to all of these processes.KSHV has been extensively studied because of its ability to cause cancer in AIDS patients. There are recent reviews that the reader is encouraged to consult for details on the role of epigenetic regulation in this virus ,70,71. EBoth HSV and EBV seem to be epigenetically regulated similarly to KSHV although the viral proteins responsible for the regulation are unique to the other viruses. In lytic infections, HSV viral DNA is chromatinized within 1–2 h of infection and the chromatin is acted upon by transcription factors from the virus and cell along with chromatin remodeling factors to initiate immediate–early gene expression . TheOur understanding of epigenetic regulation is poised for significant advancement in the next few years. One way, this will be occurring is by moving away from interpretation by association to a more mechanistic view of regulation. As described above, much of what we know about epigenetic regulation is based upon studies, which can only show that a particular epigenetic regulatory change is associated with an activating or repressive biological event. For example, a comparison between the chromatin structure of a silent gene and its active counterpart shows the changes associated with activation. This has led to some of the general regulatory rules described above, such as the observation that methylated H3K4 is associated with gene activation. Recent studies with ChIP-Seq have shown that at best this is an overly simplistic view of the role of histone modification. What is lacking in many cases is exactly how and why a particular modified histone or similar epigenetic change contributes to a biological event like the activation or repression of a specific gene. This is made more evident in the studies described in which the locations of various histone modifications are mapped to nucleosomes in chromatin. While the technology to address epigenetic mechanisms is probably available at this time, it is likely that new strategies will need to be developed to study mechanisms. This may entail new model systems, which can be exploited in vitro.A second area where progress is likely to occur is in better understanding the relationship between the location of nucleosomes containing specific histone modifications and the binding of regulatory factors. As indicated above, some of the results to date are paradoxical. For example, in SV40, T-antigen binding to Site I results in the introduction of a nucleosome which is located essentially in the same position as Site I. It is difficult to imagine how both T-antigen and the nucleosome can occupy the same location in the same chromatin given our knowledge of how each binds to DNA. Similarly, in Hepatitis B Virus, RNAPII binds to its promoter at a site, which is overlapped by a nucleosome containing H3K4me1. Again, it is not clear how the RNAPII can bind to its target DNA if the site is also being occupied by a nucleosome.Studies on the epigenetic regulation of DNA viruses will continue to be of significant interest for a number of reasons. First, their dependence on host cell proteins and processes for their molecular biology means that they can serve as useful models for cellular epigenetic events. More importantly, because of their general small size and ease of genetic and other forms of manipulation, they may serve as the chromatin substrates for the in vivo and in vitro studies needed to unravel the mechanisms involved in epigenetic regulation. Importantly the relatively small size of these viruses and the relatively large amounts of chromatin that can be obtained by infection, makes ChIP-Seq and similar NGS analyses cost effective. In addition, the high resolution, which can be obtained from the analysis of these viruses, will also be important for the mechanistic studies. As indicated above with SV40 we can obtain single base resolution in our ChIP-Seq studies. With this high resolution, it is possible to further characterize the small shifts in nucleosome location, which our group and others have recently observed.A better understanding of the mechanisms responsible for the introduction of epigenetic information and, correspondingly, the mechanisms responsible for reading this information may possibly lead to novel treatments for infections by these viruses many of which are serious human pathogens. Characterizing the mechanisms that underlie each form of epigenetic regulation and dissecting the complex interplay between viral DNA sequences may identify viral proteins and cellular contributors that will lead to new targets for therapeutic intervention.


Deinococcus bacteria requires the radiation‐stimulated cleavage of protein DdrO by a specific metalloprotease called IrrE. DdrO is the repressor of a predicted radiation/desiccation response (RDR) regulon, composed of radiation‐induced genes having a conserved DNA motif (RDRM) in their promoter regions. Here, we showed that addition of zinc ions to purified apo‐IrrE, and short exposure of Deinococcus cells to zinc ions, resulted in cleavage of DdrO in vitro and in vivo, respectively. Binding of IrrE to RDRM‐containing DNA or interaction of IrrE with DNA‐bound DdrO was not observed. The data are in line with IrrE being a zinc peptidase, and indicate that increased zinc availability, caused by oxidative stress, triggers the in vivo cleavage of DdrO unbound to DNA. Transcriptomics and proteomics of Deinococcus deserti confirmed the IrrE‐dependent regulation of predicted RDR regulon genes and also revealed additional members of this regulon. Comparative analysis showed that the RDR regulon is largely well conserved in Deinococcus species, but also showed diversity in the regulon composition. Notably, several RDR genes with an important role in radiation resistance in Deinococcus radiodurans, for example pprA, are not conserved in some other radiation‐resistant Deinococcus species.The extreme radiation resistance of  The underlying mechanisms are not fully understood, but several factors that are crucial for this extraordinary tolerance have been described, including limitation of oxidative protein damage, repair of massive DNA damage and an efficient SOS‐independent damage response pathway . Several highly induced novel genes were also identified and designated ddrA to ddrP and pprA, and the contribution to radiation tolerance in D. radiodurans was demonstrated for pprA and ddrA to ddrD  regulon  but also contain several genes that are not found in each species   , which contains the HEXXH motif suggestive of a zinc‐binding catalytic active site. The crystal structure of recA and other genes in Deinococcus was obtained only recently. First, following the genome‐wide identification of transcription start site (TSS) positions in D. deserti it was found that the RDRM sites are located in or very close to the promoters of radiation‐induced genes, indicating that an RDRM‐binding protein would function as a repressor  can activate DdrO cleavage by 22.1Deinococcus was grown at 30°C with shaking (150 rpm) in 10‐fold diluted tryptic soy broth (TSB/10) supplemented with trace elements  at 37°C unless stated otherwise. Antibiotics were used at the following concentrations for D. deserti: streptomycin, 10 μg ml−1; kanamycin, 10 μg ml−1; and for E. coli: kanamycin, 50 μg ml−1; ampicillin, 100 μg ml−1.The strains and plasmids used in this study are listed in Table 2.2C (from pET22b) and removal of the polyhistidine tag from recombinant IrrE was performed as described previously  was amplified from D. deserti  cells freshly transformed with pET SUMOddrO were grown overnight to saturation in 10 ml of LB containing kanamycin. This preculture was then diluted in 1 L of LB medium with kanamycin (in a 3‐l flask) and grown with shaking (160 rpm) at 37°C. At OD600 of 0.6–0.7, IPTG and L‐arabinose were added at a final concentration of 0.1 mmol/L and 0.2%, respectively, and the cells were then grown at 17°C for 17 hr. The induced cells were harvested by centrifugation . The cell pellets were then resuspended in 20 ml of Buffer 1 , and frozen at −20°C. For purification, cell suspensions containing the recombinant fusion protein were thawed and 100 μl of 10 mg ml−1 DNase I [DNase from bovine pancreas in 50 mmol/L MgCl2 (Sigma)] and 25 μl of anti‐protease cocktail (Sigma P8849) were added to each tube. All purification steps were done at 4°C. Cells were broken by a cell disruptor  at 2 kbar and the soluble extracts were then recovered after centrifugation at 10,000 g for 10 min at 4°C and ultracentrifugation at 150,000 g for 45 min at 4°C. The supernatant was injected at a speed of 1 ml min−1 onto HisTrapTM HP columns (1 ml) , previously equilibrated in Buffer 1 supplemented with 20 mmol/L imidazole. A step gradient of imidazole  was used for elution (20 ml was used for each fraction except for the 500 mmol/L fraction where 3 ml was used). Eluted fractions were analyzed by SDS‐PAGE to verify presence and purity of the protein. The polyHis‐SUMO‐tagged DdrOC fusion protein was found in the 500 mmol/L imidazole fraction. The polyHis‐SUMO tag was removed from the fusion protein by incubation with SUMO protease. The pET28b derivative encoding His‐tagged SUMO protease was kindly provided by Mossessova & Lima (C (in the flow‐through fractions) from uncleaved fusion protein and SUMO protease (both bound to the column). Protein concentrations were measured using a NanoVue spectrophotometer. The mass of the purified proteins was verified by electrospray ionization mass spectrometry as described previously  and DdrOa & Lima . The inc2.3C was assayed in vitro by incubating 20 μmol/L DdrOC with 5 μmol/L IrrE in a reaction mixture containing 50 mmol/L Tris‐HCl pH 7.4, 0.15 mol/L NaCl, in presence or absence of one of several metal ions  in a 20 μl final volume. All the reaction mixtures were incubated at 37°C for 15 min. After quenching by addition of NuPAGE LDS sample buffer (Invitrogen) and heating at 95°C for 10 min, the reaction products were subjected to a Novex NuPAGE 10% Bis‐Tris Gel (Invitrogen). Proteins were visualized by staining with Imperial protein stain (Pierce).Purified IrrE was first incubated with EDTA to remove divalent metal ion and inactivate the protease activity. For this, 20 μl of a 20 mmol/L stock solution of EDTA was added to 2 ml of 40 μmol/L of IrrE. Next, to remove the EDTA, 700 μl of EDTA‐treated IrrE was dialyzed in 350 ml of buffer  for 2 hr and then overnight in fresh buffer. Then cleavage of purified DdrO2.4600 0.4). Then 250 μmol/L of ZnCl2 or other metal chloride was added to the 50‐ml culture, and incubation was continued for 10 min. Preparation of cell extracts, protein separation and immunoblot analysis to analyze DdrO cleavage were performed as described previously , ddrA (Deide_09150) or ddrD (Deide_01160), or the intergenic region of the divergent ddrOC (Deide_20570) and ddrQ (Deide_20580) genes were amplified by PCR  and DdrO were present, either IrrE and DdrO were preincubated for 15 min prior to the incubation with DNA, or DdrO and DNA were incubated prior to addition of IrrE and another 15 min incubation. After adding 2 μl of 10X Orange Loading Dye, the samples were loaded onto a prerun 5% polyacrylamide gel and run for 2 hr at 70 V in TAE buffer at 4°C. The gel was then incubated for 30 min in ethidium bromide (0.5 μg ml−1) and DNA was visualized using UV.The promoter region of PCR Fig. . DNA bin2.6Growth, irradiation, RNA isolation, cDNA library construction, Illumina sequencing and RNA‐Seq analysis for strains RD42 and RD62 were performed as described previously for strain RD19 , concentrated 100X, and half of the sample was irradiated on ice to 3 kGy gamma rays  at the now dismantled radiation facilities CIGAL . The other half of the sample was not irradiated but otherwise treated in the same manner. After irradiation, cells were diluted 100X in fresh growth medium and recovered for 1 hr, harvested by centrifugation, and washed twice with 50 mmol/L Tris‐HCl pH 7.4. Cell pellets were rapidly frozen in liquid N2 and stored at −80°C. Cells were solubilized in lithium dodecyl sulfate sample loading buffer (Invitrogen) as previously described  of these genes. A conserved 17‐bp palindromic motif was searched in these extracted sequences using MEME , a platform for microbial genome annotation and comparative analysis . Together, the data do not support the previously published claim that the peptidase activity of IrrE is strictly dependent on Mn2+, but instead support that IrrE is a zinc peptidase.We previously demonstrated that the in vitro cleavage of DdrO by IrrE is inhibited in presence of the metal chelator EDTA. Here, we tested several divalent metal ions for their capacity to restore the peptidase activity of apo‐IrrE in vitro. DdrO cleavage was found in the presence of Zn+ Figure a. The add Figure b. We alsd Figure . Zn2+, ME Figure c. As forE Figure , another3.2Deinococcus to radiation is unknown. In vitro, adding zinc ions to apo‐IrrE is sufficient to induce protease activity and DdrO cleavage. The in vivo protease activity of IrrE may similarly depend on the availability of the metal cofactor. In cells, free zinc levels are low because nearly all zinc ions are bound to proteins and other molecules. IrrE is a low‐abundance protein in D. deserti   to short biotin‐labeled RDRM‐containing DNA fragments, probably because the experimental conditions were not optimal. However, we did demonstrate repression of an RDRM‐containing promoter by DdrO in E. coli  we did not observe binding of s Figure b.The IrrE‐mediated cleavage removes the last 23 residues from DdrO, which probably abolishes DdrO dimerization and stable DNA binding  , and the ddrOC deletion mutant RD62 (ΔddrOC) by transcriptomics using RNA sequencing. It should be noted that D. deserti contains two DdrO homologs, DdrOC and DdrOP3 encoded by the chromosome and plasmid P3, respectively. These proteins share 84% identity and both are cleaved by IrrE in radiation‐exposed cells , in line with results that showed that DdrOC is the major DdrO protein in D. deserti in terms of abundance  and compared with the irradiated ΔirrE strain RD42 (two comparisons IRvs42IR). In addition, if the lower amount of repressor DdrO in the ΔddrOC strain (only DdrOP3 is present) indeed causes partial derepression of the target genes, somewhat higher expression of RDR regulon genes may be observed in the non‐irradiated ΔddrOC strain RD62 compared with non‐irradiated wild‐type and ΔirrE strains (two comparisons 62NIvsNI).Each strain was analyzed after growth under standard condition  and after exposure to gamma radiation (IR). RNA sequencing data were thus obtained for six samples: NI and IR for the wild‐type, Δtkt , and most showed partial derepression in the non‐irradiated ddrOC mutant, confirming their predicted regulation by IrrE/DdrO , perhaps because their induction involves (an) additional regulatory protein(s) besides IrrE/DdrO. An RDRM site has been previously found upstream of most of the highly induced IrrE‐regulated genes , but not upstream of Deide_01090, Deide_01100, Deide_18350, Deide_09130, Deide_11446 and Deide_21600  for Deide_09130 at the first nucleotide of its ATG start codon (leaderless transcript). Transcription from this TSS was not radiation‐induced. These data suggest that Deide_09130 is not part of the operon with ddrA‐Deide_09148 and that its apparent induction is caused by read‐through from the highly induced ddrA promoter. Similarly, the induction of Deide_01100 might be caused by read‐through from the Deide_01090 promoter. Only few reads were mapped to Deide_01100 compared to Deide_01090. However, as no TSS for Deide_01100 was found, this gene might be in operon with Deide_01090, although these genes are separated by 237 base pairs.Read counts and results of the differential expression analyses for all genes are presented in Tables rO Table . Table 2ddrOC gene is highly induced in irradiated wild‐type strain. Obviously, ddrOC is not expressed in the ΔddrOC strain, because the entire ddrOC and 43 bp upstream of this gene are deleted in this strain. However, the TSS of ddrOC at position ‐131 of the translation initiation codon is still present in ΔddrOC. Differential expression analysis using the number of reads that begin at this TSS showed a more than 25‐fold higher expression in irradiated and non‐irradiated ΔddrOC compared with non‐irradiated wild‐type and ΔirrE and with irradiated ΔirrE (Table gyrA (induced at the TSS at ‐83 but not at the TSS at ‐139) and for the cinA‐ligT‐recAC operon (induced at the TSS at ‐9 but not at the TSS at ‐64), correlating with the position of the repressor‐binding motif RDRM. Highly induced IrrE‐dependent expression was also observed at a TSS that is located at ‐181 upstream of tkt (Deide_00600) within a previously identified RDRM . This could mean that the TSS at ‐181 of may not correspond to a second TSS for tkt but that it may be the TSS for a novel short transcript. No match of this transcript was detected with sequences present in the Rfam database of RNA families. Further work is necessary to characterize this radiation‐induced transcript starting at ‐181 of tkt.The rE Table . Such exrE Table  and S4 wrE Table . This reDRM Fig. . HoweverD. deserti with respect to both the number of IrrE‐regulated genes and their induction fold, and also indicate additional IrrE‐regulated genes for which an RDRM site was not found in previous bioinformatics studies.The results highlight the crucial role of IrrE in the radiation response in 3.5D. deserti wild‐type and the irrE and ddrOC mutant strains with a shotgun proteomics procedure, allowing proteome comparison of the different samples and also semiquantitation of the detected proteins by spectral counting. The strains were analyzed after growth under standard condition  and after exposure to gamma radiation (IR). A total of 237,894 MS/MS spectra could be assigned when merging all the proteomics data to 7,613 different peptide sequences after querying the database containing the annotated proteins of D. deserti. In total, 906 proteins were validated with at least two different peptides and compared in terms of abundances . Thirteen of these proteins were induced in an IrrE‐dependent manner (Tfold > 1.5 in the comparison irradiated wild‐type versus irradiated ΔirrE), including ten proteins encoded by genes of the predicted RDR regulon, of which eight proteins were also found derepressed in non‐irradiated ΔddrOC compared with non‐irradiated wild‐type (Tfold > 1.5). Compared to previous proteome studies, the RDR regulon member Deide_20580, hitherto annotated as hypothetical protein, was detected here for the first time, thus validating its existence as a protein. Deide_20580 is encoded by the gene adjacent and divergently oriented to ddrOC, but the function of this 83‐amino‐acid‐residue protein is unknown. Normalized spectral abundance factors (NSAF) were calculated to estimate the relative quantities of the proteins (Table C and RecAP), DdrB, DdrD, GyrA, GyrB, PprA and SSB are the most abundant highly radiation‐induced proteins. It should be noted that the NSAF and Tfold values for RecAC and RecAP in the tables were calculated by taking into account only the spectra corresponding to peptides that distinguish between RecAC and RecAP. Many spectra for RecA peptide sequences that are common to RecAC and RecAP isoforms were also obtained, and their quantities in the different samples are in line with the IrrE/DdrO‐regulated expression of RecA (data not shown). Considering all RecA peptides, the relative abundance of total RecA protein is about twice as high as indicated in the tables.Table Based on the NSAF values, members of the RDR regulon were found most abundant among the identified highly radiation‐induced proteins Table , showing3.6Deide_01090 (DinB family protein) and Deide_18350 (Holliday junction DNA helicase RuvB). Moreover, these novel RDRM sites  overlap with the promoter sequences , correlating with DdrO functioning as a repressor also for these genes. The IrrE‐dependent regulation and RDRM sites thus identified Deide_01090 and Deide_18350 as new members of the RDR regulon in D. deserti. RDRM sites were not found upstream of Deide_11446 and Deide_21600, indicating that these genes might not be regulated directly by DdrO or that their upstream region might contain a weakly conserved RDRM that was not detected in these searches.The RNA sequencing and proteome shotgun analysis confirmed IrrE‐regulated expression of predicted RDR regulon genes, and also revealed IrrE‐dependent radiation‐induced expression of genes for which an RDRM site was not found in previous studies. To investigate this further, the nucleotide sequences upstream of the induced genes were analyzed for the presence of conserved motifs using the online tool MEME  and possesses a probable operon with recA preceded only by ligT (2′‐5′ RNA ligase) instead of the cinA‐ligT‐recA operon present in others. Interestingly, a second recA is present in D. peraridilitoris, similar to D. deserti that has two additional recA genes. Homologs of the D. deserti RDR regulon genes Deide_20580, Deide_01090, Deide_04721 and the five‐gene operon Deide_18730 to Deide_18690, all encoding proteins of unknown function, are present in several other Deinococcus species.The IrrE and DdrO proteins are highly conserved in different Deinococcus species: ddrA, ddrB, ddrC, ddrD, ddrO, Deide_20580, pprA, cinA , extra recA, ssb, gyrA, gyrB, uvrA, uvrB, ruvB, Deide_04721, Deide_18730. Interestingly, two RDRM sites were found upstream of each ddrA, including D. geothermalis ddrA for which an RDRM was not previously reported   regulon . Of the genes common to the predicted RDR regulons of D. deserti, D. radiodurans and D. geothermalis  was not found induced after irradiation. However, the RDRM upstream of Deide_00600 appeared to be involved in IrrE‐dependent regulation of a probably noncoding transcript of unknown function. Transcriptomics and proteomics also led to the identification of additional members of the RDR regulon that were not predicted previously by bioinformatics, showing the power of both bioinformatics and experimental data to identify regulon members. The majority of the highly induced genes belong to the RDR regulon, underscoring the importance of the IrrE/DdrO‐regulated response to radiation.Transcriptomics and proteomics confirmed the IrrE‐dependent upregulation of the D. deserti, D. radiodurans, and D. geothermalis, the RDR regulon is largely well conserved in other, more recently sequenced Deinococcus bacteria, but some unexpected differences were also found. At least 10 genes or operons are common to the predicted RDR regulons in the seven analyzed genomes: ddrO, ddrB, ddrC, recA operon, ssb, gyrA, gyrB, ruvB, uvrA, and uvrB. These 10 genes encode DNA repair proteins, repressor DdrO, and uncharacterized protein DdrC. Remarkably, ddrA, ddrD, and pprA, which were shown to contribute to resistance to gamma radiation, UV and/or mitomycin C in D. radiodurans . It will be interesting to unravel the function of the uncharacterized RDR members that are present in all or most Deinococcus species .Besides None declared. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


These results provided the first analysis of mSTING promoter and demonstrated that transcription factor GATA1 and Sp3 positively regulate the basal transcription of the mSTING gene.Stimulator of Interferon Gene (STING) is a key mediator of innate immune signaling. STING plays a pivotal role in the pathogenesis of many diseases including infectious diseases, auto-immune diseases and cancer. Many studies have been carried out recently in the field of STING-regulated pathway, however, rarely of transcriptional mechanisms. To characterize the murine STING (mSTING) promoter, we cloned a series of different nucleotide sequences of the 5′-flanking region of the mSTING gene. Transient transfection of promoter-reporter recombinant plasmids and luciferase assay illustrated the region (−77/+177) relative to the transcription start site (TSS) of the mSTING gene was sufficient for full promoter activity. This region contains GATA1, IK2, Sp1/Sp3 and STAT putative transcription factor binding sites. Mutation of GATA1 or Sp1/Sp3 sites led to obvious decrease of the mSTING promoter activity. Overexpression of GATA1 and Sp3 enhanced the mSTING promoter activity, whereas knockdown of GATA1 and Sp3 by a siRNA strategy significantly reduced the transcription activity. Chromatin immunoprecipitation assays demonstrated that GATA1 and Sp3 interact with the mSTING promoter  During infection, nucleic acids from invading pathogens are recognized by TLRs and RLHs, which then elicit a series of signaling events leading to the production of pro-inflammatory cytokines, type I interferons (IFNs) and chemokines3.The innate immune system is the first line of defense against invading pathogens, which is initiated by different germline-encoded pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), Nod-like receptors (NLRs) and RIG-I-like receptors (RLRs)8. In 2008, the discovery of a new sensor, named as STING, helped to understand the signaling pathway of immune-stimulatory DNA to mobilize immune responses9. STING, also known as MITA, ERIS, MPYS, TMEM173, is expressed ubiquitously in multiple human tissues and cell lines13. Recent researches showed that STING plays a key role in response to viral and bacterial pathogens, to self-DNA during autoimmune disease, and in DNA adjuvancy16. Once DNA is accumulated in cytoplasm, STING is activated by interaction with upstream DNA sensors and leads to binding and phosphorylation of TANK binding kinase 1 (TBK1). The STING-TBK1 complex phosphorylates IRF3, then translocated to the nucleus to induce transcription of type I interferon (IFN), as well as multiple interferon-stimulated genes (ISG)17. Unexpectedly, a recent study showed that STING can interact with conserved hemagglutinin fusion peptide (FP) of influenza A virus (IAV), a kind of RNA viruses, to evoke innate immune response18. What’s more, up to the present, many studies have linked STING to many physiological and pathological responses involving innate and adaptive immunity, and placed this innate immune adaptor at the center of both protective and detrimental processes in vivo19. In the anti-tumor immunity, a protective role for STING during cancer immunotherapy has been revealed in mouse tumor models. STING in dendritic cells is activated by the recognition of tumor cell DNA, leading to IFN-β induction and adaptive immunity21. However, the STING-signaling is generally suppressed in cancers, for example, in colorectal carcinoma. Loss of STING signaling impedes DNA damage response accountable for generating key cytokines that facilitate tissue repair and anti-tumor-T cell priming22. Elevation of the expression and activity of STING improve the anti-tumor effect in response to standard therapies and immunotherapies. In addition, other studies have linked STING to MHC class II signaling. That MHC class II complexes mediate cell death signaling required STING-dependent activation of the extracellular signal-regulated kinase signaling pathway12.Intracellular nucleic acid especially DNA is an important pathogen-associated molecular patterns (PAMPs) during infection with viruses, intracellular bacteria and protozoan. Many cytosolic DNA sensors acted as PPRs, such as IFI16, DDX11, Mre11, DNA-PK, cGAS, were discovered in the past decades9. Many studies have focused on the field of STING signaling pathway in the past several years, however, rarely of its transcriptional mechanisms. Here we first cloned the murine STING gene promoter region and investigated the molecular mechanisms involved in the mSTING transcriptional activity. The results showed that the proximal promoter of mSTING is located within the region −77/+177 nt relative to TSS and transcription factor GATA1 and Sp3 positively regulate the mSTING promoter activity.The STING homologues exhibit high identity in many species, such as Homo sapiens, Mus musculus, Bos Taurus and Susscrofa. STING gene encodes a protein of 379 amino acid (aa) in human and 378 aa in mouse and shares 81% similarity and 68% identity between human and murineBased on the NCBI database, sequence alignment was applied for the mSTING gene to identify the full sequence of the STING gene(chr18:35733678~35740554) and promoter prediction. Based on Encyclopedia of DNA Element (ENCODE) at UCSC , the region around TSS showed highly affinity of DNase I hypersensitive sites in several murine cell lines and tissues , siRNA-Sp3 (si-Sp3) or negative control (NC) into NIH3T3 cells individually. As showed in Fig. in vivo, whereas non-specific IgG antibody failed to precipitate protein bound this sequence in vivo. Meanwhile, the mSTING primers targeted on the mSTING exon 6 could not amplified any sequences using DNA precipitated by anti-GATA1 or anti-Sp3 protein as the template. These data suggested transcription factor Sp3 and GATA1 are capable of binding to the proximal promoter of the mSTING gene in vivo.To examine whether GATA1 and Sp3 bind to the mSTING promoter region, Chromatin immunoprecipitation (ChIP) assay was performed in NIH3T3 cells. GATA1 and Sp3-associated DNA fragments were immunoprecipated using an antibody that recognized the specific protein subunit, respectively. Normal rabbit IgG was used as a negative control. Then, the DNA precipitated in the complexes was subjected to PCR with primers flanking the region containing the GATA1 or Sp3 binding site. As shown in Fig. STING is a central and multifaceted mediator in innate immune response to cytosolic nucleic especially cytosolic DNA. STING can act both as a direct sensor of CDNs (cyclic dinucleotides) and as an adaptor for the recruitment of downstream signaling components to orchestrate innate immune defenses against various pathogens, abnormal auto-antibodies and cancers. The characterization of transcriptional regulators influencing the STING expression might help to understand these relative diseases’ pathogenesis and progression and provide new target to elucidate therapeutic strategies against these diseases. In our study, we first identified the promoter of the mSTING gene and found the functional proximal minimal promoter of the mSTING gene located from −77 to +177 nt relative to the TSS and the basal transcriptional activity of the mSTING gene is positively regulated by transcription factor GATA1 and Sp3.23. GATA1, the first recognized member of the GATA family (GATA1-6), activates target genes mainly involved in hematopoietic development of erythroid, magakaryocytes, eosinophils, and mast cells24. Somatic mutations in the GATA1 gene leading to an N-terminal truncated ‘short’ protein (GATA1s) occurred in nearly all Down syndrome patients with acute megakaryoblastic leukemia (DS-AMKL) and transient myeloproliferative disorder (DS-TMD)26. GATA1s mice (knockin mice with exclusively produce GATA1s) revealed critical difference in the expression of type I IFN-response genes compared with wild type mice. Andrew J. Woo’s study showed that loss of type I interferon signaling contributes to GATA1s-induced MkP hyperproliferation, and exogenous IFN-α inhibited hyperproliferation of GATA1s-containing Mks27. In our study, we proved that GATA1 positively regulates the expression of the mSTING gene. These results provided some evidences that GATA1 play important roles in immune system.The GATA family of transcription factors are highly conserved DNA-binding proteins which have zinc finger DNA domains to recognize the sequences of (A/T)GATA(A/G)29. Usually co-expression of Sp1 and Sp3 compete for the same binding sites in vivo and Sp3 acts as an activator or as a repressor depended on Sp1-mediated activations. IRF-3 is expressed ubiquitously in a number of tissues and plays an important role in virus-mediated induction of type I interferon30. Xu et al. have shown that Sp1 and Sp3 both positively regulate the basal expression of human IRF3 gene by interact with the IRF-3 promoter31. In contrast, in normal human prostate epithelial cells, Sp1 positively regulates 15-lipoxygenase 2 (15-LOX2) gene expression, Sp3 reduces the promoter activity of 15-LOX2 gene32. Interestingly, in our study, the results showed that Sp1 has no effect on the promoter activity of the mSTING gene whereas Sp3 enhances the mSTING gene expression in NIH3T3 cells. However, the reason why Sp3, instead of Sp1, is the major Sp protein activating the STING promoter region still needs to be confirmed, and whether this regulator mechanism is a cell-type-specific manner still remains unknown.The transcription factor Sp3 is a constitutively expressed member of the Sp family of transcription factor involved in the expression and regulation of many genes, including housekeeping genes, tissue-specifically expressed genes, viral genes, and cell cycle-regulated genes. Sp1, another member of Sp family, shares over 90% DNA sequence homology in the DNA binding domain. They can recognize and bind to the same DNA element with similar affinity33. In addition to protein structured differences, the basal transcriptional regulation mechanism of STING was also different between human and murine. We have demonstrated the minimal promoter of human STING is located in the region −124/+1 relative to TSS. Instead of transcription factor GATA1 and Sp3, CREB and c-Myc are involved in the regulation of human STING transcription34. All those studies contribute to understand the interspecies differences and may provide some notes when we try to transform achievements from animal experiments to clinical experiments.Although homologues of STING exhibit high sequence similarity in murine and human, some species-specific features lead to interspecies differences in regulation mechanisms and ability to recognize some different ligands. For instance, 10-carboxymethyl-9-acridanone (CMA), a potent type I IFN inducer in mice, directly bound to mSTING protein and triggered a strong antiviral response through the STING-TBK1-IRF3 signaling pathway but failed to activate the hSTING gene because of its inability to bound to the C-terminal ligand binding domain of the hSTING geneIn conclusion, this study showed that the mSTING proximal promoter is located within the region of −77 to +177 nt relative to TSS and that transcription factor GATA1 and Sp3 sustain basal transcription activity of the mSTING gene.Mouse embryonic fibroblast cells (NIH3T3) and human embryonic kidney 293 (HEK293) cells were obtained from the American Type Culture Collection (ATCC) (http://www.atcc.org) and cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were incubated at 37 °C in 5% carbon dioxide with humidified atmosphere.KpnI restriction sites (underlined) and protective bases were introduced to the 5′ end of the forward primer, and HindIII restriction site (underlined) and protective bases were added to the 5′ end of the reverse primer. The PCR was carried out using F1 and R1 as primers . Primers were designed by Primer Primier 5.0 software, in which Kpn I and Hind III  and then subcloned to promoter-less reporter gene vector pGL3-Basic prepared by Kpn I and Hind III double restrictive digestion. The recombinant plasmid was tested and sequenced, and the positive cloned plasmid was named as pSTING-1005. Similarly, the 5′ deletion clones were amplified with PCR by the reverse primerR1, which paired with the forward primers listed in Table The PCR products were digested with The expression plasmids of GATA1, pcDNA empty vector were stored by our group. The expression plasmids Sp1 and Sp3 were kindly provided by Dr. Guntram Suske. GATA1 siRNA, Sp1 siRNA, and Sp3 siRNA and the negative control (NC) were designed and synthesized in Genepharma Company . Sequences targeted in the GATA1, Sp1 and Sp3 mRNA, as well as the negative control sequence were below: si-Sp1: 5′-UUGAGUCACCCAAUGAGAA-3′; si-Sp3: 5′-UCAUUCCUGGCUCUAAUCAA-3′; si-GATA1: 5′-UGGCGGAGGGACAGGACA-3′; NC: 5′-UUCUCCGAACGUGUCACGUTT-3′.The promoter region (−77/+52 nt) of the mSTING gene were predicted by online software TFSEARCH ver 1.3 (http://mbs.cbrc.jp/research/db/TFSEARCH.html). Oligonucleotides with site-specific mutations at the critical nucleotides necessary for transcription factor  according to the manufacturer’s suggestion. Cells were vaccinated in a 96-well culture plates and when these cells had grown to70–80% before transfection. For luciferase assays, 200 ng of each of the luciferase recombinant plasmids was cotransfected into NIH3T3 cells with 2 ng of control pRL-TK plasmids as an internal control. For overexpression or knockdown by siRNA, expression plasmid (200 ng) or siRNA (50 nM) of GATA1, Sp1 or Sp3 was individually cotransfected into NIH3T3 cells with 100 ng of pSTING-254 luciferase recombinant plasmids together with 3 ng of control pRL-TK plasmids as an internal control. Cells were harvested after 24 hours. Quantification of firefly and Renilla luciferase activities of at least three independent transfections was measured with the Dual Reporter assay system  using FB12 luminometer . The relative luciferase activities (RLA) were calculated by normalizing the fluorescence luciferase with internal standard Renilla luciferase.Transient transfections were carried out in NIH3T3 or HEK293 cells using LipofectamineTotal RNA was extracted from NIH3T3 cells using TRIzol reagent  followed by chloroform-isopropanol extraction and ethanol precipitation. Then total RNA was used as the first strand to synthesis cDNA by reverse transcription. Subsequently, the quantification of gene transcripts was performed by real-time PCR using SYBR Green I dye  and ABI 7300 real-time PCR system (Applied Biosystems Inc.). The forward and reverse primer sequences were designed using Primer Primier 5.0 software and listed in Table g for 20 min at 4 °C, and protein quantification was performed with the BCA protein assay kit . For western blotting, 20 μg total protein were prepared with 5× SDS sample buffer and resolved by 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membrane  using standard procedures. After blocking in 5% skim milk in Tris-buffered saline–Tween (TBST), incubation for overnight at 4 °C with primary antibodies against GAPDH, β-Tubulin, Sp1, Sp3 , GATA1 and STING  and then with HRP-conjugated secondary antibodies  was performed. Subsequently, Chemoluminescense signals of membranes were quantified with ECL reagent  and visualized by enhanced chemiluminescence . Anti-GAPDH or β-Tubulin was used as loading control.The whole-cell extracts were prepared by washing cells twice with ice-cold 1× PBS and lysed in lysis buffer with the addition of Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktails and PMSF  for 30 min at 4 °C on a rocking platform before scraping and transferring to tubes. The lysates were cleared by centrifugation at 13,000 7 cells were needed for ChIP assay. NIH3T3 cells were fixed with 1% formaldehyde for 10 min at room temperature. After fixation, cells were harvested by centrifugation at 4 °C. The cell pellets were resuspended in nuclear lysis buffer and sonicated using six 15 sec pulses with 50 second rest in between pulses at 5% of maximum output strength on a Sonicator Ultrasonic Processor . The chromatin was immunoprecipitated with anti-IgG (Millipore USA), anti-Sp3 and anti-GATA1 antibody. After reverse cross-linking and DNA purification, DNA from input or immunoprecipitated samples were assayed by PCR with the appropriate promoter primers listed in Table ChIP assays were performed with the Magna ChIP™ kit  following the manufacturer’s instructions. A total of 1 × 10Data were presented as means ± SD and represented at least three independent experiments, and differences between groups were analyzed by ANOVA using SPSS 18.1 software. For all statistical comparisons, a p < 0.05 was considered statistically significant.


MYB10 in pear (PcMYB10) and Arabidopsis (AtMY75) elevated anthocyanin levels. Introduction of the R6 motif into the promoter region of an anthocyanin biosynthetic enzyme F3′5′H of kiwifruit imparts regulation by MYB10. This results in elevated levels of delphinidin in both tobacco and kiwifruit. Finally, an R6 motif inserted into the promoter the vitamin C biosynthesis gene GDP-L-Gal phosphorylase increases vitamin C content in a MYB10-dependent manner. This motif therefore provides a tool to re-engineer novel MYB-regulated responses in plants.In apple, the MYB transcription factor MYB10 controls the accumulation of anthocyanins. MYB10 is able to auto-activate its expression by binding its own promoter at a specific motif, the R1 motif. In some apple accessions a natural mutation, termed R6, has more copies of this motif within the MYB10 promoter resulting in stronger auto-activation and elevated anthocyanins. Here we show that other anthocyanin-related MYBs selected from apple, pear, strawberry, petunia, kiwifruit and Arabidopsis are able to activate promoters containing the R6 motif. To examine the specificity of this motif, members of the R2R3 MYB family were screened against a promoter harboring the R6 mutation. Only MYBs from subgroups 5 and 6 activate expression by binding the R6 motif, with these MYBs sharing conserved residues in their R2R3 DNA binding domains. Insertion of the apple R6 motif into orthologous promoters of  TF proteins possess distinct functional domains, such as a DNA binding domain and a transcriptional activator domain. The DNA binding domain is conserved within the separate classes of TF and contain trans-acting motifs that bind to the cis-acting sequences of target gene promoters. TFs are identified by their distinct DNA binding domains, such as the large family of R2R3 domain MYB TFs (Gene transcription is partly controlled by transcription factors (TFs), often in combination(s) and within hierarchical networks . These T MYB TFs . Activat MYB TFs . Transcr MYB TFs . As TFs  MYB TFs .Identifying regulatory elements in plant promoter sequences is critical to understanding the specificity of interaction between TFs and target gene promoters, as well as understanding gene regulatory networks. The most common regulatory elements are between 6 and 15 nucleotides, which are well-conserved between species. These elements provide sites for the binding of different classes of TF proteins onto the regulatory sequences of genes to provide regulation of a particular biosynthetic pathway or transcriptional cascade. These have been characterized at the bioinformatic and functional level, and can be viewed in various databases including TRANSFAC, AGRIS, and PLACE . Well-stcis-elements known to associate with MADS-box binding  driving the apple MYB10 gene generates increased anthocyanin in petals  is responsible for anthocyanin accumulation in the apple skin, while MYB10 regulates flesh color in some cultivars . Previoug assays . This isg assays  and crabg assays . The R6 n petals .proMYB10-R6. We show that the introduction of an R6 motif into promoters of MYB10 orthologs can initiate auto-regulation in species as diverse as pear and Arabidopsis. Engineering of this cis-element can change anthocyanin composition, harnessing the activity of different biosynthetic genes to produce a different metabolic profile. We then engineered the promoter of an enzyme completely unrelated to anthocyanin, the biosynthesis of ascorbic acid . Some of the MYBs tested, particularly PhDPL, were able to activate the R6 promoter to a greater extent than MYB10 from apple. In the same experiment, MdMYB8, an R2R3 MYB from apple, which is not implicated in anthocyanin regulation (We have previously reported that apple MYB10 can regulate its own transcription by binding to a characterized motif (R1) in its own promoter . A naturreporter . Expressgulation , failed MdMYB10 promoter were screened with a panel of apple R2R3 MYBs. To ensure that we tested MYBs across a range of subgroups  in the presence of MdbHLH3, suggesting that recognition of the R6 motif was restricted to flavonoid-related MYBs . These MYBs are from subgroup 5 (MdMYB9 and 11), representing the proanthocyanidin MYB clade , subgroup 6 (MdMYB10 and 110) representing the anthocyanin-associated clade  and a putative ortholog of AtMYB5 (MdMYB12), which regulates seed mucilage and seed-coat pigmentation , revealed that the flavonoid related MYBs shared a potential amino acid signature in their R2R3 domain consisting of a Lysine or Arginine residue at position 25 and a Lysine, Arginine, or Glutamine residue at position 72, relative to MdMYB10 sequence . We also included in our analysis the sequences of other MYBs not necessarily related to flavonoid biosynthesis . In these, the signature (K/R)25-(X)x46-(K/R/N)72 was not apparent at these positions, supporting a specificity linked to flavonoid related MYBs.Protein alignment of the R2R3 domain of all the MYBs tested in the transient assay . Transient assays in N. benthamiana revealed that both pear and Arabidopsis MYB promoters harboring the R6 motif were strongly activated by MdMYB10, PcMYB10, or AtMYB75 . This activation was dependent on a bHLH, either from apple (MdbHLH3) or Arabidopsis (AtbHLH2/EGL3). For the engineered promoters of either pear or Arabidopsis, the level of activation was similar when co-infiltrated with MdMYB10 or with their own MYB activator, PcMYB10 or AtMYB75, respectively.The specificity of the activation of the R6 motif to MYB10 orthologs and other flavonoid-related MYBs suggests that this motif could be used to elevate plant anthocyanin levels in species other than apple. To test this, the motif was inserted into the promoter of MYB10 orthologs from a close apple relative, European pear (PcMYB10) and an unrelated species, Arabidopsis (AtMYB75). The promoter of PcMYB10 naturally harbors a single repeat of the R1 motif at a similar distance to the start codon as that seen in the apple PcMYB10 and AtMYB75), rather than a luciferase reporter. Three constructs were transformed in Arabidopsis: the native MYB75 promoter driving MYB75 genomic DNA (termed pro:MYB75MYB75), the engineered MYB75-R6 promoter driving MYB75 (termed pro:MYB75MYB75-R6), and a pro:MYB7535S construct  previously shown to elevate the anthocyanin content in Arabidopsis . However, this is not as pigmented as the pro:MYB7535S lines . Expression analysis of these lines revealed an eightfold increase in MYB75 expression in the pro:MYB75MYB75-R6 lines compared with pro:MYB75MYB75 . Analysis of anthocyanins in these lines revealed that the total anthocyanin content measured in each line was related to the level of MYB75 transcription .The engineered promoters harboring the R6 motif were then used to drive the expression of their own respective genes . The expression of PcMYB10 was elevated in the presence of the R6 motif compared with the wild-type line . These results showed that the insertion of the R6 motif in the promoter of AtMYB75 or PcMYB10 is able to generate an auto-regulatory allele, leading to an ectopic accumulation of anthocyanins in the plant.The pear Actinidia sp.), delphinidin derivatives have been identified in two pigmented species, A. melanandra and A. arguta var. purpurea (A. deliciosa) and yellow or red (Actinidia chinensis) fruit.We examined the potential of using the R6 motif to modify a known biosynthetic pathway by modifying the expression of key genes. The hue of anthocyanin pigments depends on their level of hydroxylation and the position of the hydroxyl group on the B-ring . These hpurpurea , but notF3′5′H gene was isolated from genomic DNA of the yellow kiwifruit and fused to the luciferase reporter gene, with or without a R6 motif inserted 183 bp upstream of the start codon . Using a transient assay in N. benthamiana, apple MdMYB10 and kiwifruit AcMYB110 . The engineered R6 promoter was then fused to the genomic DNA of the F3′5′H gene to generate the F3′5′pro:F3H-R6′5′H construct, which was transiently expressed together with MYB110 in tobacco leaves (N. tabacum). Anthocyanin measurements revealed that MYB110 induced a 6.7-fold increase of the Dp/Cy (derivatives) ratio when the R6 motif was inserted in the F3′5′H promoter compared with the native promoter . MYB110 alone induces synthesis of approximately equal amounts of cyanidin  and delphinidin derivatives (Dp- rutinoside) in tobacco leaves. The higher Dp/Cy ratio obtained with MYB110 plus F3′5′pro:F3H-R6′5′H construct was due to a reduced amount of cyanidin derivatives, while content of delphinidin derivatives were similar. This could suggest that the enhanced F3′5′H activity competed with the endogenous F3′H of tobacco, hence reducing the amount of substrate available for cyanidins.The promoter region of the AcMYB110  were ablF3′5′H promoter was engineered with additional copies of the R6 motif, generating promoters harboring 6, 12, or 30 units of the R1 sequence . These promoters, fused to the luciferase reporter or to the F3′5′H genomic coding sequence, were tested in N. benthamiana and tobacco. The luciferase reporter assay suggested that maximum activation is obtained with 12 to 30 repeats, while the Dp/Cy ratio measured in tobacco leaf showing 12 repeats gave the highest ratio .We also assessed whether the number of repeats had an effect on the magnitude of promoter activation. The kiwifruit Agrobacterium carrying cloned genes into A. eriantha fruits  . Co-infiltration of the F3′5′pro:F3H′5′H construct slightly enhanced delphinidin derivative levels. However, a significant increase of delphinidins was measured when the engineered F3′5′pro:F3H-R6′5′H was co-infiltrated, yielding to a Dp/Cy ratio 8 times higher than with the native promoter .To validate this result in kiwifruit tissue, the same constructs were assayed by injection of gure 7B) . Fruits L-galactose phosphorylase, encoded by the GGP gene  has been shown to be the main control point of the pathway (via a highly conserved uORF within its 5′ UTR region that represses its translation under high ascorbate concentration (Ascorbate (or vitamin C) is an essential metabolite for most living organisms. The GDP- pathway . This gentration .A. eriantha), fused to the LUC reporter gene and the R6 motif was inserted upstream of the 5′ UTR . Transient expression in N. benthamiana revealed that MdMYB10 was able to activate the GGP promoter harboring the R6 motif . To test for any potential increase in ascorbate levels, further assays were performed in N. benthamiana using the CaMV35S promoter driving GGP and a version of the GGP promoter engineered with two copies of the R6 motif fused to the GGP cDNA (named pro:GGPGGPwt-R12). These were co-infiltrated in the presence of either the kiwifruit MYB110 or the apple MYB8 as a control. Ascorbate content of the infiltrated patch revealed that the GGP construct harboring the R12 motif (pro:GGPGGPwt-R12) did not increase the ascorbate content . This is likely due negative feedback repression by ascorbate involving the uORF in the 5′ UTR of the GGP promoter  was able to induce over-expression of GGP in the presence of MYB110, resulting in a fivefold increase in ascorbate content in tobacco leaves . In the presence of control MdMYB8, no increase in ascorbate concentration was observed. The 35S-driven construct was able to elevate ascorbate levels, irrespective of MYB co-infiltration.The GGP promoter sequence was isolated from kiwifruit , alter anthocyanin profile , or enhance other metabolites .The transcriptional regulation of endogenous genes and pathways by TFs is one key approach for plant biotechnology. The promoters of targeted genes must respond to the TF, which is dependent on the presence of cription . Enginees debate . Here weDPL, in particular, was able to trans-activate the apple promoter to a high level. Corresponding cis-elements in these species, which resemble R1 or R6-like motifs, were seen in promoters of apple, pear and Prunus species, but less evident outside the Rosaceous family . Activation required the co-infiltration of a bHLH partner.Apple MYB10 is a member of a distinct clade of dicot R2R3 MYBs that activate the expression of anthocyanin biosynthetic genes. MYB10 is able to auto-activate its expression by binding its own promoter at an R1 motif, with a natural mutation of this motif, termed R6, resulting in stronger auto-activation. MYB10 orthologs from Arabidopsis, petunia and kiwifruit, representing the Brassicaceae, Solanaceae and Actinidiaceae families, also activate R6. Petunia MYB BoMYB2 causes ectopic anthocyanin accumulation in the curd (Prunus cerasifera) PcMYB10.6 is constitutively up-regulated and is responsible for this phenotype (P. persica) where PpMYB10.4  is mis-regulated 72 is present in MYBs that trans-activated the R6 motif. This suggests evolution of key amino acids specifying DNA motif recognition. The mechanism for this should be examined in further studies.Previous studies show the specificity of certain subgroups of R2R3 MYBs for regulation of specific flavonoids, such as in Arabidopsis , peach  and reviFigure 5). The stable R6-containing lines showed increases in anthocyanin, which was confirmed by HPLC analysis. The transcription level of AtMYB75 was also clearly elevated in these lines. Transformation of pear with an engineered version of the PcMYB10 gene containing the R6 motif produced trees with ectopic anthocyanin accumulation in the leaves. Both PcMYB10 transcription and anthocyanin concentration were elevated, compared with undetectable levels in WT tissue. This is the first reported case of manipulation of pear pigmentation using transformation.We tested R6 in stable transformations of both pear and Arabidopsis to confirm their effect on anthocyanin biosynthesis. The Arabidopsis promoter containing R6 fused to the AtMYB75 coding sequence was transformed into Arabidopsis . We compared the effect of infiltrating AcMYB110 alone or with the endogenous kiwifruit F3′5′H promoter driving the F3′5′H, or with the R6-containing F3′5′H promoter driving the F3′5′H. There was an increase in total anthocyanin concentration with F3′5′pro: F3H-R6′5′H together with a dramatic shift from cyanidin derivatives to delphinidin derivatives. Several studies have targeted the content of cyanidin and delphinidin derivatives in plants in order to modify color, which is usually achieved by either overexpressing or silencing the F3′5′H enzyme, such as in carnations  were transformed using the floral dip method . ResultaNicotiana benthamiana and N. tabacum were grown in a glasshouse at 22–25°C with natural light and supplemental lighting to extend the day length to 16 h.in vitro in apple multiplication media. Freshly opened leaves from 4 weeks old plants in tissue culture were used as explants for Agrobacterium-mediated transformation based on the apple protocols , MdMYB3 (ADL36760.1), MdMYB5 (ADL36769.1), MdMYB9 (ABB84757.1), MdMYB12 (ADL36755.1), MdMYB28 (KY623714), MdMYB47 (ABK20308.1), MdMYB88 (ADL36771.1), MdMYB93 (ADL36772.1).Apple MYB10-R1C vector  as a traC vector . Other c, MdMYB8 , MdMYB11MdMYB110 , PcMYB10650/EGL3 , PhDPL , the PCR product was digested by DraI, the 194 bp fragment was gel purified, treated by T4 DNA polymerase and ligated into pGreen0800LUC-AtMYB75pro at the HindIII site located 489 bp upstream of the start codon to generate the AtMYB75-R6pro-LUC construct. The AtMYB75 promoters with and without the R6 insert were then excised from the pGreen0800-LUC (KpnI/NcoI) and used to replace the 35S promoter in the pGreenII 62-SK-AtMYB75 (35S-AtMYB75) to generate the R0-AtMYB75 and R6-AtMYB75 constructs.Promoters were engineered using standard restriction cloning of the PCR amplified R6 fragment from apple. A 1227 bp fragment of the AtMYB75 promoter was cloned into pGreen0800LUC in which the onstruct  using prPcMYB10 promoter and the full-length PcMYB10 gene, including 3.3 kb of promoter and 1.5 kb of terminator, were amplified from ‘Williams Bon Chretien’ pear genomic DNA using primers FW02/RV02 and FW02/RV03, and cloned into pGreen0800LUC and in pGEMTeasy, respectively. The 194 bp R6 fragment isolated as previously was ligated at the BsgI restriction site located at position -275 within the PcMYB10 promoter in the pGreenLUC and in the pGEMTeasy constructs. The full-length PcMYB10 alleles with and without the R6 insert were then sub-cloned at the NotI site into the pART27 binary vector to generate the R1-PcMYB10 and R6-PcMYB10 constructs.A 3.3 kb fragment of the F3′5′H gene, including 1906 bp of promoter and 780 bp of terminator was amplified from kiwifruit (A. chinensis) genomic DNA using primers FW03 and RV04 and cloned into pENTR-D/TOPO following instructions from the manufacturer . The plasmid was linearised at position -215 in the F3′5′H promoter by inverse PCR using primers FW04 and RV05 and one, two and five copies of the R6 fragment, isolated as described earlier, was ligated within the promoter, hence generating new R6-, R12-, and R30- F3′5′H versions. The CaMV35S promoter region and nopaline synthase 3′ region were deleted from the pHEX2 vector (F3′5′H were recombined by LR reaction following manufacturer instructions (Invitrogen). The promoter region of the four F3′5′H alleles was amplified using primers FW03 and RV06 and cloned at the BamHI-NcoI sites fused to the LUC reporter in the pGreen0800LUC vector.A full-length 2 vector  to generGGP promoter into pGreen0800LUC has been described previously (HpaI restriction site located at position -694 within the GGP promoter. Positive clones were sequenced and a clone containing two tandem copies of the R6 fragment was selected (R12). The GGP promoter containing the R12 domain was fused to the GGP coding region by overlap PCR extension. The GGP promoter was amplified using primers OE3/OE4 and the GGP coding sequence was amplified from the pHEX2-GGP construct  following instructions from the manufacturer.Cloning of the kiwifruit eviously . The R6 onstruct  using OE0 vector  at the KThe phylogenetic analysis was conducted using the PHYLIP suite of programs . Full-leN. benthamiana and transient expression experiments in N. tabacum of the F3′5′H and MYB constructs were performed as previously described  construct shown previously to synergistically enhance the ascorbate production by GGP . After the samples were centrifuged  the samples were analyzed by UHPLC as detailed in Supplementary Data Sheet Nicotiana tabacum leaf samples and A. eriantha fruit samples expressing F3′5′H constructs were harvested, snap-frozen in liquid nitrogen and freeze-dried overnight, then re-suspended in methanol + 0.1% HCl and incubated at room temperature for 2 h and analyzed by UHPLC (Supplementary Data Sheet RNA from the 27 transgenic Arabidopsis samples and the 15 pear samples (fully expanded mature leaves) used for anthocyanin extractions were extracted by using Spectrum Plant Total RNA Kit . First-strand cDNA synthesis was carried out by using RT Primer Mix according to manufacturer’s instructions . Real-time qPCR DNA amplification and analysis was carried out using the LightCycler 480 Real-Time PCR System , with LightCycler 480 software version 1.5. The LightCycler 480 SYBR Green I Master Mix (Roche) was used, and 10 μl of total reaction volume applied in all the reactions following the manufacturer’s method. qPCR conditions were 5 min at 95°C, followed by 40 cycles of 5 s at 95°C, 5 s at 60°C, and 10 s at 72°C, followed by 65–95°C melting curve detection. The qPCR efficiency of each gene was obtained by analysing the standard curve of a cDNA serial dilution of that gene. Sequence of the primers used are provided in Supplementary Table AA, RE, CB, and RH conceived the project and its components. CB, KL-W, WL, and SD cloned constructs and performed transient transformations. ST generated pear lines. CB and KL-W conducted gene expression analysis. TM, WL, and YP grew Arabidopsis lines and performed HPLC. AA and RE performed bioinformatics. CB, AA, and RE wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The regulation of mRNA stability has emerged as a critical control step in dynamic gene expression. This process occurs in response to modifications of the cellular environment, including hormonal variations, and regulates the expression of subsets of proteins whose levels need to be rapidly adjusted. Modulation of messenger RNA stability is usually mediated by stabilizing or destabilizing RNA-binding proteins (RNA-BP) that bind to the 3′-untranslated region regulatory motifs, such as AU-rich elements (AREs). Destabilizing ARE-binding proteins enhance the decay of their target transcripts by recruiting the mRNA decay machineries. Failure of such mechanisms, in particular misexpression of RNA-BP, has been linked to several human diseases. In the adrenal cortex, the expression and activity of mRNA stability regulatory proteins are still understudied. However, ACTH- or cAMP-elicited changes in the expression/phosphorylation status of the major mRNA-destabilizing protein TIS11b/BRF1 or in the subcellular localization of the stabilizing protein Human antigen R have been reported. They suggest that this level of regulation of gene expression is also important in endocrinology. Transcriptional regulation of the cellular responses to hormones has been the primary focus of many endocrinological research studies during the past decades. Although the transcriptional mechanisms that regulate the production of specific mRNAs are undoubtedly important, it has become increasingly evident that processes regulating the stability of mRNAs also represent critical steps in the control of dynamic gene expression. In particular, acute changes in gene expression are now recognized to be controlled by RNA-binding proteins (RNA-BP) and microRNAs through their binding to target transcripts and their positive or negative regulation of mRNA turnover. Because the ability to respond to rapid changes in ACTH levels is essential for maintaining steroid hormone homeostasis, posttranscriptional mechanisms are expected to be involved in ACTH action. The pleiotropic effects exerted by ACTH on adrenocortical cell functions are regulated through a multiplicity of mechanisms. Through binding to its adenylate-cyclase-coupled receptor MC2R, ACTH stimulates the release of cAMP and the activation of the cAMP-dependent protein kinase A (PKA), which in turn phosphorylates and regulates a number of specific substrates including the steroidogenic acute regulatory protein (StAR)  and the cis elements mainly located in the 3′-UTR of mRNAs and trans-acting factors. These latter factors comprise a number of RNA-BP that specifically bind distinct cis elements, form multimolecular scaffolds that favor or prevent the subsequent recruitment of the mRNA deadenylation and mRNA degradation machineries  that are often arranged in mRNA 3′-UTRs as repeated AUUUA pentameric sequences that eventually overlap (http://brp.kfshrc.edu.sa/ARED/) . Eu. Eucis es Figure . The mos overlap . More tha/ARED/) . Many ofDrosophila proteins embryonic lethal abnormal vision) family  identified so far, some are destabilizing the target mRNA by recruiting deadenylases and exonucleases and some are stabilizing them by protecting them from these degradation enzymes. The competition between both types of proteins for a given ARE will ultimately determine the fate of the target mRNA. The best characterized stabilizing ARE-BP is human antigen R (HuR), which is a member of the ELAV (homologs of the ) family . HuR is ) family . Upon ce) family . Binding) family , 14. A lsyndrome . Deletiosyndrome , 17. Thisyndrome . Structusyndrome , 20. Othzfp36-L1 gene transcription in bovine adrenocortical cells (BAC) through phosphorylation of CREB transcription factor and CREB-mediated activation of TIS11b promoter (zfp36-L1 gene. TIS11b mRNA is also rapidly stimulated by 8-bromo-cAMP in murine adrenocortical Y-1 cells or Leydig MA-10 cells (TIS11b, also named BRF1 or ZFP36-L1, was identified in the adrenal cortex through a differential display RT-PCR analysis of ACTH-stimulated versus non-stimulated adrenocortical cells . HuR andpromoter . A highl10 cells , suggest10 cells .StAR mediates intramitochondrial cholesterol transport in most steroidogenic tissues in response to hormonal changes (Two major target transcripts of TIS11b have been identified in the adrenal cortex. The first one is the message encoding the angiogenic cytokine VEGF . Knowing changes , whereas changes . Followivia protein phosphatase PP2A and allows the recruitment of the mRNA decay ribonucleases. A new ACTH-regulated phosphorylation site recently identified in TIS11b seems, however, to mediate a different biological response. ACTH-mediated activation of PKA was reported to induce the phosphorylation of TIS11b on two serine residues, S54 and S334 (ARE-binding proteins are also distal targets of several signaling pathways. TTP and TIS11b are phosphorylated by a variety of protein kinases. However, the impact of these phosphorylations on their mRNA-destabilizing activity is still a matter of debate, with some studies indicating that phosphorylations of TTP decrease its affinity for ARE-rich mRNAs  and otheand S334  (Figure and S334 . These oOverexpression of ARE-containing transcripts encoding factors promoting growth, inflammation, angiogenesis, and invasion has been observed in carcinogenesis . These aStAR gene. This has prompted extensive investigations to elucidate the mechanisms controlling RNA processing, mRNA nuclear export and localization, mRNA stability, and turnover, in addition to translational rates and posttranslational events. The regulation of mRNA stability has emerged as a critical control step in determining the cellular mRNA level, which is regulated through specific RNA sequence elements–protein interactions. In this context, study of the hormonal control of mRNA stability regulatory proteins and their activity in adrenal cortex function is just beginning. Considering that acute ACTH treatment affects a large number of transcripts, it seems very likely that mRNA stability regulations might play an important role in these transient gene expressions. These mechanisms are expected to also operate in response to other cAMP-mobilizing hormones in their respective target organs. For example, parathyroid hormone has been shown to decrease sodium/hydrogen exchanger 3 mRNA stability through the action of the destabilizing protein KSRP in kidney epithelial cells (Transcriptional regulation has been considered the primary control point of protein production in eukaryotic cells. However, there is growing evidence of pivotal posttranscriptional regulation for many genes, including those involved in differentiated functions of the adrenal cortex such as the al cells . ImportaAll authors listed have contributed to the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.