Egr1 expression both in cultured neurons and in vivo. Since Egr1 is an immediate early gene involved in memory formation, we wondered whether other early genes involved in memory were regulated by APP and we studied molecular mechanisms involved. By comparing prefrontal (PF) cortex from wild type (APP+/+) and APP knockout mice (APP−/−), we observed that APP down regulates expression of four immediate early genes, Egr1, c-Fos, Bdnf and Arc. Down regulation of Egr1, c-Fos and Bdnf transcription resulted from a decreased enrichment of acetylated histone H4 on the corresponding gene promoter. Further characterization of H4 acetylation at Egr1 and c-Fos promoters revealed increased acetylation of H4K5 and H4K12 residues in APP−/− mice. Whereas APP affected Egr1 promoter activity by reducing access of the CREB transcription factor, its effect on c-Fos appeared to depend on increased recruitment of HDAC2 histone deacetylase to the gene promoter. The physiological relevance of the epigenetic regulation of Egr1 and c-Fos gene transcription by APP was further analyzed following exposure of mice to novelty. Although transcription of Egr1 and c-Fos was increased following exposure of APP+/+ mice to novelty, such an induction was not possible in APP−/− mice with a high basal level of expression of these immediate early genes. Altogether, these results demonstrate that APP-mediated regulation of c-Fos and Egr1 by different epigenetic mechanisms is needed for their induction during exposure to novelty.We previously demonstrated that APP epigenetically regulates  Egr1 is an IEGs member of early growth response family of zinc fingers transcription factors, widely studied for its role in reconsolidation of memory and its ability to establish long term spatial localization memories Egr1 is needed for late-phase LTP, is involved in long-term memory formation and controls neuronal function in the hippocampus One of the first steps involved in memory formation is the rapid induction of immediate early genes (IEGs) transcription in the brain. c-Fos protein in neurons is induced by a wide range of sensory stimuli c-Fos in the establishment of neuronal plasticity by regulating downstream gene expression c-Fos deficient animals show spatial and associative learning deficits correlated with decrease in synaptic plasticity c-Fos gene sharply increases after exposure to novelty. Both Egr1 and c-Fos mRNA levels are significantly increased 30–45 minutes after exposure to novelty, in particular in the CA1 and CA3 regions of the hippocampus as well as in the PF cortex Expression of Egr1 and c-Fos, Arc protein is not a transcription factor but an effector synaptic protein involved in multiple neuronal pathways Arc induction occurs in the hippocampus and the cortex following exploration of a novel environment Arc expression both at the mRNA and protein levels Contrary to Bdnf is a member of neurotrophin family involved in neuronal growth and survival Bdnf mRNA are produced by alternative splicing of a primary transcript, and 9 alternative promoters control Bdnf gene transcription, but exon IV promoter is preferentially induced by neuronal activity Bdnf gene transcription in neurons Egr1 gene We previously demonstrated that the amyloid precursor protein (APP) of Alzheimer disease regulates, at the epigenetic level, the transcription of the Egr1 and c-Fos expression in mouse PF cortex, by inhibiting CREB recruitment and improving HDAC2 recruitment on the corresponding gene promoters. A low constitutive level of Egr1 and c-Fos expression mediated by APP is needed for their induction during exposure of mice to novelty.Here we show for the first time that APP fosters a low level of ad libidum and were housed under controlled temperature (22°C) and with a 12 h light cycle (starting at 7 am). One week before experiments, mice were moved to the experimental platform. As previously described, the behavioral test of exposure to novelty was performed in a square openfield (60×60×40 cm) with gray plastic walls. Mice were able to explore this environment during 15 minutes and sacrificed by CO2 inhalation after a resting period of 30 minutes. Another group of mice were directly sacrificed without exposure to the open field. After brain dissection, PF cortex and hippocampus were directly placed in liquid nitrogen, frozen at −80°C or directly crushed and used in further experiments. All manipulations on mice have been approved by the local ethics committee of the catholic University of Louvain and follow the European legislation.Five months old mice C57Bl/6J APP+/+ and APP−/− were used in this study. Mice were obtained from The Jackson laboratory and backcrossed for more than five generations in the CD1 genetic background. All animals had access to food Egr1 Forward: TCCTCTCCATCACATGCCTG, Egr1 Reverse: CACTCTGACACATGCTCCAG, c-Fos Forward: GATGTTCTCGGGTTTCAACG, c-Fos Reverse: GGAGAAGGAGTCGGCTGG. GAPDH Forward: ACCCAGAAGACTGTGGATGG, GAPDH Reverse: ACACATTGGGGGTAGGAACA, Arc Forward: GCTGAGCTCTGCTCTTCTTCA, Arc Reverse: GGTGAGCTGAAGCCACAAAT Bdnf Forward: GCGGACCCATGGGACTCT, Bdnf Reverse: CTGCTGCTGTAGTGACCGA.Total RNA was purified using Trizol method . Reverse transcription (RT) and quantitative (q) real time PCR (q-RT PCR) were performed with the iScript cDNA synthesis Kit and the iQ SYBR Green supermix using a iCycler MyIQ2 multicolor Real-Time PCR detection system (Biorad). The relative amplification of cDNA fragments was calculated by the 2-ΔΔCt method. q-RT PCR primer sequences used were as follows: ChIP was performed using the EZ-ChIP Assay kit (Millipore) according to manufacturer instructions and as described previously Egr1 Forward: GTGCCCACCACTCTTGGAT, Egr1 Reverse: CGAATCGGCCTCTATTTCAA, c-Fos Forward: GAAAGCCTGGGGCGTAGAGT, c-Fos Reverse: CCTCAGCTGGCGCCTTTAT, Arc Forward: CAGCATAAATAGCCGCTGGT, Arc Reverse: AGTGTGGCAGGCTCGTC, Bdnf exIV Forward: GCGCGGAATTCTGATTCTGGTAAT, Bdnf exIV Reverse: GAGAGGGCTCCACGCTGCCTTGACG. GAPDH Forward: AGAGAGGGAGGAGGGGAAATG, GAPDH Reverse: AACAGGGAGGAGCAGAGAGCAC. The quantification method used is based on the ratio between immunoprecipitated chromatin and input chromatin.qPCR primers were as follows: Nuclear extracts of mouse PF cortex were prepared in 0.25 M sucrose buffer . Samples were centrifuged 10 min at 250 g and nuclear fraction was resuspended in Laemmli buffer. All samples were sonicated before protein assay  and Western blotting was performed on 20 µg of protein lysates. Membranes were incubated overnight at 4°C with the primary antibodies; anti-HDAC2 1∶1500 (Abcam), anti-H3 1∶10000 (Millipore), anti-tubulin 1∶4000 (Sigma), anti-CREB and phospho-CREB 1∶1000 (Millipore). Washes with PBS-Tween (0.005%) were followed by incubation with secondary antibody (1∶10 000 anti-mouse or anti-rabbit IgG)  coupled to horseradish peroxidase and revealed by ECL. For quantification, the membranes were stripped and reincubated with an anti-tubulin or an anti-H3 antibody. Immunoreactive bands were quantified with an electrophoresis Gel Doc 2000 imaging system coupled to a Quantity one software (Bio-Rad).All results were expressed as mean ± standard deviation (SD) values. Statistical significance was determined by student’s t-test for two-group comparisons or one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test for multi-group comparison.Egr1 mRNA levels in PF cortex of APP−/− mice, confirming our previous results c-Fos, Arc and Bdnf mRNA levels in APP−/− mice. Levels of IEGs mRNA were normalized to GAPDH mRNA levels, which were not regulated by APP, as 3 other housekeeping genes  (c-Jun and Homer-1a) was not affected by APP  . In addid by APP .These results therefore suggest that APP is able to regulate transcription of several IEGs involved in memory formation.Egr1 gene transcription by APP is independent of the APP intracellular domain (AICD) but occurs at the epigenetic level Egr1 promoter in APP−/− mice, confirming our previous results c-Fos   . Therefo−/− mice . To testc-Fos gene promoter also contains a single CRE site  c-Fos gene transcription by APP occurs by a different mechanism, since the same recruitment of CREB on the c-Fos gene promoter was measured in both APP+/+ and APP−/− mice but less HDAC2 was found on the c-Fos gene promoter in APP−/− mice. APP decreased CREB recruitment on Egr1 gene promoter and increased HDAC2 recruitment on c-Fos gene promoter, without any modification of either CREB and HDAC2 expression or CREB phosphorylation. The presence of two CRE sites in the Egr1 gene promoter could explain why APP-mediated regulation of this gene is more dependent on CREB recruitment.Surprisingly, regulation of Our results indicate that APP fosters a low expression of a group of IEGs involved in memory formation. Consequently, overexpression of APP could have important consequences on memory formation. Interestingly, it was previously demonstrated that in APP transgenic mice, overexpression of mutated APP decreases the basal levels of IEGs mRNA and impairs the proper induction of IEGs transcription upon exposure to novelty Basal expression of IEGs is important for normal synaptic activity, but their rapid induction is needed to activate transcription of many genes playing a key role in establishment of long term memory Figure S1Housekeeping genes and other IEGs expressions are not regulated by APP.GAPDH mRNA levels were quantified by q-RT PCR in APP+/+ and APP−/− PF cortex (n = 6) and normalized versus Actin B) q-RT PCR method was used to assess mRNA levels of the housekeeping genes Actin, peptidylprolyl isomerase A (Ppia) and β-glucuronidase (Gusb) in APP+/+ and APP−/− PF cortex (n = 6). C) mRNA levels of the IEGs c-Jun and Homer-1a were quantified by q-RT PCR in in APP+/+ and APP−/− PF cortex (n = 6). Values were normalized to the GAPDH mRNA, and expressed as percentage of APP+/+, mean ± SD. Primers sequences used were as follows: Ppia FOR: CAGACGCCACTGTCGCTTT; Ppia REV: TGTCTTTGGAACTTTGTCTGCAA, Gusb FOR: ACTGACACCTCCATGTATCCCAAG, Gusb REV: CAGTAGGTCACCAGCCCGATG, c-Jun FOR: TGAAAGCTGTGTCCCCTGTC; c-Jun REV: ATCACAGCACATGCCACTTC, Homer 1a FOR: GAAGTCGCAGGAGAAGATG, Homer1a REV: TGATTGCTGAATTGAATGTGTACC.(TIF)Click here for additional data file.Figure S2Analysis of histone acetylation by ChIP assays on GAPDH promoters. ChIP experiments were performed on chromatin obtained from APP+/+ and APP−/− PF cortex. Immunoprecipitation was completed with antibody recognizing normal mouse IgG as negative control, anti H3Ac, H2BAc and H4Ac. The quantification of immunoprecipitated DNA and the normalization versus total DNA (input) was assessed by real-time qPCR with primers designed on GAPDH. All results were obtained from at least 3 or more mice per group and per antibody, and are expressed as mean ± SD.(TIF)Click here for additional data file.Figure S3Tip60 is enriched in KAI1 promoter. Tip60 binding to KAI1 gene promoter in APP+/+ and APP−/− PF cortex was assessed by ChIP using anti-Tip60 antibody, with primers designed in KAI1 promoter region. Primers sequences used were as follows: KAI1 FOR: ACCGTTAGGCAGCGCCGTGAG; KAI1 Rev: CTTGGGAAGGCGGTGCGCTC. IgG was used as negative control. Results show a significant enrichment of Tip60 in APP+/+ mice, **p<0.01. Enrichment values were normalized to input values and are the average of three or more experiments per group. Results are expressed as mean ± SD.(TIF)Click here for additional data file.


UCP3) is mainly expressed in muscle. It plays an important role in muscle, but less research on the regulation of cattle UCP3 has been performed. In order to elucidate whether cattle UCP3 can be regulated by muscle-related factors, deletion of cattle UCP3 promoter was amplified and cloned into pGL3-basic, pGL3-promoter and PEGFP-N3 vector, respectively, then transfected into C2C12 myoblasts cells and UCP3 promoter activity was measured using the dual-Luciferase reporter assay system. The results showed that there is some negative-regulatory element from −620 to −433 bp, and there is some positive-regulatory element between −433 and −385 bp. The fragment (1.08 kb) of UCP3 promoter was cotransfected with muscle-related transcription factor myogenic regulatory factors (MRFs) and myocyte-specific enhancer factor 2A (MEF2A). We found that UCP3 promoter could be upregulated by Myf5, Myf6 and MyoD and downregulated by MyoG and MEF2A.Uncoupling protein 3 ( UCP3) is a member of the family of uncoupling proteins (UCPs), which are members of the mitochondrial anion carrier family. UCPs consist of seven members: UCP1, UCP2, UCP3, UCP4, BMCP1 , StUCP (plant UCP homologs have been identified in Solanum tuberosum) and AtUCP  [UCP3 was first discovered and described in 1997 [UCP1, UCP3 was initially implicated in thermoregulation [UCP3) gene is primarily expressed in skeletal muscle and up-regulated by fatty acids [UCP3 is primarily expressed in skeletal muscle, but is also found in brown fat and heart tissue [UCP3 protein is one of the most important target proteins involved in skeletal muscle energy metabolism substance, and plays key regulatory roles in mitochondrial fatty acid oxidation [MyoD controls UCP3 promoter activity through a noncanonical E box site located close to the transcription initiation site [MyoD response element is located at different positions in rats compared to mice/humans, which may cause species-specific differences in UCP3 expression [Uncoupling protein 3  Profiling assay combined with bioinformatics analysis, and we identified several transcription factor binding sites for UCP3. We investigated whether the UCP3 promoter activity could be increased by myogenic regulatory factors (MRFs) family and myocyte-specific enhancer factor 2A (MEF2A) transcriptional factors. These results of cattle UCP3 promoter provide a platform for further researche on promoter activity and expression regulation. The results are meaningful in the research of muscle-specific promoters and the mechanism of cattle’s muscle development and growth.Guanling cattle are a yellow-coated breed that was developed in China . In thisUCP3 gene expression in the longissimus dorsi, adipose tissue, hind shin, heart, liver, and small intestine tissues were recorded using qRT-PCR. The result showed that expression of the UCP3 was at a high level in longissimus dorsi and hind shin tissues. The lowest level was in small intestine , relative to pGL3-basic) A,B. ThisL3-basic . In contL3-basic . These rUCP3 promoter, it is regarded as the core promoter of UCP3 and is consistent with the result of pGL3-basic-ucp3-pro.In this study, plasmid PEGFP-N3-ucp3-pro and PEGFP-N3 transfection C2C12 cells after 48 h were observed with an inverted fluorescence microscope to determine the fluorescence peak of the recombinant plasmid expression in C2C12 cells. The results show that Eukaryotic expression vector PEGFP-N3-ucp3-pro was successfully constructed . Green fUCP3 promoter using promoter-binding TF profiling Assay II detects the experimental results by multifunctional microplate chemiluminescence detection function. The data obtained by the experimental methods deals with the data analysis processing value. The processing value represents the intensity of luminosity. In essence, it represents the probe number after the nuclear extracts corresponding transcription factor binding probe has been eluted. Indirectly, it indicates whether the promoter region contains the transcription factor binding sites. The results from the data processing point of view could determine that the UCP3 promoter region had MEF2, MyoD, MZF, pax-5, PPAR, SATB1, TFIID, HOX4C, Nkx2-5, Nkx3-2, Pax3, MEF1, Snail, NRF2 (ARE) transcription factor binding sites  polyclonal loci of the plasmid. Coding area sequence of the MRFs family and MEF2A were cloned through double enzyme digestion and sequenced. The result shows that the eukaryotic expression vector pcDNA3.1(+)-MRFs family and pcDNA3.1(+)-MEF2A were constructed successfully . In a word, the results showed that additional SV40 promoter can strengthen the activity of UCP3 promoter, thereby optimizing the question about low activity of UCP3 promoter in skeletal muscles. The result of PEGFP-N3-ucp3 recombinant vector transfection into C2C12 cells was in agreement with the result of pGL3-promoter-ucp3 transfection into C2C12 cells.The cloned 5′-upstream regions of the Guanling cattle in C2C12 ,23. DeleUCP3 promoters such as MyoD, MZF, sox-5, TATA, Nkx2. The result of the software also includes MyoD, MZF1 and Nkx-2. We have shown that the UCP3 promoter of Guanling cattle have MyoD, TFIID, Pax3, MEF1 and MEF2 transcription factor binding sites by promoter-binding TF brofiling Assay II study. Our experimental and bioinformatics analysis revealed that the Guanling cattle UCP3 promoter region had MyoD, TFIID, Pax3, MEF1, MEF2 transcription factor binding sites. Several studies have reported that the muscle-specific promoter contained Trex/MEF-3 components, MEF-1, Pax3/7, SRE, MEF-2, CACCC boxes and other transcriptional regulatory components [UCP3 transcription in skeletal muscle [Pax3 is a key regulator of MyoG during development [Pax3, and its close homolog Pax7, give rise to a population of adult muscle stem cells [MEF2 was considered to be a conservative member of the vast majority of muscle-specific genes [MEF2 and the transcription factor containing the basic helix-loop-helix (bHLH) could coordinate muscle genes expression and regulation of the initial myogenic differentiation [MyoD1, which bound to this canonical E-box during C2C12 differentiation [Previous studies had shown that transcription factors combined with these special sequences can open or close a set of specific gene expressions. The interaction of regulatory sequence and transcription factors plays a key role in gene expression . Bioinfomponents . Multipll muscle . Pax3 iselopment ,26. The em cells ,28. MEF2ic genes ,31,32,33ntiation . In addintiation .UCP3 promoter for a possible regulation of MRFs family and MEF2A transcription factor, Myf5, Myf6, MyoD, MyoG and MEF2A were selected as the most likely transcription factors to be involved in this study. C2C12 myoblasts were co-transfected with the MRFs family transcription factor or MEF2A transcription factors expression vectors and pGL3-basic-ucp3-pro reporter vectors. The results showed that Myf5, Myf6 and MyoD contributed to the up-regulation of the UCP3 expression in C2C12 myoblasts, which was consistent with the present results that MyoD was required to activate the promoter of human UCP3 [UCP3 promoter from cattle have been identified including MyoD, which is also present in mouse [MyoG and MEF2A had down-regulation effects on the UCP3 promoter region. Interestingly, the effects of MRFs family and MEF2A were different in C2C12 cell line, Myf5, My6 and MyoD increased the dual luciferase activity, but myogenin and MEF2A had no effect. These results indicated that both the MRFs family and MEF2A are necessary, but, together, they were not sufficient enough transcription factors to induce UCP3 promoter activity. The result is consistent with previous findings [To explore the role of in mouse . Conversfindings .UCP3 promoter regions respond to the Myf5, Myf6 and MyoD transcription factors, and the promoter-binding TF profiling assay shows that these transcription factors bind directly to the UCP3 promoter. We only focus the UCP3 promoter in this study, so we can not exclude the possibility that the intoronic region  is associated with the regulation of UCP3 gene [In summary, the results indicated that this difference was most likely conferred by different ways in which CP3 gene ,37.n = 3) with similar genetic backgrounds in Guanling county of Guizhou province were reared under the same experimental conditions. At a mean age of 18 months, the animals were slaughtered using standard commercial procedures. We collected longissimus dorsi, adipose tissue, hind shin, heart, liver, and small intestine tissue samples. The tissue samples were placed in the RNAlater RNA stabilization solution  immediately after collection. The samples were frozen in liquid nitrogen, and stored at −80 °C.Guanling cattle .Total RNA was isolated from each tissue sample using the TRIzol reagent , according to the manufacturer’s instructions. An aliquot containing 1 μg of total RNA was used for the synthesis of complementary DNA (cDNA) by reverse transcription using TransStartUCP3 were designed to have matching melting temperatures. The primer sequences are listed in UCP3 analysis of the longissimus dorsi, adipose, hind shin, heart, liver, and small intestine tissue samples were used in the quantitative reverse transcription and polymerase chain reaction (qRT-PCR) experiments. The first strand of the cDNA was synthesized from 1 µg of total RNA using the TransScript II One-Step gDNA Removal and cDNA Synthesis SuperMixkits  with the oligo (dT) 20 primer (TransGen Biotech). The real-time PCR reactions were performed in a final volume of 20 µL using the Trans Start Green qPCR SuperMix UDG kit (TransGen Biotech), according to the manufacturer’s instructions. The mRNA expression were normalized to that of the β-actin, RPL4 and 18SRNA gene. The real-time PCR assays were performed using a CFX96 real-time PCR system . Thermal cycling was performed using an initial denaturation step of 95 °C for 1 min, followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min. A melting curve was constructed using annealing temperatures from 58 to 95 °C to verify the specificity of the amplified product. The data were analyzed using the 2Ct−ΔΔ method.The primer pairs of UCP3 promoter  according to the manufacturer’s instructions. Based on the sequence of UCP3 gene of cattle (UCSC NM_174210), a pair of PCR primers were designed using Primer Premier 5.0  to amplify 1080, 980, 814, 624, 437 and 389 bp sequence of promoter . Six frag cattle . The PCRg cattle . The PCRUCP3 promoter simultaneously, Promoter-Binding TF Profiling Assay II was used according to the protocol provided by Signosis Inc. . Nuclear proteins were isolated from longissimus dorsi using the reagents and protocol provided by a Nuclear Extraction Kit . Androgen receptor (AR) was used to normalize the readings for as a blank control (http://diyhpl.us/~bryan/irc/protocol-online/protocol-cache/TFSEARCH.html) and ALGGEN ROMO  were used to predict the transcription factor binding sites of UCP3 promoter region.For monitoring the activation of multiple TFs of the  control . LuminesMyoD, Myf5, Myf6, MyoG and MEF2A were designed according to the sequence of NCBI were amplified from the cDNA of longissimus dorsi . Then they were confirmed by dual-enzyme digestion and sequencing. Recombinant expression vectors were pcDNA3.1(+)-MyoD, pcDNA3.1(+)-Myf5, pcDNA3.1(+)-Myf6, pcDNA3.1(+)-Myogenin and pcDNA3.1(+)-MEF2A respectively.RNA was isolated from longissimus muscle tissue of Guanling cattle using Trizol (Invitrogen). First strand cDNA was synthesized by the Reverse transcription kit . The primers of us dorsi . The MRFUCP3 promoter fragments was excised with NheI and XhoI restriction enzyme, and ligated into the pGL3-Basic, pGL3-promoter and PEGFP-N3 , respectively. Then they were confirmed by dual-enzyme digestion and sequencing.Purification of 2 at 37 °C). C2C12 myoblasts were seeded into 24-well plates at a density of 1 × 105/well. C2C12 myoblasts reached 80% confluence after 18–24 h. Subsequently, C2C12 myoblasts were cultured in OPTI-MEM (500 µL/well) containing of 2 µL of lipofectamine 2000, 0.8 µg of the pGL3-Basic-ucp3-pro and pGL3-promoter-ucp3-pro recombinant vcectors and 0.06 µg of the internal control vector pRL-TK/luciferase reporter plasmid, C2C12 myoblasts was cultured in OPTI-MEM (500 µL/well), after 5 h, the OPTI-MEM medium was removed and replaced by DMEM medium containing 10% FBS. After 24 h transfection, cells were harvested, luciferase activity was measured using the Dual-Luciferase® Reporter Assay System , and then the activities of firefly luciferase in pGL3 and Renilla luciferase in pRL-TK were analyzed.The C2C12 myoblasts were obtained from the Cell Bank of the Chinese Academy of Sciences . C2C12 myoblasts cells were cultured in DMEM  medium with 10% fetal bovine serum (FBS)  under normal culture conditions -MyoD, pcDNA3.1(+)-Myf5, pcDNA3.1(+)-Myf6, pcDNA3.1(+)-Myogenin and pcDNA3.1(+)-MEF2A, 2 µL of Lipofectamine 2000 using Lipofectamine 2000  according to the manufacturer’s protocol, and the pcDNA3.1 vectors were cotransfected with pGL3-basic-ucp3-P1, which was the control. After 5 h, the OPTI-MEM medium was replaced by DMEM medium containing 10% FBS. After 24 h transfection, luciferase activity was measured using the same techniques as those described for C2C12 myoblasts cells.Cotransfection of eukaryotic expression vectors of p < 0.05 was considered statistically significant.Statistical analyses were performed using SPSS17.0. Data  were presented as the mean ± standard error of the mean. Statistical differences between conditions were assessed using one-way ANOVA, UCP3 promoter led to tissue-specific expression in the muscle. The activity of six different lengths of UCP3 promoter were determined using the dual luciferase reporter gene. The results suggest that there may be positive regulatory elements in the region of the UCP3 promoter from −433 to −385 bp, and there may be negative regulatory element in the region from −620 to −433 bp. Double luciferase assay and PEGFP-N3 recombinant vector results suggested that the region of UCP3 promoter from −385~+3 bp may be the core promoter region. We successfully screened out that the UCP3 promoter region of Guanling cattle have transcription factor binding sites which include MyoD, TFIID, Pax3, MEF1 and MEF2 binding sites. Co-transfection experiments of the transcription factors found that Myf5, Myf6 and MyoD have a certain positive regulatory role with regard to activity of the UCP3 promoter. The result indicated that the UCP3 promoter may play an important role in the muscle tissue.The results showed that


APETALA3 (AP3)-like PeMADS2∼5 and one PISTILLATA (PI)-like PeMADS6, specify the spectacular flower morphology in orchids. The PI-like PeMADS6 ubiquitously expresses in all floral organs. The four AP3-like genes, resulted from two duplication events, express ubiquitously at floral primordia and early floral organ stages, but show distinct expression profiles at late floral organ primordia and floral bud stages. Here, we isolated the upstream sequences of PeMADS2∼6 and studied the regulatory mechanism for their distinct gene expression. Phylogenetic footprinting analysis of the 1.3-kb upstream sequences of AP3-like PeMADS2∼5 showed that their promoter regions have sufficiently diverged and contributed to their subfunctionalization. The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage. The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal. Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal. All these results suggest that the regulation via the upstream sequences and increased H3K9K14ac level may act synergistically to display distinct expression profiles of the AP3-like genes at late floral organ primordia stage for Phalaenopsis floral morphogenesis.Five B-class MADS-box genes, including four  Arabidopsis thaliana and Antirrhinum majus, the development of different floral organs is controlled by several classes of floral-organ identity genes AP2 from A. thaliana, belong to the MADS-box family, with a highly conserved 180-bp sequence of the MADS domain that can bind to the conserved CArG-box [CC(A/T)6GG] sequence In Arabidopsis, including transcriptional regulation on the upstream sequences or intron regions by transcription factors, with feedback and feed-forward loops, and epigenetic regulation by small RNAs cis-acting elements on the B-class APETALA3 (AP3) and PISTILLATA (PI) promoters are responsible for their expression in petal and stamen FLOWERING LOCUS C (FLC) AGAMOUS (AG) Various regulatory strategies have been reported for the expression of MADS-box genes in Orchidaceae is one of the largest families of flowering plants. The high species diversity in orchids is largely due to their adaptation to specialized insect pollination Phalaenopsis orchids, four AP3-like and one PI-like B-class MADS-box genes, PeMADS2∼6, have been isolated and characterized for their roles in flower morphogenesis AP3-like PeMADS2∼5. The first, occurring early in the evolutionary history of Orchidaceae, resulted in AP3A and AP3B clades, and the second resulted in four subclades, AP3A1 (PeMADS3), AP3A2 (PeMADS4), AP3B1 (PeMADS2), and AP3B2 (PeMADS5) in situ hybridization revealed that the four AP3-like PeMADS2∼5 genes are located on different chromosomes of P. equestris, so the four orchid AP3 paralogs may have been resulted from genome duplication PeMADS2∼5 were detected ubiquitously, and then they are constrained to distinctively expressed organs at the late floral organ primordia stage and floral bud stage: PeMADS2 mainly expresses in sepal and petal, PeMADS3 predominantly expresses in petal and lip, PeMADS4 exclusively expresses in lip and column, and PeMADS5 is mainly expressed in petal PI-like PeMADS6 is ubiquitously expressed in sepal, petal, lip, and column In PeMADS2∼6 in Phalaenopsis orchids and used phylogenetic footprinting to identify conserved motifs among these promoter sequences. We analyzed the promoter activity of the upstream sequences of PeMADS2∼6 for driving GUS and luciferase gene expression in various floral organs. In addition, we examined the regulatory effects of the intron region, DNA methylation, and histone modification for their association with the high expression level of PeMADS4 in lip.Although the five B-class MADS-box genes play important roles in the perianth development in orchids, the regulatory strategies for their distinct expression profiles in various floral organs have not been characterized. In this study, we identified the upstream promoter sequences of PeMADS genes were isolated from P. equestris with red sepal, petal and orange lip P. aphrodite subsp. formosana with white sepal, petal and yellow lip was purchased from Taiwan Sugar Corp.  and used in particle bombardment experiments because the white sepal and petal made it easier for GUS staining. All plant materials were grown in the greenhouse at National Cheng Kung University  under natural light and controlled temperature from 23°C to 27°C.All upstream sequences of PeMADS2∼6 were isolated by use of the Universal GenomeWalker Kit . The desired DNA fragment was obtained by two successive PCR-based rounds of screening the GenomeWalker libraries and checked by agarose gel electrophoresis. The major bands were recovered from gels with use of the Gel DNA Fragment Extraction Kit , and cloned into the pGEM-T Easy Vector . We randomly selected 10 to 12 colonies for sequencing. The promoter sequences were compared to all known DNA sequences with use of the default settings of BLASTN from NCBI (www.ncbi.nlm.nih.gov).Genomic DNA was extracted from young flower buds by the cetyltriammonium bromide (CTAB) method PeMADS3∼5 genes, we used BAC clones constructed from P. equestrisPeMADS genes with the gene-specific probes used in previous study HindIII and separated by electrophoresis in 0.7% agarose gel. The resolved bands corresponding to promoter sequences with hybridized signals were recovered with use of the Gel DNA Fragment Extraction Kit (Geneaid) and cloned into the pGEM-T Easy vector (Promega). We randomly selected 10 to 12 colonies for sequencing. The promoter sequences characterized are deposited at the NCBI site under the accession numbers: PeMADS2 promoter (KJ127932), PeMADS3 promoter (KJ127933), PeMADS4 promoter (KJ127931), PeMADS5 promoter (KJ127934), and PeMADS6 promoter (KJ127935).When genome walking could not extend the upstream regulatory sequences for cis-acting Regulatory DNA Elements, http://www.dna.affrc.go.jp/PLACE/index.html) to predict the putative CArG box having 10-nt matches with the C(A/T)8G sequence 6GG We used the PLACE software  35S promoter with the serial deletion fragments of promoter sequences. All constructs were confirmed by sequencing to eliminate possible PCR-introduced mutations. Both the pBI221 vector containing the CaMV 35S promoter-GUS fusion (pBI221) and pBI221 vector containing a promoterless GUS cassette (pBI-PL) were recruited and considered as positive and negative controls, respectively.All the promoter sequences of e primer . The res µm in diameter by coprecipitation as described The promoter deletion-GUS fusion plasmids were isolated by use of the High-Speed Plasmid Mini kit (Geneaid) and coated on gold particles 1.6Histochemical staining of GUS activity was performed as described PeMADS4 and PeMADS6 promoters in pBI221 with SphI and BamHI, and then ligated into pJD301, containing a firefly  luciferase gene, to replace the CaMV 35S promoter for the serial pJD-Pe4p and pJD-Pe6p constructs. pJD301_R, with Renilla luciferase gene driven by the CaMV 35S promoter, was an internal control to normalize transfection efficiency.Serial-deleted promoter fragments were obtained by digestion of Renilla luciferase activity. For each analysis, two independent buds were bombarded and analyzed, and the bombardments were repeated three times independently. Statistical analysis was performed by T-test, and the differences were considered significant at p<0.01.On day 2 after bombardment, each sample was ground, and then 1 X Passive Luciferase Buffer  was added. Luciferase activity was measured by use of the dual-luciferase reporter assay system (Promega) with a Lumat LB 9507 Luminometer , a 10-sec pre-measurement delay and a 10-sec measurement period for each assay. The relative luciferase activity was calculated as the ratio of firefly to P. equestris was digested with restriction enzymes HpaII and MspI or DraI and HpaII, resolved on 0.8% agarose gel, and transferred to nylon filters  by use of a vacuum transfer system (Amersham Pharmacia Biotech). The recognition sites of HpaII and MspI are both CCGG, but digestion of HpaII is blocked by cytosine methylation and MspI is blocked by only cytosine methylation within the first cytosine (CpCGG). Two probes of PeMADS4 for methylation Southern blot assay were used: probe 1 contained the promoter and 5′ UTR sequences, and probe 2 contained the 5th intron regions. The primers were listed in 32P-labeled probes prepared by a PCR strategy.Genomic DNA isolated from sepals, petals, lips, and columns of µg genomic DNAs from the petal and lip of P. equestris were treated with conversion reagents and then cleaned up as described in the manufacturer's instruction. The DNA was served as a template for PCR amplification with the incubation at 94°C for 5 min, thermocycling for 35 cycles , and finally at 72°C for 7 min with the primers listed in Escherichia coli. We randomly selected 10 colonies for sequencing and analysis.Bisulfite sequencing analysis was carried out with the EpiTect Bisulfite kit . 2-P. equestris treated with formaldehyde. Chromatin was sheared to an average length of 500–1500 bp by sonication and immunoprecipitated with the antibodies anti-H3K4me3 , anti-H3K9me2 , or anti-H3K9K14ac . The immunocomplexes were harvested with Protein G agarose beads (Millipore) and heated at 65°C for 5 hours to release DNA cross-linked to the immunoprecipitated proteins. The DNA cross-linked to the immunoprecipitated proteins was analyzed by real-time PCR with the primers listed in ChIP assay was as described ACTIN2 for H3K4me3 and H3K9K14ac or Ta3 for H3K9me2 The DNA template was mixed with 2X SYBR Green PCR master mix  in an ABI Prism 7000 sequence detection system (Applied Biosystems), and each sample was analyzed in triplicate. Reactions involved incubation at 95°C for 10 min, and thermocycling for 40 cycles (95°C for 15 s and 60°C for 1 min). After amplification, melting curve analysis was used to verify amplicon specificity and primer dimer formation. The amount of DNA after ChIP was quantified and normalized to an internal control PeMADS promoter regions were cloned from genomic DNA by use of GenomeWalker. The PeMADS2∼6 promoter fragments obtained were 3,249, 1,293, 422, 2,121, and 1,514 bp, respectively r region , Table 28G sequence to further examine the CArG box PeMADS2 promoter region; two at nucleotides −1,097 and −443 in the PeMADS3 promoter region; six at nucleotides −3,258, −2,980, −2,656, −2,523, −559, and −65 in the PeMADS4 promoter region; and two at nucleotides −1,041 and −862 of the PeMADS5 promoter region; one at nucleotide −774 in the PeMADS6 promoter region (We used the PLACE database with a standard of 10-nt match with the C(A/T)r region , Table 2PeMADS2∼5 by phylogenetic footprinting, a method for discovering regulatory elements in a set of regulatory regions Arabidopsis and Orchis italica and for bZIP genes in rice and sorghum PeMADS3 and PeMADS4 in different order 6GG motifs    and DraIaI/HpaII . Probe 1 and the 5th intron regions of PeMADS4 , Expansin (OnExpansin), and three trypsin inhibitor (OnTI1∼3) in leaves and flowers of Oncidium Gower Ramsey, and cytokinin oxidase (DSCKX1) in protocorm-like-bodies of Dendrobium Sonia, respectively PeMADS2∼6 for driving GUS and luciferase reporter genes by particle bombardment, and the ubiquitous expression in all floral organs may be caused by the high copies of bombarded plasmid DNA and/or the naked DNA lack of chromatin modification. However, the serial deletion sequences of PeMADS4 promoter showed a higher luciferase activity in lip and column than in sepal and petal in contrast to the more-or-less similar lucifease activity detected in all four floral organs driven by PeMADS6 promoter. Moreover, we further examined the DNA methylation and histone modification within the translation start site of PeMADS4 to verify the regulatory strategy for its differential expression pattern. All these results suggested that the transient expression assay by particle bombardment accompanying with DNA methylation and histone modification analyses provides a basic information about the regulatory strategies of these PeMADS2∼6 genes exhibiting distinct expression profiles.Stable transformation of PeMADS4 conferred higher luciferase activity in lip and column, which meant that the PeMADS4 promoter was regulated by a lip- and column-specific transcription factor. Several transcription factors have been shown to dominantly express in lip, such as MADS, ARF, C3H, HB-other, YABBY, ZF-HD, bZIP, CO, TALE, HD-ZIP, MYB, and AP2-like families cis-acting regulatory elements on the upstream region of PeMADS4 were analyzed by PLACE software, and the CARGCW8GAT for MADS and MYBCOREATCYCB1 or MYBST1 for MYB-binding motifs were predicted within this sequence (S2 Table). It is possible that unidentified motifs were resided in the 375-bp fragment of PeMADS4 promoter for the interaction with above mentioned transcription factors. The exact transcription factors responding for the activation on the 375-bp fragment of PeMADS4 promoter were required for further studies.The 375-bp promoter sequence of AP3-like genes, PI-like genes showed uniformly and highly conserved expression patterns in all four floral organs in several species of various subfamily of Orchidoideae, such as Cypripedioideae, Epidendroideae, Orchidoideae, and Vanilloideae OrcPI is a PI-like MADS gene from Orchis italica, a species of Orchidoideae, and expresses in young inflorescences and all floral organs OrcPI with 1324-bp, 854-bp, 577-bp, and 356-bp upstream sequences can drive the GUS expression in petal tissue in the white Rosa hybridPeMADS6 and OrcPI could not be aligned together, even for their minimal promoter with 208- and 356-bp fragments, respectively. However, it is intriguing that two 11-bp motifs were detected between nucleotide −249 and −173 bp of PeMADS6 promoter and between −937 and −641 bp of OrcPI promoter by using the BLAST2 algorithm . Whether this element plays any roles in the ubiquitous expression of the PI-like genes in orchid flowers awaits further studies.Comparing the differential expression patterns of FLC locus and thereby controlling flowering time, in contrast to the activated state of FLC chromatin with active histone marks, H3K4me3 and H3ac, before prolonged cold exposure ATX1 results down-regulation of AP1, AP2, PI, and AG, but not of AP3 and SEP3AG by regulating H3K27me3 emf2 mutant results ectopic overexpression of AG, AP3, AP1, PI, SEP2, and SEP3PeMADS4 gene may enhance the exclusive gene expression in lip to decipher the lip morphogenesis in Phalaenopsis orchids, although its mechanism is still needed to be investigated.The epigenetic regulation is dynamic and varies between cell types and in response to development stages or environmental stimuli Arabidopsis, the promoter sequences of AP3 and PI have been used for the floral organ-specific expression in the transgenic approach. The 288-bp promoter sequence of AP3 with a petal-specific domain PI with expression in petal and stamen PeMADS2∼5 drove the expression of GUS and luciferase reporter genes in the whole flower but not exclusively in distinct floral organs. Thus, the amplified upstream regulatory sequences of PeMADS2∼5 could be used as flower-specific, but not practically for floral organ-specific promoters in the application for transgenic plants.In S1 FigureFunctional analysis of serial deletions of PeMADS2 promoter. (A) Serial deletion constructs of PeMADS2 promoter. (B–Q) Histochemical assay of flower organs bombarded with serial deletions of PeMADS2 promoter shown in the order of pBI-Pe2p-2224 (B-E), pBI-Pe2p-1823 (F-I), pBI-Pe2p-1312 (J-M), pBI-Pe2p-750 (N–Q), and pBI-Pe2p-291 (R–U). Constructs were bombarded into four independent floral buds, and results are representative of three independent bombardment experiments. Scale bar  =  0.5 mm.(TIF)Click here for additional data file.S2 FigureFunctional analysis of serial deletions of PeMADS3 promoter. (A) Serial deletion constructs of PeMADS3 promoter. (B–Q) Histochemical assay of flower organs bombarded with serial deletions of PeMADS3 promoter shown in the order of pBI-Pe3p-1007 (B–E) and pBI-Pe3p-407 (F–I). Constructs were bombarded into four independent floral buds, and results are representative of three independent bombardment experiments. Scale bar  =  0.5 mm.(TIF)Click here for additional data file.S3 FigureFunctional analysis of serial deletions of PeMADS5 promoter. (A) Serial deletion constructs of PeMADS5 promoter. (B–Q) Histochemical assay of flower organs bombarded with serial deletions of PeMADS5 promoter shown in the order of pBI-Pe5p-1507 (B-E), pBI-Pe5p-1053 (F–I), pBI-Pe5p-441 (J–M), and pBI-Pe5p-122 (N–Q). Constructs were bombarded into four independent floral buds, and results are representative of three independent bombardment experiments. Scale bar  =  0.5 mm.(TIF)Click here for additional data file.S4 FigureAlignment of the promoter sequences of PeMADS6 and OncPI.(TIF)Click here for additional data file.S1 TablePrimers used in this study.(DOC)Click here for additional data file.S2 TableCis-acting regulatory elements on the upstream region of PeMADS4.(DOC)Click here for additional data file.


In response to genotoxic stress the TP53 tumour suppressor activates target gene expression to induce cell cycle arrest or apoptosis depending on the extent of DNA damage. These canonical activities can be repressed by TP63 in normal stratifying epithelia to maintain proliferative capacity or drive proliferation of squamous cell carcinomas, where TP63 is frequently overexpressed/amplified. Here we use ChIP-sequencing, integrated with microarray analysis, to define the genome-wide interplay between TP53 and TP63 in response to genotoxic stress in normal cells. We reveal that TP53 and TP63 bind to overlapping, but distinct cistromes of sites through utilization of distinctive consensus motifs and that TP53 is constitutively bound to a number of sites. We demonstrate that cisplatin and adriamycin elicit distinct effects on TP53 and TP63 binding events, through which TP53 can induce or repress transcription of an extensive network of genes by direct binding and/or modulation of TP63 activity. Collectively, this results in a global TP53-dependent repression of cell cycle progression, mitosis and DNA damage repair concomitant with activation of anti-proliferative and pro-apoptotic canonical target genes. Further analyses reveal that in the absence of genotoxic stress TP63 plays an important role in maintaining expression of DNA repair genes, loss of which results in defective repair. TP53, TP63 and TP73, which play critical roles in development, growth control, differentiation, cellular homeostasis and response to genotoxic and other types of stress (reviewed in (Trp53 knockout mice and patients with germline TP53 mutations (Li-Fraumeni syndrome—LFS) . While ome—LFS)  are highTP53 family members share highly conserved core DNA binding and oligomerization domains, which facilitate tetramerization and DNA binding to TP53 response elements consisting of two decamers ‘RRRCWWGYYY’ separated by spacers of variable length . Target The transcriptional program regulated by TP53 represents a potent tumour suppressive pathway, which regulates expression of a network of genes in response to a range of stresses to induce cell cycle arrest, DNA repair, senescence or apoptosis . As suchExpression of TP53 responsive genes can further be influenced by expression of the TP53 family members TP63 and TP73 and recent evidence suggests that all three family members can in certain contexts act as tumour suppressors or oncogenes dependent on the expression of other family members and their isoforms (reviewed in ). This iUnlike TP53 the role of TP63 in cancer is less clear. It is mutated in ∼7% of squamous cell carcinomas  and overThe pervading model for the functional interplay between TP53 and TP63 is based largely on a relatively small number of canonical p53 activated anti-proliferative and pro-apoptotic target genes. The ΔNTP63α isoform opposes TP53- and TP73-mediated activation of anti-proliferative and pro-apoptotic target to promote proliferation and survival . For insThe extent of this interplay is highlighted by recent studies from our laboratory and others, identifying dual roles for ΔNTP63α in the regenerating epidermis opposing TP53 activity to maintain proliferative capacity , whilst Here we report the first genome-wide analysis of TP53 and TP63 binding sites in human keratinocytes. Using integrative analyses, we reveal that in response to genotoxic stress, changes in TP53 and TP63 bound cistromes result in induction or repression of a large network of target genes, the dynamics of which depends on the nature of the genotoxic stress.Collectively, through direct effects of TP53 binding or indirect inactivation of TP63 this results in global repression of cell cycle and DNA damage repair genes concomitant with activation of anti-proliferative and pro-apoptotic canonical target genes. Importantly, our analyses reveal a role for TP63 in the constitutive maintenance of DNA repair genes, highlighting the importance of defining the TP53 /TP63 network in order to better understand the biological implication of de-regulation which frequently occurs in cancer.Primary neonatal Human Foreskin Keratinocytes (HFKs) were isolated as described previously  and cultet al.  in a Diagenode Bioruptor. Resulting chromatin supplemented with complete protease inhibitor cocktail (Roche) was incubated with 50 μl of protein-G Dynabeads (Invitrogen) pre-blocked and bound with 5 μg monoclonal antibodies  and incubated for 16–18 h at 4°C. ChIPs were washed five times with 1 ml Radioimmunoprecipitation assay buffer (RIPA) buffer and once with 1 ml TE buffer, eluted incubated overnight at 65°C to reverse cross-links, digested with RNAseA (Ambion) and proteinase K (Invitrogen) and purified previously described and libraries prepared for sequencing as previously described  and subsequently filtered to remove polymerase chain reaction (PCR) duplicates. Reads were mapped to the GRCh37/hg19 reference genome using Burrows Wheeler Alignment (BWA) allowing for gapped alignment and maximum five alignment locations for each read.P = 1e−5, shiftsize = 100)  , used toe = 100) . See Supe = 100)  accessioResulting peak .bed files and .bam read files were used as input for DiffBind ,37 to dehttps://github.com/rgejman/wigreader) and visualized using the Integrative Genomics Viewer (www.r-project.org).For visualization IGVtools  from coms Viewer . Sitepros Viewer  was usedRead counts across binding sites were generated using custom scripts  and heatmaps visualized using Java TreeView .de novo motifs, Meme-ChIP  or termination sites extended by 25 kb downloaded from the University California Santa Cruz (UCSC) browser  within tSeqminer (Version 1.3)  was usedGene ontology analysis was conducted using DAVID  and termRNA extraction was carried out using Trizol (Invitrogen) according to the manufacturer's instructions. For quantitative RT-PCR, RNA was reverse transcribed to cDNA using the transcriptor high-fidelity cDNA synthesis kit (Roche), with random hexamers according to the manufacturer's instructions. Amplification of PCR products was monitored using Lightcycler 480 SYBR Green I Master (Roche) according to the manufacturer's instructions and fluorescence monitored on a Roche 480 Lightcycler and melting curve analysis also performed. In brief, cDNA samples were diluted 1:50 and quantified compared to a standard dilution series using the absolute relative quantitation method. The cycling conditions were as follows: initial denaturation 95°C for 10 min 45 cycles of 95°C, 15 s; 58°C, 15 s; 72°C, 20 s. Expression levels were assessed in triplicate, normalized to RPLPO and 18S control levels (for primer sequences see Supplementary file 8). Primers for TP53 and TP63 isoforms are previously published .For microarray analysis RNA was further purified using RNAeasy columns (Qiagen) according to manufacturer's instructions, before submission to Almac Diagnostics for analysis on the Affymetrix Exon ST 1.0 array platform.P < 0.05 or expression <1. Fold change and log2 fold change for all samples was then calculated by comparing each sample relative to the mean of the no siRNA and scrambled untreated controls. Results have been submitted to NCBI Gene Expression Omnibus  . Batch effects were removed and differential gene expression comparing tumour with normal was determined using a three-way ANOVA model incorporating tumour/normal, site and HPV status . Pyeon et al. included 14 normals and 40 HNSCC cases. Data from Thurlow et al. was pre-filtered to remove duplicates and retain only samples from only oral cavity, oropharynx and larynx .For head and neck squamous cell carcinoma (HNSCC) array analysis raw .cel files were downloaded for Thurlow me.vice)  and for  . Cells were fixed with 4% paraformaldehyde at the indicated time points, permeabilized in 0.1% Triton X-100/PBS and blocked in 10% foetal bovine serum/PBS. Cells were stained for γ-H2AX (Millipore) and 53BP1 (Millipore), anti-TP63 , TP53 , primary antibodies overnight at 4°C, washed and stained with goat anti-rabbit Alexafluor 488 or anti-mouse 568 Fab′2 fragment secondary antibodies (LifeTechnologies) washed and mounted in Prolong Gold (LifeTechnologies) containing 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Cells were visualized and foci counted using a Nikon Eclipse Ti fluorescence microscope, using a ×60 objective.Cells were fixed and stained for PI as previously described  and analCDKN1A , but with only modest effects on cell cycle . This enrichment of TP63 and TP53 around TSS is further supported by more detailed analysis of genomic location of subsets of sites identified in each of the treatment antibody combinations measured . This indicates that constitutive TP63 and TP53 sites in cells treated with genotoxic agents are highly enriched in 5′UTRs, bi- and uni-directional promoters.To generate robust consensus peaksets, we considered only peaks detected in both replicates of each ChIP/treatment combination using the MACS peak-calling algorithm  Supplem and D m and F. BComparison of the subsets of peaks identified in different treatment conditions reveals 2962 TP53 sites constitutively bound by TP53 in untreated HFKs. The number identified increases concomitant with TP53 stabilization to 6745 sites upon adriamycin treatment and to 11 560 upon cisplatin treatment Figure  and F, tTaken together, this suggests that differential dynamics of TP53 and TP63 binding depend on nature and intensity of genotoxic stress.JAG2, RHOC and RRM2B genes, respectively , whereaComparison of normalized TP53 signal intensity across all 12 287 TP53 sites reveals that, similar to occupancy-based analyses, TP53 binding is increased to the greatest extent following cisplatin treatment Figure . The sigde novo TP53-like binding motifs or half sites , adriamycin (326) or 3868 overlapping damage-induced TP53 binding events each of which is significantly enriched for s Figure . This hiTo identify genes whose expression could be influenced by the TP53 and TP63 cistromes, we first annotated peaks to REFSEQ genes identifying 9087 and 13 403 genes within 25 kb of a TP53 or TP63 binding event respectively regardless of treatment . Therefore, TP53 and TP63 have the potential to directly influence expression of a large proportion of the genome. This threshold was initially chosen since data from our laboratory and others has revealed potential for regulatory events to occur in more distal regions for TP63, TP53 and other factors such as estrogen receptor ,55,57.To gain a global perspective of the downstream effects of these binding events we integrated binding data with mRNA expression data generated by exon-array profiling, carried out concomitantly with one of the ChIP-seq replicates. Specifically, the same batch of HFKs cells were transfected with siRNA targeting TP53, TP63, scrambled control or untreated control. These cells were then treated in parallel with the cells used for ChIP-seq with cisplatin or adriamycin for 24 h and RNA extracted and submitted for Affymetrix Exon Array analysis. Results were analysed using the Altanalyze software package  at an inP < 1e−16) are also within 25 kb of 961 TP53 binding sites, potentially representing direct TP53-induced target genes . In both treatments, induction of a significant proportion of these genes (700 adriamycin and 463 cisplatin) was prevented in TP53 siRNA depleted cells (1.5-fold change in opposite direction) , indicating that this induction is TP53-dependent. A highly significant proportion of these genes  of these TP53-induced genes are within 25 kb of constitutive TP53 binding sites in untreated cells, 123 of these sites are also associated with at least one additional DNA-damage-induced TP53 site  associated with TP53 binding was significantly less than that induced by adriamycin (457), , perhaps reflecting the differing binding dynamics and/or biological effects of the doses and time points chosen . Cumulatively, these 772 TP53-damage-induced targets associated with TP53 and/or TP63 binding Figure  are enride novo damage-dependent events at more distal binding sites . This was particularly apparent for cisplatin treatment, which resulted in TP53-dependent down-regulation of a larger total number of genes (1160) than associated with adriamycin treatment (557) . Like induced genes, the majority of TP53 sites within 25 kb of repressed genes are also bound by TP63 in one of the conditions measured  were also within 25 kb of a TP53 site, considering both genotoxic agents Figure , rather d Figure . Importad Figure  genes weOur global analyses indicate that TP53 can positively and negatively influence the expression of a large number of genes in response to DNA damage and that TP63 potentially plays an important role in regulating these events. Specifically, our results suggest that activation events are likely mediated directly by TP53, through a combination of increased binding, activation of pre-bound protein, or loss of repression mediated by TP63.Furthermore, analysis of all TP53 and TP63 sites associated with induced genes indicates they have higher levels of H3K4me3 in NHEK cells , indicatRAD51B, MSH2 and XRCC4) and (iii) those with TP63 binding only within 25 kb   Figure .RRM2B and DDB2 promoters and a weaker binding to the XPC promoter  correlates with ChIP-seq results, with induced binding of TP53 to r Figure . Interesr Figure . To corrr Figure . TP53 der Figure  and D. Tr Figure .RAD51B, MSH2 and XRCC4)  and are less strongly bound as measured by fold enrichment and peak height  Figure , indicatInterestingly, TP63 mRNA and protein levels were both observed to be reduced in a TP53-dependent manner upon adriamycin and cisplatin treatment since TP53 depletion resulted in higher expression of TP63 Figure , quantitRRM2B promoter in the absence of genotoxic insult. This is not the case for all TP53-activated targets, since XPC and DDB2 are not activated upon TP63 depletion , whereas those induced by DNA damage are weaker and more diverse and less well conserved. This suggests that induced TP53 binding is a distinct event, which correlates with recent evidence indicating that co-operative interaction between TP53 monomers is required for binding to low affinity sites associated with pro-apoptotic genes  and thatThe majority of TP53 sites we identified are bound by TP63 in untreated cells, irrespective of whether TP53 is constitutively bound or TP53 binding is induced by genotoxic stress. However, the converse is not true for TP63 sites and a large proportion of sites are not bound by TP53 in any of the conditions measured. Surprisingly, comparison of the changes in TP53 and TP63 binding upon cisplatin and adriamycin reveals substantial differences in the dynamics of both TP53 and TP63 binding, suggesting different mechanisms of regulation. Specifically, cisplatin treatment results in a substantial increase in both the number of sites and the amount of TP53 bound compared with adriamycin treatment.de novo by genotoxic stress are generally weaker. Interestingly, these constitutive TP53 binding sites include canonical TP53 target genes CDKN1A, and RRM2B. Additionally, these canonical TP53/TP63 genes are up-regulated in a TP53-dependent manner in TP63 depleted cells, implying that removal of TP63-mediated repression is sufficient to activate TP53-mediated transcription. Irrespective of their constitutive or DNA-damage-induced nature, the majority of TP53 sites associated with DNA-damage-induced genes are also bound by TP63 in untreated cells and these are frequently associated with promoter proximal elements. Our genome-wide analyses identify >500 such ‘canonical’ targets and the results suggest and that >30% of TP53-dependent DNA damage-activated genes are associated with at least one constitutive TP53 binding site. This suggests that TP53 is pre-bound where it is poised for rapid activation in response to a stress signal, which is held in check through repression mediated by TP63 in unstressed cells.This is in contrast to the effects on TP63 binding, which is greatly reduced on adriamycin treatment, compared with cisplatin treatment. Importantly, we also observed a significant number of strongly bound constitutive TP53 and TP63 sites in untreated cells, whereas TP53 sites induced Interestingly, ATM has been shown to both activate TP53 through serine-15 phosphorylation  and concTP63 C40 enhancer region  or repressed through a TP53-mediated alteration of TP63 activity . This along with our genome-wide data indicates that in response to high levels of genotoxic stress TP53 activation results in a transcriptional shut down of a large number of DNA repair proteins in coordination with repression of cell cycle progression related genes. These binding events are opposed by TP63 and interestingly, TP53 repressive events are more frequently associated with more distal enhancer-like regions, the majority of which are bound by TP63 suggesting that these repressive effects may be influenced through modulating enhancer activity. This may be similar to recently described repressive effects elicited by TP53 binding to enhancer regions to repress, pluripotency genes in mouse embryonic stem cells .RRM2B, DDB2 and XPC)  ,59,69, wInterestingly, expression of DNA repair genes BRCA2, Rad51 and Mre11 have recently been shown to be activated by TP63 and TP73 in mouse embryonic fibroblasts (MEFS)  and it wTogether, our analyses reveal that through differential binding events, TP53 and TP63 coordinate expression of an extensive network of genes in response to genotoxic stress resulting in both transcriptional activation and repression. Loss of these repressive functions of TP53 or its counteraction through elevated TP63 expression has the potential to contribute to tumour growth and survival, in addition to loss of TP53's tumour suppressive pro-apoptotic and anti-proliferative functions. In support of this, when we compared our TP53/TP63 regulated genes with microarray data from tumours and matched normal in two independent head and neck squamous cell carcinoma cohorts , we founto better determine how this network is de-regulated in cancer . This iiewed in  and thatiewed in ,76. As aSupplementary Data are available at NAR Online.


Bisphenol A (BPA), originally developed as a synthetic oestrogen, is nowadays extensively used in the production of polymeric plastics. Under harsh conditions, these plastics may release BPA, which then can leach into the environment. Detectable concentrations of BPA have been measured in most analysed samples of human serum, plasma, or urine, as well as in follicular fluid, foetal serum, and amniotic fluid. Here we summarize the evidence about adverse BPA effects on the genetic and epigenetic integrity of mammalian oocytes. We conclude that increasing evidence supports the notion that low BPA concentrations adversely affect the epigenome of mammalian female germ cells, with functional consequences on gene expression, chromosome dynamics in meiosis, and oocyte development. Specific time windows, during which profound chromatin remodelling occurs and maternal imprints are established or protected, appear particularly vulnerable to epigenetic deregulation by BPA. Transgenerational effects have been also observed in the offspring of BPA-treated rodents, although the epigenetic mechanisms of inheritance still need to be clarified. The relevance of these findings for human health protection still needs to be fully assessed, but they warrant further investigation in both experimental models and humans. According to the “Developmental Origin of Health and Disease” hypothesis (DOHaD) that was proposed over a decade ago , 2, life in utero undernourishment alters the germline DNA methylome of F1 adult males in a locus-specific manner. Although altered DNA methylation did not persist in F2 tissues, dysregulated expression of genes neighbouring affected loci was observed, suggesting the possibility of intergenerational transmission of environmentally induced disease not mediated by Mendelian inheritance . Ne. Ne28]. β [ in vitro with BPA [Exposure of mice, from midgestation until birth, to daily doses of 400 ng BPA resulted in synaptic abnormalities and increased rates of recombination between homologous chromosomes in the oocytes. Interestingly, these effects resembled those observed in mice homozygous for a targeted disruption of the gene encoding for oestrogen receptor β . Increasβ  and in hwith BPA . Moreovewith BPA . It is kwith BPA ). The stIn addition to effects on chromosome synapsis in the oocytes, BPA was shown to induce also alterations of follicle maturation. Normally, at birth, the oocyte nests break down and primordial follicles are formed by recruitment of a single layer of flattened granulosa cells around the dictyate-arrested oocytes. Primordial follicles will develop to the secondary, tertiary, and finally large antral stage only from puberty onwards, when folliculogenesis proceeds under the influence of gonadotropic hormones . Mature follicles contain multilayered outer mural granulosa cells and layers of cumulus granulosa cells surrounding a fully grown, meiotically and developmentally competent oocyte, within a large fluid-filled space, the antrum. The follicle is surrounded by a basal membrane and by layers of luteal cells that are involved in steroidogenesis. Growth of the oocyte and full development of the follicle is a lengthy process that takes about 120 days in humans. in utero exposure [ In vitro experiments supported the notion that BPA impairs follicular development. Germ cell nest breakdown and primordial follicle assembly were significantly reduced when newborn mouse ovaries were exposed to 10 or 100 μM BPA in culture medium [μg/mL BPA (440 μM) inhibited follicle growth and induced atresia in a mouse follicle in vitro model [μM BPA led to reduced granulosa cell proliferation and arrest of some oocytes at meiosis I [ in vitro treatment of human oocytes with low BPA concentrations was shown to increase the frequency of oocyte degeneration [In rhesus monkeys, chronic exposure to BPA during pregnancy, leading to serum concentrations of 2.2–3.3 ng/mL, caused in the offspring a significant increase in the frequency of abnormal follicles containing multiple oocytes . Disturbexposure . In vitre medium . Similarro model , by mechro model . Continueiosis I , whereasneration .Since the entire pool of primary oocytes that can ever be ovulated is already formed in the foetal ovary, disturbances in oocyte meiosis and follicle formation, recruitment and survival, induced by any mechanism, can contribute to premature ovarian depletion, a clinically recognized condition in women . In addition, the fidelity of chromosome segregation at first and second meiosis might be compromised when the oocyte pool becomes prematurely depleted. Whether BPA exposure may influence follicle pool size in humans is still controversial. High urinary BPA levels were associated with reduced antral follicle counts in a cohort of 209 women undergoing infertility treatments , whereasOnce follicles have developed to the large antral, Graafian stage, release of one  or of multiple (in multiovulatory species like rodents) oocytes from meiotic arrest occurs under the influence of gonadotropins and the LH surge, downstream from signalling by complex and redundant pathways. Resumption of meiosis I, normally, results in gene expression changes and molecular signalling in cumulus cells and oocyte via epidermal growth factor-like hormones and critical changes in the concentration of cyclic nucleotides. Fully grown, developmentally competent oocytes then become transcriptionally quiescent, and their chromatin is remodelled to surround the nucleolus in a characteristic fashion . Followi in vitro maturation of oocytes, induced meiotic arrest, spindle abnormalities and misalignment of metaphase II chromosomes. These experimental results supported the hypothesis that a sudden, unexpected increase of aneuploid oocytes that had previously occurred in the mouse colony had been caused by accidental release of BPA from damaged plastic bottles and cages. An independent study, conducted under similar exposure conditions to verify these findings, showed BPA induction of subtle spindle abnormalities in metaphase II oocytes, but not aneuploidy [In 2003, it was reported that low, chronic BPA doses might induce aneuploidy in oocytes exposed prior to resumption of meiosis . Oral treuploidy . Similareuploidy . Differeeuploidy . Other seuploidy , supportIn vitro experiments in mouse oocytes showed that high concentrations of BPA induced spindle aberrations, chromosome congression abnormalities, and meiotic arrest, but not aneuploidy [ in vitro to 20, 200 ng/mL, or 20 μg/mL BPA  [euploidy , 52, sug, 88 μM) .Fertilization triggers release of metaphase II oocytes from second meiotic arrest. This entails second polar body extrusion and completion of oocyte second meiosis during which sister chromatids separate from each other (reviewed in ). There Oogenesis, from primordial germ cell differentiation to fertilization, and preimplantation embryonic development entail profound epigenetic changes . After gAvy) mutant mouse model. The Avy allele carries an Intracisternal A Particle (IAP) retrotransposon insertion upstream of the locus. The Agouti gene is usually expressed during a narrow window in embryogenesis and codes for a signalling molecule that can lead to either production of black eumelanin or yellow pheomelanin from a hair-cycle-specific promoter in exon 2 [Avy allele, transcriptional control of the Agouti coding sequence is driven by promoter elements in the retrotransposon, containing 9 CpGs, whose methylation level can vary among individual isogenic mice [The first report showing BPA epigenetic effects came from studies in the agouti viable yellow  or phytoestrogen prevented hypomethylation, resulting in more offspring with brown or mottled brown coat colour and normal weight [When l weight . AlthougIgf2r) and the paternally expressed gene 3 (Peg3) are first imprinted in female mice after birth when follicles and oocytes start to develop in the ovary. Chao and coworkers exposed CD-1 mice to low doses of BPA (20 or 40 μg/kg b.w.) either by daily hypodermal injections from postnatal day 7 to postnatal day 14 or by intraperitoneal injections administered each fifth day between postnatal days 5 and 20 [ Igf2r and Peg3 differentially methylated regions, but also lowered the transcription of DNA methyltransferase Dmnt1, Dmnt3a, Dmnt3b and Dnmt3l genes in the oocytes. Since the oestrogen receptor (ER) may recruit coactivator complexes with histone acetyltransferase or methyltransferase activities to activate downstream target genes [α mRNA and protein levels at the highest BPA dose. ER inhibitor ICI182780 abolished the reduction in Dnmt gene expression in the ovary of BPA-exposed mice. These observations suggested that ER signalling mediated the epigenetic effects induced by BPA in the oocytes [The insulin-like growth factor II receptor  during late stages of meiosis and oocyte growth, from 2 weeks prior to mating until day 9.5 of gestation, resulted in significant alterations in the expression of imprinted genes Peg3, Snrpn, H19/Igf2, and Kcnq1 in embryonal and placental tissues and affected foetal, placental, and postnatal development. The higher BPA dose disrupted the parental specific, monoallelic expression of the Snrpn, Igf2 and Kcnq1ot1 genes in a tissue-specific manner, and resulted in the biallelic expression of the paternally expressed Snrpn gene in the placenta, suggesting that maternal imprinting of Snrpn was disturbed before fertilization or that Snrpn was susceptible to loss of imprinting during early embryogenesis. Expression of the normally repressed maternal allele of Kcnq1ot1 in the placentas ranged from 12.3 to 72.3% of total expression, whereas no differences were found between control and BPA-treated mice on the Kcnq1ot1 maternal allele expression in the embryo [ Snrpn and Kcnq1ot1 loci in the placenta. To determine whether the altered expression pattern of imprinted genes was linked to abnormal DNA methylation, DNA methylation was analysed from the placentas and embryos of mice exposed to the high BPA dose during oocyte growth and early embryogenesis. A small but significant (P < 0.05 through ANOVA) reduction in the mean methylation level of the Snrpn imprinting control region was detected by pyrosequencing; furthermore, the decrease of methylation was attributed to the normally hypermethylated maternal allele by bisulfite mutagenesis sequencing, which allows assaying allele-specific methylation levels. Pyrosequencing analysis of the H19/Igf2 imprinting control region in BPA-exposed embryos revealed also a slightly, but significantly, reduced average methylation of the 6 analysed CpG sites (P < 0.001). Analysis of global DNA methylation by Luminometric Methylation Assay (LUMA) in control and BPA-exposed samples found a significant difference in the placentas, but not in 9.5-day embryos, after exposure to the high dose only (P < 0.05). Exposure of females to the high BPA dose only from day 5.5 to day 12.5 of gestation, that is, outside of the critical windows of DNA methylation acquisition in the oocytes and epigenetic reprogramming in embryos, did not significantly affect expression of imprinted genes. As might be expected from the important role of imprinted genes in placental development, aberrant imprinting induced by BPA exposure was associated with abnormal placental phenotypes [ per se and/or disturbance of imprint maintenance were due to direct effects on the early embryo/placenta, or were preprogrammed in the oocyte, prior to conception.Further evidence for BPA effects on methylation in differentially methylated regions of imprinted genes during oogenesis and early embryogenesis came from . Exposure embryo , suggestenotypes . In concμg/kg or 50 mg/kg BPA in diet induced nonmonotonic dose-dependent alterations of DNA methylation patterns in liver. Altered methylation was predominantly found within CpG island shores, and, overall, several hundred novel BPA-sensitive methylation sites were identified involving pathways in metabolism and stimulus response [Other studies, although not specifically focused on oocytes, support the hypothesis that BPA exposure may affect methylation of cytosines in DNA of imprinted and nonimprinted genes, outside and within coding regions. A genome-wide analysis showed that perinatal exposure to 50 response .μg/kg b.w.) altered the epigenome in the forebrain of the offspring, inducing hypomethylation at NotI locus, and deregulation of gene expression [In another study it was shown that exposure of pregnant mice throughout gestation to low doses of BPA (20 pression . Transgepression .F3 generation [μg/kg b.w.) deregulated glucose homeostasis in the F2 generation was observed; decreased global methylation and differential methylation of a specific CpG site in the glucokinase promoter in the F1 sperm suggested that the F2 phenotype could be caused by epigenetic alterations induced in the male paternal germline by BPA prenatal exposure [An epigenetic impact of BPA was demonstrated also on male germ cells. Male offspring of rats perinatally exposed to BPA had reduced sperm counts and other changes in phenotypes not only in the first but also in the neration , 10. Indneration , 72. Wheexposure .Finally, exposure to BPA appears to affect DNA methylation also in humans; a study in human foetuses found an organ-specific association between changes of global DNA methylation and BPA exposure ; a cross in vitro experiments using the same preantral follicle culture model in which nonlinear negative effects had been shown on spindle integrity, chromosome congression, and meiotic progression [ in vitro to 3 or 300 nM BPA for 12 days, during which they matured under the influence of follicle stimulating hormone up to the large antral stage, when stimulation of ovulation by recombinant hCG and recombinant EGF caused resumption of oocyte maturation and development of oocytes to metaphase II, at day 13 of culture [ Snrpn, Igf2r, Mest genes and in the paternally imprinted H19 gene were analysed using limiting dilution bisulfite pyrosequencing [An impact of BPA on the oocyte epigenome was confirmed bygression . Follicl culture , 76. Fol culture . Thus, tquencing . A cut-oChanges of posttranslational histone modifications had been previously imputed to BPA , and altP < 0.05) compared to the control and 300 nM BPA group [ Mest, 7.4% in Igf2r, and 4.8% in Snrpn alleles. No BPA effect was detected on the paternally imprinted H19 allele. Single changes in cytosine methylation, presumably not relevant for gene expression, were not significantly affected by BPA exposures. The observations suggest that low, chronic BPA exposure during oocyte growth can either adversely influence maternal imprinting per se or affect imprint stability. The particular sensitivity of the maternal Mest allele to epimutations by low BPA concentrations may relate to influences of BPA on bidirectional signalling between the oocyte and its surrounding cumulus granulosa cells by gap junctional communication, as altered methylation of Mest was also detected in mouse oocytes of connexin 37 deficient transgenic mice [Only the low BPA concentration (3 nM) caused a slight but significant acceleration of follicular growth. Overall, 7.5% of all analysed maternally imprinted alleles were abnormally demethylated in the group exposed to 3 nM BPA, a percentage significantly higher (PA group . The spenic mice .P < 0.001) (Figures μm to 1.22 ± 0.3 μm (After exposure to 3 nM BPA, congression failures and loosely aligned chromosomes at the metaphase II plate were observed Figures . Concomi Figures , whereas Figures . Interes± 0.3 μm . H3K9 is± 0.3 μm , therebyBy now there is increasing evidence that low BPA concentrations adversely affect the epigenome of mammalian female germ cells, with functional consequences on gene expression, and oocyte development and quality. There are specific time windows, during which profound chromatin remodelling occurs and maternal imprints are established or protected that appear particularly vulnerable to epigenetic deregulation by BPA: these correspond to primordial germ cell formation and oocyte meiotic prophase in the foetus, oocyte maturation after puberty, and early preimplantation development after fertilization. Transgenerational effects have also been observed in the offspring of BPA-treated rodents, although the epigenetic mechanisms of inheritance still need to be clarified. Thus BPA exposure might have long-lasting consequences on the female reproductive health, ranging from reduced fertility to offspring defects. Finally, the studies on histone posttranslational modifications suggest that BPA can also predispose the oocytes to altered chromosome behaviour in meiosis, particularly at low concentrations. The relevance of these findings for human health protection still needs to be fully assessed, but they warrant further investigation in both experimental models and humans.