Phalaenopsis bellina is a scented orchid emitting large amount of monoterpenes. GERANYL DIPHOSPHATE SYNTHASE (PbGDPS) is the key enzyme for monoterpene biosynthesis, and shows concomitant expression with the emission of monoterpenes during flower development in P. bellina. Here, we identified a dual repeat cis-element in the GDPS promoter that is critical for monoterpene biosynthesis in Phalaenopsis orchids. A strong correlation between the dual repeat and the monoterpene production was revealed by examination of the GDPS promoter fragments over 12 Phalaenopsis species. Serial-deletion of the 2-kb GDPS promoter fragments demonstrated that the integrity of the dual repeat was crucial for its promoter activities. By screening the Arabidopsis transcription factors (TFs) cDNA library using yeast one-hybrid assay, AtbZIP18, a member of group I of bZIP TFs, was identified to be able to bind the dual repeat. We then identified PbbZIP4 in the transcriptome of P. bellina, showing 83% identity in the DNA binding region with that of AtbZIP18, and the expression level of PbbZIP4 was higher in the scented orchids. In addition, PbbZIP4 transactivated the GDPS promoter fragment containing the dual repeat in dual luciferase assay. Furthermore, transient ectopic expression of PbbZIP4 induced a 10-fold production of monoterpenoids in the scentless orchid. In conclusion, these results indicate that the dual repeat is a real TF-bound cis-element significant for GDPS gene expression, and thus subsequent monoterpene biosynthesis in the scented Phalaenopsis orchids. Phalaenopsis species are widespread in the tropical Asia regions and includes approximately 56 native species   . These s violace . Both P. ocimene , 2011b,  alcohol . The VOC acetate .Monoterpenoids, the most abundant constituent in volatile terpenoids , are invPhalaenopsis orchids, PbGDPS is characterized as the key enzyme to provide precursors for monoterpene biosynthesis in P. bellina   pathway in the plastid. The short-chain prenyltransferases, GDPS, is responsible for the head-to-tail condensation of IDP and DMADP to generate the direct substrate GDP for monoterpene synthases . In Phal bellina . Interes bellina . Express (D + 5) .HDS) promoter is identified from the MEP pathway in Catharanthus roseus (SmGGPPs) promoter is analyzed in Salvia miltiorrhiza (GbMVD) from MVA pathway is examined in Ginkgo biloba (CPS), epi-cedrol (ECS), β-farnesene (FS), and amorpha-4,11-diene synthase (ADS), and one monoterpene synthase gene (LIS) are studied in Artemisia annua  for GaWRKY1 binding (Arabidopsis sesquiterpene synthase genes TPS21 and TPS11 is recognized by MYC2 involved in gibberellin and jasmonate induction (AaTPS1) in the scented kiwifruit Actinidia arguta for AaNAC2, 3 and 4 binding, which are mutated in the scentless A. chinensis (TPS10) for ZmEREB58 binding in Zea mays , P. aphrodite subsp. formosana (abbreviated as P. aphrodite), P. bellina, P. cornu-cervi var. red (abbreviated as P. cornu-cervi), P. equestris ‘RO-5’, P. equestris ‘WY-7’, P. javanica, P. lueddemanniana, P. mannii, P. schilleriana, P. I-Hsin Venus ‘KHM2212’ (abbreviated as P. I-Hsin Venus), and P. Meidarland Bellina Age ‘LM128’ (abbreviated as P. Meidarland Bellina Age). These individual plants were collected from various orchid nurseries across Taiwan , P. equestris (25%), P. venosa (18.75%), P. violacea (15.63%), P. lueddemanniana (3.13%); P. Meidarland Bellina Age - P. bellina (50%), P. violacea (24.22%), P. venosa (15.63%), P. amboinensis (3.91%), P. lueddemanniana (3.13%) via ORCHIDEYA.CA1. All the plant materials were grown in the greenhouse at National Cheng Kung University (NCKU) under natural light and surrounding temperature from 27 to 30°C in spring and summer with 75–85% humidity.Ten native and two cultivar hybrids were used in the study . Standard PCRs were applied to amplify the N-terminal region of GDPS (∼400-bp) with the primer designed based on PbGDPS genomic sequence . Reverse transcription to cDNA involved use of SuperScript III . Primers were designed by using Primer Express 3.0 . Quantitative RT-PCR (qRT-PCR) was performed by using the ABI StepOne Plus Quantitative real-time PCR instrument and SYBR Green kit  as described . Mean and standard error were calculated from triplicate repeats.Total RNA was extracted from the flowers on D + 5 stage of the 12 escribed . All expPbGDPS (denoted as PbGp-2010) and its nine serial deletion fragments   were amplified from the genomic DNA of P. bellina. Further serial-deletion fragments   were amplified for detail analysis of the region between -836 bp to -710 bp. Specific primers with the restriction endonuclease sites of BamH I and Nco I were designed to amplify these truncated fragments. The amplified fragments were double-digested with the restriction enzyme BamH I and Nco I, and cloned into the corresponding enzyme digestion sites of pJD301(f) to drive the firefly  luciferase gene , containing the Renilla luciferase gene driven by cauliflower mosaic virus (CaMV) 35S promoter. For normalization, the luciferase activity of the reporter construct was divided by that of the internal control. The involvement of internal control reduced experimental variability resulted from differential bombardment efficiency and transformation efficiency among various experimental groups. The amount of the reporter plasmid and the internal control was 10 and 0.1 μg, respectively. At least six individual flowers of P. I-Hsin Venus were employed for replicates. Luciferase activity of each sample was measured , D + 3, D + 5, and D + 7, which corresponds to the four periods of the floral monoterpene emission pattern, including onset, increase, peak and decline , by using those in Arabidopsis as queries with E-value cutoff of 1e-5 . After sequence confirmation, these promoter fragments were cloned into pJD301(f) to drive the firefly luciferase gene. Coding sequences for both PbbZIP4 and PbbZIP26 were amplified with gene-specific primers from full-bloom flowers of P. bellina and cloned into pBI221 and under the control of CaMV 35S promoter. Three separate plasmids, including pBI221 with PbbZIP4/26, pJD301(f) with promoter fragments, and internal control pJD301(R), were co-bombarded into P. aphrodite floral tissues at a ratio of 1.5: 1.5: 0.15  as described previously  and the compounds were identified by GC/HRMS as described above. Total RNA was isolated from the infiltrated tissues following the protocol of RNeasy Plant Mini Kit . Reverse transcription to cDNA and quantitative RT-PCR was performed as described above.The coding sequence of d stages . The empGDPS promoters were isolated from the scentless P. aphrodite, namely PaGDPSpA and PaGDPSpB . Compared to the GDPS promoter from the scented P. bellina (PbGDPSp), two GDPS promoters identified from P. aphrodite, PaGDPSpA and PaGDPSpB contained an 11-bp deletion and a 75-bp deletion, respectively . PaGDPSpB also had two 14-bp insertions in addition to numerous nucleotide substitutions. By performing the luciferase promoter assays in planta, PaGDPSpA showed the similar promoter activity as PbGDPSp either in scented or scentless flowers, while PaGDPSpB revealed very low promoter activity even in the scented P. bellina floral tissues. These results indicated that the lack of the 75-bp region in the PaGDPSpB is detrimental for its activity . Further sequence analysis of the PbGDPS promoter showed that a second 75-bp repeat is present downstream from the original 75-bp repeat and formed a dual repeat consisted of the two 75-bp units. The first and second 75-bp units were then denoted as ‘R1’, and ‘R2’, respectively, located from -859 to -710 nt upstream from the ATG .Previously, two individual 1-kb fragments of PaGDPSpB lacked the entire R1 unit, and PaGDPSpA harbored a 11-bp deletion in the center of R1, which was defined as R1-b subunit . The region (25-bp) prior to the R1-b was denoted as R1-a, and those (39-bp) behind was R1-c, and the corresponding divisions in R2 were denoted as R2-a, R2-b and R2-c . The dual repeat structure was schematically represented in Figure 1, and the sequence of the dual repeat is in Supplementary Figure GDPS promoters of the scent P. bellina and the scentless P. aphrodite resided in the dual repeat, and this is well correlated with the monoterpene phenotype.The e region 5-bp prioPaGDPS and PbGDPS from scented and scentless Phalaenopsis orchids, we hypothesized that the dual repeat is associated with the monoterpene production. To confirm this, another 10 frequently used breeding parents of Phalaenopsis orchids . Intriguingly, the GDPS gene was present in all of these orchids regardless of being scent or scentless phenotype . It is plausible that the defects are resided in the promoter region . We then amplified the dual repeat on GDPSp and a polymorphism of the dual repeat fragment length was detected among the 12 Phalaenopsis orchids . The four scented orchids with monoterpene production contain the complete dual repeat . In contrast, the amplified dual repeat fragments of the other orchids were reduced to various extents with various deletions in the dual repeat region. These 12 fragments were cloned and sequenced. Deletions in the dual repeats were detected between nucleotides 11 and 110, which appear to cause defects in GDPS promoter activities in the orchids without monoterpene production . Strikingly, most defects occurred in the R1 region .The presence of the GDPS for 12 Phalaenopsis orchids . The four orchids emitting monoterpenoids especially both P. Meidarland Bellina Age and P. bellina showed higher GDPS expression levels than the others . Taken together, we concluded that the integrity of the dual repeat in the GDPS promoter is strongly correlated with its elevated expression and thus the monoterpene production.We then examined the expression levels of GDPS, the ∼2-kb promoter fragment (denoted PbGp-2010) upstream from the start site of PbGDPS was isolated and subjected to serial deletions. The activity of PbGp-2010 and the nine truncated promoter fragments were evaluated in P. I-Hsin Venus flowers via particle bombardment for dual luciferase assays. It was legitimate that we should examine PbGDPS promoter activity in the original species P. bellina. However, the supply of P. bellina flowers fell short of demand for experiments as P. bellina commonly produces only one flower per 20 days. Instead, P. I-Hsin Venus, the offspring of P. bellina emitting similar scents, was micropropagated to large quantities with the identical genetic background and would help to reduce variation.To investigate the role of the dual repeat in the promoter activity of PbGp-859, which showed approximately threefold increase as compared to that PbGp-784, and fivefold increase as compared to that of PbGp-710 . Thus, the cis-element responsible for high promoter activity was between nucleotide (nt) -859 and nt -710 (150-bp), in which the dual repeat located. Further dissection of PbGp-710 to generate PbGp-584, PbGp-410, PbGp-354, PbGp-297, and PbGp-216 showed that no extra enhancers were present at the downstream of the dual repeat . Compared with PbGp-859, the promoter activity of PbGp-2010 and PbGp-1076 was much decreased , suggesting the presence of repressor elements at the upstream region of PbGp-859. These results verified that the dual repeat plays a crucial role for PbGDPS promoter activity.The highest luciferase activity was observed for cis-element in the 150-bp dual repeat region for transcriptional regulation, a more detailed analysis was performed based on the subunit division, namely R1a, R1b, R1c, R2a, R2b, and R2c. These subunits were serial deleted to generate a series of truncated promoter constructs. The luciferase activities were reduced further with gradual deletions of the subunit, indicating the essential nature of the complete dual repeat for full promoter activity of PbGDPS .To further define the trans-activators, Y1H screening was performed using P. bellina floral cDNA as preys. However, the dual repeat bait produced extensive background growth of yeast, and it could not be eliminated even under the addition of inhibitor 3-Amino-1,2,4-triazole (3-AT). Alternatively, Y1H screening against prey library composed of approximately 1,350 TFs of Arabidopsis thaliana was performed  and three of them encoded bZIP family proteins including AtbZIP18, AtbZIP29, and AtbZIP30. The other one candidate AtAGL81, a MADS-box TF, failed to be isolated from P. bellina floral transcriptome.To identify the candidate upstream erformed . The duaP. bellina floral transcriptomic data  showed nine proteins belong to group I, and can be divided into three subgroups  according to their phylogenetic relationship with the Arabidopsis ones . We selected subgroup i including AtbZIP18 for further analysis since AtbZIP29 and AtbZIP30 (subgroup ii) were repeatedly isolated in other unrelated screenings and therefore considered as sticky factors in the Y1H system. No PbbZIPs were classified into subgroup iii . Multiple alignments showed that five PbbZIPs namely PbbZIP4, PbbZIP10, PbbZIP26, PbbZIP29, and PbbZIP32 in the subgroup i shared 83–86% identity with AtbZIP18 in the basic region .The three bZIP TFs belong to the group I of bZIP family composed of a conserved bZIP domain for DNA binding and a leucine zipper motif for dimerization . The P. GDPS was much higher in the scented P. bellina than in the scentless P. aphrodite. In addition, PaGDPSpA was a functional promoter since it showed similar promoter activity as PbGDPSp in the scented P. bellina flower tissues . Both results indicated that the down-regulation of the corresponding upstream activators of GDPS was responsible for the extremely low GDPS expression in P. aphrodite .Previously, we found that the promoter activity of GDPS was evaluated by comparing their gene expression between P. bellina and P. aphrodite . Among them, bZIP4, bZIP26, bZIP29, and bZIP32 showed higher expression in P. bellina than in P. aphrodite. However, both bZIP29 and bZIP32 also expressed in floral bud of P. aphrodite but still GDPS did not express , suggesting that both bZIP29 and bZIP32 did not transactivate PaGDPSpA promoter. Both PbbZIP29 and PbbZIP32 thus were excluded for further analysis.The possibility of the five bZIPs regulating PbGDPS promoter . Here, to further examine the transactivating ability of PbbZIP4 and PbbZIP26 on various GDPS promoters, dual luciferase assay was performed in the floral tissues of the scentless P. aphrodite. In the presence of PbbZIP4, it enhanced the promoter activities of both PbGDPSp and PaGDPSpA, but revealed no effects on PaGDPSpB . This was consistent with the previous results, in which the activities of both PbGDPSp and PaGDPSpA were higher than PaGDPSpB in the scented P. bellina floral tissues . In contrast, PbbZIP26 did not transactivate PbGDPSp, PaGDPSpA, or PaGDPSpB . These results indicated that PbbZIP4 was able to distinguish the GDPS promoter containing the full dual repeat (PbGDPSp), or near full-dual-repeat (PaGDPSpA), from the solely one repeat unit (PaGDPSpB).Previously, we have shown that PbbZIP4 was able to transactivate cis-element and trans-factor for GDPS promoter were both identified in the scented P. bellina. To investigate how these two factors affected the monoterpene phenotype among the 12 Phalaenopsis orchids, we examined the expression levels of bZIP4  to establish its correlation with the presence of the dual repeat on GDPS promoter  and GDPS expression levels  for monoterpene production . Interestingly, bZIP4 expressed to various extents among these orchids . Strikingly, only the orchid plants concomitantly harbored bZIP4 expression and the dual repeat on GDPS promoter exhibited high GDPS expression, and thus produced monoterpenes, including P. Meidarland Bellina Age, P. bellina, P. lueddemanniana, and P. I-Hsin Venus .The bZIP4 expression levels but without the dual repeat showed low GDPS expression, and thus did not emit monoterpenes, including P. javanica, P. amboinensis, P. mannii, and P. cornu-cervi . In contrast, another four species with extremely low expression levels of bZIP4, together with their incomplete dual repeat, contributed to their scentless phenotype, including P. schilleriana, P. aphrodite, P. equestris ‘RO-5’, and P. equestris ‘WY-7’ . Collectively, these results indicated that not only the dual repeat in GDPS promoter but also the TFs are crucial for monoterpene production in Phalaenopsis orchids.However, orchid species with similar or even higher Phalaenopsis orchids is with low efficiency. In addition, Phalaenopsis orchids have a long-life cycle with the regeneration time is about 2–3 years, especially if we want to examine the floral phenotype. Instead, the transient ectopic expression system in floral tissues has been successfully established for the study of three MYB TFs regulating the pigmentation patterning in Phalaenopsis orchids  as compared to the GUS control . Furthermore, the raise in the levels of monoterpenes in the infiltrated tissues was indeed resulted from the large increase of PbbZIP4 transcripts . Therefore, we concluded that PbbZIP4 was involved in the scent production in Phalaenopsis orchids.So far, the stable genetic transformation for  orchids . Thus, tPhalaenopsis orchids. We found that a dual repeat of cis-element on the GDPS promoter played a vital role for floral monoterpene production. The orchids without monoterpene production harbored defective dual repeat and noticeably, most occurring in the R1 region. Serial deletion analysis showed that the removal of R1 unit decreased the promoter activity by 67%, and the further deletion of R2 unit caused an additional 16% reduction, which implies that the R1 unit is crucial for high levels of GDPS promoter activity and that the R2 unit is required for the 100% transactivation activity.In this study, we studied the molecular mechanism determining the scent phenotype in GDPS promoter fragments isolated from the 10 native Phalaenopsis species shared extremely high similarities , which indicates the conservation of the GDPS promoter sequences among the Phalaenopsis orchids. The 10 native Phalaenopsis orchids belong to two-pollinia Polychilos and subgenus Phalaenopsis , it will be interesting to study whether they have the dual repeat as well, just similar to the cases in Capsella , an enzyme representing a branch point for carotenoid biosynthesis, are important for its promoter activity  on the promoter of Tryptophan decarboxylase, the first step in the indole alkaloids biosynthesis pathway, is strongly associated its expression levels   to interact with tra 2.0)  and P. es genome .cis-elements on the dual repeat was examined by PlantPan analysis (cis-elements, suggesting that the dual repeat could be a novel cis-acting sequence. As the length of the dual repeat is 150-bp, it is possible that other types of TFs may be involved in the scent regulating phenotype, and it awaits further analysis.The presence of analysis . SeveralThe molecular markers to distinguish scent traits have been well developed in rice , and alsPhalaenopsis orchids and two cultivars emitting monoterpenes showed the 150-bp fragment length of dual repeat, which indicates its potential to be developed as a molecular marker for scent trait. After the link between the dual repeat and the monoterpene emission further verified in more Phalaenopsis species and cultivars, the dual repeat could be applied as a molecular marker for early characterization of the scent phenotype in the seedlings and thus benefiting the breeding of scented Phalaenopsis orchids.Here, PCR amplification of the dual repeat showed fragment length polymorphism. The two native Phalaenopsis orchids. C-MY, NM, and MO-T performed the yeast one hybrid screening analysis and provided valuable comments on the manuscript. H-HC supervised and complemented the writing. All authors read and approved the submitted version.Y-CC, W-CT, W-HC, and H-HC conceived the research plans. Y-CC performed most of the experiment, analyzed the data, and wrote the article with contributions of all the authors. Y-CH and C-YH assisted in the identification of the dual repeat and the analysis in the gene expression and scent compounds in 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.


PIWIL2 gene, is widely expressed in various types of tumors and may promote tumorigenesis. However, the mechanisms underlying the activation of expression of PL2L60 remain unknown. In this study, an intragenic promoter responsible for the activation of PL2L60 within the human PIWIL2 gene has been identified, cloned and characterized. The promoter of PL2L60 is located in the intron 10 of the host gene PIWIL2. Bioinformatic and mutagenic analysis reveals that this intragenic promoter within the sequence of 50 nucleotides contains two closely arranged cis-acting elements specific for the hepatic leukemia factor (HLF) in the positive strand and signal transducer and activator of transcription 3 (STAT3) in the negative strand. Chromatin immunoprecipitation analysis demonstrates that both the HLF and polymerase II (Pol II), a hallmark of active promoters, directly bind to the sequence, although STAT3 does not. Knockdown of HLF and STAT3 alone or both by RNA interference significantly reduced both promoter activity and the PL2L60 protein expression, although there is no additive effect. The expression of PL2L60 proteins was enhanced when host gene Piwil2 was genetically disrupted in a murine cell model. Taken together, we have identified a PL2L60-specific intragenic promoter in the host gene of PIWIL2, which is interdependently activated by HLF and STAT3 through steric interaction. This activation is dependent on cellular milieu rather than the integrity of host gene PIWIL2, highlighting a novel, important mechanism for a cancer-causing gene to be activated during tumorigenesis.PIWIL2-like (PL2L) protein 60 (PL2L60), a product of aberrantly activated  PIWIL2 (piwi-like RNA-mediated gene silencing 2) gene in the tumorigenic status [PIWIL2  is a member of P-element-induced wimpy testis/Argonaute (PIWI/AGO) gene family [PIWIL2 is usually expressed in testis [The phenomenon of aberrant activation has been observed in c status , 2. The e family –6, mediae family , 7–12. Tn testis –6, but cn testis . It playn testis , 14, tran testis , 16, chrn testis , 18, bion testis  and trann testis , includin testis , cervican testis , gastricn testis ,acute myn testis , colorecn testis , colon cn testis , 25, ovan testis  and testn testis , 27, etcPIWIL2 gene rather being directly produced from short form of mRNAs transcribed by putative intragenic promoters in the host gene of PIWIL2 [in vitro through up-regulation of STAT3 and BCL2 genes. It can also coordinate with NF-κB to promote tumorigenesis, probably representing a common pathway for the development of tumors in various types of tissues [Piwil2 by homologous recombination did not abrogate the expressions of PL2L60 and PL2L80 in the testis [PL2L60 and PL2L80 might be transcribed by alternative promoters in the host gene Piwil2.We and others have found that PIWIL2 has multiple variants including PL2L80, PL2L80A, PL2L60, PL2L60A, PL2L50 and PL2L40. Some of the variants appear to be transcribed by intragenic promoters rather than a canonical promoter , 2, 27. f PIWIL2 , 27. Sinf PIWIL2 , 13, 28, tissues , 28. Imp tissues . Interes+ RNA (meRNA) from host gene, although the function of meRNA remains unclear [Alternative promoters may exist in intergenic or intragenic regions , 31. Int unclear .PIWIL2, which is responsible for the transcription of mRNA encoding PL2L60 proteins in testicular cells and tumor cells [PL2L60-specific promoter may be bound by multiple transcription factors such as the hepatic leukemia factor (HLF) and signal transducer and activator of transcription 3 (STAT3) that might be not tightly bound to cis-acting elements. The cellular milieu rather than the integrity of host genes may determine the activation status of intragenic promoter. Our results suggest that activation of intragenic promoter might be a driving force for aberrant activation of host genes, representing an important mechanism underlying the development and tumorigenesis.In this study, we have cloned and characterized a human intragenic promoter that resides in the intron 10 of the host gene or cells , we refePL2L60 gene encoding PL2L60 proteins is located in the region of the intron between exon 10 and 11 of PIWIL2 gene. This promoter is supposed to initiate transcription of PL2L60 mRNA (AK027497), which can be translated into an about 60 kDa protein (PL2L60) [PL2L60 within the host gene of PIWIL2. Based on the bioinformatic analysis, we defined a potential promoter region of PL2L60 within the region consisting of the intron 10 and partial sequence of exon 11 in PIWIL2 [th nucleotide (nt) of exon 11 to exon 23 of PIWIL2 gene  located at the 36th base of exon 11 of PIWIL2 . Since the vector V-3688/−2280 with high luciferase activity was far away  from the putative TSS (pTSS) of PL2L60, it might be associated with the activity of an intragenic enhancer, which usually regulates neighboring or distant genes but might act as a promoter to transcribe meRNAs of host genes . Thus, wTo further validate the results above, the fragment -3688/−2680 in forward or reverse direction was cloned into the upstream (EV-UF and EV-UR) or downstream (EV-DF and EV-DR) of luciferase gene in the pGL3-enhancer vectors, which were absent of promoter. As shown in Figure PIWIL2 gene.To further exclude the enhancer activity, the fragment -3688/−2680 was inserted into the up or down stream of pGL3-promoter and the vectors of PV-UF, PV-UR, PV-DF and PV-DR were constructed. Both PV-UF and PV-UR did not significantly enhance the activity of pGL3-promoter vector after transfected into HEK-293T cells. Interestingly, PV-DF and PV-DR significantly inhibited the pGL3-promoter activity Figure , implicaPL2L60 gene, which might serve as a suppressor as well for canonical PIWIL2 promoter.Taken together, the results shown above confirm that the fragment -3688/−2680 is truly a promoter specific for PL2L60-specific promoter, we further determined the binding sites specific for transcription factors (TFs) that transactivate PL2L60 gene. First, we refined the promoter activity in the region between -3688 and -2680 upstream of the pTSS of PL2L60. The fragment -3688/−2680 was further divided into of three fragments, which were amplified by PCR  site with low affinity in the complementary chain of the fragment -2380/2330  and HLF siRNAs, respectively, and simultaneously transfected with pGL3-basic reporter vectors harboring the fragment -2380/−2330. As a result, the mRNAs . This may be related to the steric interaction between HLF and STAT3, which was required for transactivation of PL2L60, because the cis-acting elements for STAT3 and HLF were located in two complementary DNA chains, respectively, but closely rearranged  murine embryo fibroblasts (MEFs), using Gene-Exon-Mapping Reverse transcription polymerase chain reaction (GEM RT-PCR) assay [mili−/− MEFs, in which the Piwil2 gene was genetically disrupted via homologous recombination targeting between the exon 2 and exon 5 [Piwil2 gene expression. Although full length Piwil2 transcripts were not detected, the transcripts truncated at 5′-end spanning from exon 6 to exon 23 were detectable. The truncated transcripts were absent of exons 1 - 5. As controls, full length Piwil2 transcripts (exons 1-23) were detected in the wild type (wt) MEFs and testicular cells of wt mice  assay . A seriae Figure . Howeverpression . Consists Figure . Importae Figure . As we hF Figure . The res80 mRNAs . These rPIWIL2), we examined the expression of PL2L60 proteins after knockdown of putative PL2L60 mRNAs in human tumor cell lines, using the siE21 siRNAs, which targeted exon 21 of PIWIL2, PL2L80 and PL2L60 transcripts. On the other hand, to verify the mRNAs knocked down by siE21 were mainly PL2L60-specific, we used siE7 siRNAs, which targeted exon 7 of PIWIL2 and PL2L80 transcripts but had no effect on PL2L60 transcripts, and siNC (negative control siRNA) as controls  surrounding the pTSS of the putative PL2L60–specific promoter [To refine the promoter , pGL3-baTo determine the cis-acting and trans-acting elements of the promoter, a number of deletional mutants were generated from the region of -3688/−2280, from which only 50 nt in the region of -2380/−2330 responsible for promoter activity were identified. In this 50-nt-long fragment, six potential cis-acting elements were predicted, and two of them were verified by site-directed mutagenesis as cis-elements specific for transcription factors (TFs) HLF and STAT3, respectively Figure .PL2L60 was further verified by the specific recruitments of HLF and Pol II to the region, although STAT3 binding to its cis-element was not confirmed by ChIP assay. It is likely that the STAT3 has lower affinity for its cis-elements than HLF and fails to be precipitated by anti-STAT3. However, knockdown of STAT3 significantly reduced both luciferase activity of the promoter and PL2L60 protein expression . The phenomenon suggests that HLF and STAT3 might interdependently transactivate y Figure , providi factors , 8. It wPL2L60 gene at present study because of a lack of positive result from ChIP analysis, the regulatory function of STAT3 on PL2L60 promoter is highly appreciated. It has been demonstrated that the over-expression of PL2L60 in cancer cells is associated with up-regulation of STAT3 mRNAs [STAT3 has been considered to play a critical role in cell differentiation, survival, proliferation, anti-apoptosis and angiogenesis [STAT3 [PL2L60-specific promoter, which is associated with increased expression of PL2L60 .We have previously reported that disrupting ern blot . In thiss Figure  as obser tissues . The obspression , 27. Mors Figure , suggests Figure . This isPIWIL2 gene reveals a novel, important mechanism by which PIWIL2 plays differential roles in the development and tumorigenesis [PL2L60-specific intragenic promoter reveals that (a) activation of an intragenic promoter may be associated with aberrant activation of a host gene, resulting in a protein product with the function being opposite to that of host gene; (b) intragenic promoter may be activated by multiple specific TFs through steric interaction; (c) intragenic promoters are usually inactive in somatic cells under physiological conditions but may be over-activated by pathological stimuli, such as stress and carcinogenic agents; (d) the relationship between canonical and intragenic promoters might be mutually regulated, because pGL3-promoter activity was significantly inhibited when PL2L60-specific promoter was inserted into downstream of the promoter in either directions  traPL2L60-specific intragenic promoter is distinct from the intragenic enhancer as well as canonical promoter. The finding is supported by the following additional evidences: first, the PL2L60-specific intragenic promoter was activated in both pGL3-basic and pGL3-enhancer luciferase report vectors unidirectionally but not in the pGL3-promoter vector  to distinguish the mRNAs transcribed by canonical promoters or meRNAs transcribed by enhancer-derived alternative intragenic promoters. The protein products of amRNA are opposite to the proteins products of host gene's mRNAs. For an example, the host gene o PIWIL2 .PIWIL2 gene in somatic cells. A PL2L60-specific promoter within the PIWIL2 gene has been identified, cloned and characterized, which is located between exon 10 and 11 of PIWIL2 gene and can transcribe amRNA, distinct from the recently reported meRNA transcribed by an intragenic enhancer-derived alternative promoter. The intragenic PL2L60-specific promoter can be activated by HLF and, likely, STAT3, probably through the interdependent steric interaction. Its activation is not dependent on the integrity of host gene but may be enhanced by mutations of host genes. Our study has confirmed that the intragenic promoter may contribute to aberrant activation of a host gene, a potentially important mechanism for activation of a cancer gene in tumorigenesis. The finding gives rise to a novel insight into the mechanisms mediating cancer development as well as into therapeutic strategies for cancers.In summary, we have revealed a mechanism underlying the aberrant activation of One sample of testicular parenchyma  was obtained from a patient with prostate cancer underwent orchiectomy at the Department of Urology, Renji Hospital, with the permission of Institutional Review Board of Renji Hospital. Animal experiments were conducted based on Animal Use Protocols approved by the Institutional Animal Care and Use Committee (IACUC), Renji Hospital.−/− B6 mice were used at the age of 8 −12 wk. The mili−/− mice, which were obtained from Dr. Haifan Lin's laboratory [−/− MEF (mouse embryonic fibroblasts) and mili+/+ MEFs, which were established in our laboratory [2 and 95% air.Male C57BL/B6 (B6) mice and miliPL2L60-specifc intragenic promoter fragments, covering nucleotide sequence from +155 to −13707 , upstream of the reporter firefly  luciferase gene. Luciferase activity was determined from the fragments by transfection of the vectors into HEK-293T cells, using jetPRIME® transfection reagent (Polyplus-transfection SA), according to manufacturer's instruction. Biological and technical triplicates were used to ensure reproducibility. Cells were lyzed 48 h after the transfection and the activity of both firefly and Renilla reniformis luciferases was assessed using Dual Luciferase Reporter Assay System  and Tecan GENios Pro Luminometer , according to the manufacturers’ instructions.Six pairs of primers that contain restriction sites were synthesized for amplifying six putative o −13707 . All theTo further refine the regions with promoter activity, vectors of PGL3-5.1, PGL3-5.2.1 and PGL3-5.2.2 were constructed with three subfragments of 729bp, 329bp and 400bp, respectively, which were derived from a promoter-containing fragment. The vector PGL3-5.2.1 showing promoter activity was further used as template to serially construct a number of vectors with 50 bp or more increments . These vectors were transfected into HEK-293T cells to screen promoter activity within a 50-nt fragment.In the experiments described above, each fragment was purified using the QIAquick Gel Extraction Kit  according to the manufacturer's recommendations and sub-cloned into the pGL3-basic luciferase reporter vector . All constructs were verified by DNA sequencing. All the primers used for amplifying the fragments are shown in To distinguish whether the activity of an identified DNA fragment was associated with a promoter or an enhancer, a serial of vectors was constructed with a fragment (−3688/−2280) showing high luciferase activity. The fragment was cloned into pGL3-basic vectors or pGL3-enhancer vectors up and down stream of the luciferase gene as well as in forward and reverse direction, respectively, for validation of promoter activity. On the other hand, the fragment was cloned into pGL3-promoter vectors in the same way as above to exclude enhancer activity of the fragment. The vectors transfected into HEK-293T cells for luciferase activity assay as described above.5 cells/well) and co-transfected with 1 μg of Firefly luciferase reporter vectors of interest and 20 ng of the Renilla luciferase expression vectors (pRL-TK), using jetPRIME® transfection reagent (Polyplus-transfection SA) 24 h later. After 48 h of transfection, the cells were lysed with passive lysis buffer , and Luciferase activity was determined in cell extracts using the Dual-Luciferase Reporter Assay kit . Data were expressed as the ratio of Firefly to Renilla luciferase activities.HEK-293T Cells were plated into 6-well plates . For real-time PCR, a total of 500 ng of RNAs was used for reverse transcription with the Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit (Thermo). SYBR Green PCR real-time PCR Master Mix  was used according to the manufacturer's instructions. First, the reaction was incubated at 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 1 min, followed by 95°C for 15 s. The relative changes in mRNAs levels were normalized for β-actin mRNAs in the same samples. The primers used are summarized in de novo isolated testicular cells and splenocytes, as previously described [For RT-PCR, total RNA was extracted from cell lines or escribed , 8. BriePiwil2 were designed (http://www.ensembl.org). The upstream of each pair of primers overlapped the downstream of previous one. PCR was performed as described previously [To identify Piwil2 variants, GEM RT-PCR was performed as previously described . Five tidesigned , which ceviously .PIWIL2 (siE7), Exon 21 of human PIWIL2 (siE21) and negative control (NC) were purchased from Shanghai Gene Pharma. Transient transfections were carried out according to the manufacturer's instructions by using jetPRIME® transfection reagent (Polyplus-transfection SA). HEK-293T cells were plated into 6-well plates at 2 × 105 cells/well for 24 h prior to treatment with siRNAs  for STAT3, HLF, Exon 7 of human h siRNAs . TransfeAs previously described with modification , the celTMChromatin Immunoprecipitation Kit  was used for the ChIP assay. Briefly, 5 × 106 cells were fixed with 1% formaldehyde in PBS for 10 min to cross-link DNA with associated proteins and the cells were then washed and harvested in PBS. The pelleted cells were lysed on ice in SDS lysis buffer and then sonicated on ice for 10 seconds, 10 times . The sheared DNA was incubated with normal rabbit IgG  as a negative control, Polymerase II (Pol II) antibodies  as a positive control, and human STAT3 antibodies , or human HLF antibodies  overnight at 4°C, followed by 1 h of incubation with DNA/protein A-agarose beads. 10% of the sample was kept as input. Protein A-agarose beads pellets were sequentially washed with a low salt buffer, a high salt buffer, a LiCl washing buffer, and TE buffer. Protein-DNA complexes were eluted in a buffer containing 1% SDS and 0.1 M NaHCO3. Cross-linking was reversed with 200 mM NaCl overnight at 65°C, followed by incubation in a buffer containing 40 mM Tris-HCl, pH 6.5, 10 mM EDTA, and 20 μg of proteinase K for 2h at 55°C. DNA was purified with the TIANquick Midi Purification Kit . Prepared samples were amplified using specifically designed ChIP primers for DNA-PCR covering the region of interest  was considered statistically significant, and p < 0.01 (**) or < 0.001 (***) was considered as highly significant.


Coronaviruses (CoV) comprise a large group of emerging human and animal pathogens, including the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) strains. The molecular mechanisms regulating emerging coronavirus pathogenesis are complex and include virus–host interactions associated with entry, replication, egress and innate immune control. Epigenetics research investigates the genetic and non-genetic factors that regulate phenotypic variation, usually caused by external and environmental factors that alter host expression patterns and performance without any change in the underlying genotype. Epigenetic modifications, such as histone modifications, DNA methylation, chromatin remodeling, and non-coding RNAs, function as important regulators that remodel host chromatin, altering host expression patterns and networks in a highly flexible manner. For most of the past two and a half decades, research has focused on the molecular mechanisms by which RNA viruses antagonize the signaling and sensing components that regulate induction of the host innate immune and antiviral defense programs upon infection. More recently, a growing body of evidence supports the hypothesis that viruses, even lytic RNA viruses that replicate in the cytoplasm, have developed intricate, highly evolved, and well-coordinated processes that are designed to regulate the host epigenome, and control host innate immune antiviral defense processes, thereby promoting robust virus replication and pathogenesis. In this article, we discuss the strategies that are used to evaluate the mechanisms by which viruses regulate the host epigenome, especially focusing on highly pathogenic respiratory RNA virus infections as a model. By combining measures of epigenome reorganization with RNA and proteomic datasets, we articulate a spatial-temporal data integration approach to identify regulatory genomic clusters and regions that play a crucial role in the host’s innate immune response, thereby defining a new viral antagonism mechanism following emerging coronavirus infection. Rhinolophus), the SARS-CoV was thought to have adapted to Palm civets and raccoon dogs in open markets, before finally colonizing human populations [The severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002/2003, most likely in the Guangdong Province, China. From the initial outbreak, SARS-CoV rapidly spread across the globe causing 8000 infections and ~800 deaths in 28 countries; mortality rates approached 50% in aged individuals ,2,3. Froulations . More reulations ,7.In 2012, the antigenically distinct Middle East respiratory syndrome coronavirus (MERS-CoV) emerged and it is continuing to cause an ongoing outbreak in Saudi Arabia, the Arabian Peninsula and eastern Africa. Currently, 1626 infections in 26 countries have been reported with a mortality rate of ~30% . Most ofBoth pathogens cause severe lower respiratory tract infections, with the most severely impacted individuals developing acute respiratory distress syndrome (ARDS), a severe end-stage lung disease with poor treatment options and high fatality rates. Although asymptomatic infections were rare during the SARS-CoV epidemic, MERS-CoV infections frequently result in asymptomatic infections leading to asymptomatic spread. In general, the molecular mechanisms governing virus pathogenesis and disease severity remain understudied and vaccines and therapeutics are still not available ,10.Coronaviruses are enveloped RNA viruses, containing the largest currently-known single-stranded, positive-sense RNA genome, which ranges in length from 25.5 to 32 kb. The viral particles range from 70 to 120 nm and are surrounded by ‘spike’-shaped glycoproteins, which give the viruses their characteristic ‘corona-like’ appearance in the electron microscope. Coronaviruses encode for 7 to 14 open reading frames (ORFs). ORF1 compromises approximately two thirds of the genome at the 5′-end and consists of two overlapping ORFs, ORF1a and ORF1b. Both ORFs are translated into large polyproteins, the precursor of at least 16 nonstructural proteins (nsps) that encode the viral replication machinery, and other important functions in virus–host interaction, like innate immune antagonism and pathogenesis. By ribosomal frameshifting, ORF1 is expressed as two polyproteins designated ORF1a and ORF2b, which are processed by a papain-like protease (PLpro) and the viral main protease (3CLpro), into at least 16 proteins. Unique among RNA viruses, coronaviruses encode a proof-reading complex, consisting of nsp10/nsp14, nsp12 (replicase) and perhaps nsp16, which regulates fidelity . DownstrInnate immunity is one of the earliest barriers to coronavirus infection. Following infection, pathogen recognition receptors (PRRs) such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), Toll-like receptors  and other sensing molecules recognize pathogen-associated molecular patterns (PAMPs) in viral components, such as viral structure proteins or viral nucleic acid. Successful recognition initiates a signaling cascade that activates an antiviral state in the host. Several main players of innate immunity, such as signal transducer and activator of transcription 1 (STAT1), myeloid differentiation primary response gene 88 (MyD88), TLR4, TLR7 and TLR3/TIR-domain-containing adapter-inducing interferon-β (TRIF), function to dampen infection severity during coronavirus infection in vivo ,15,16,17Like other viral pathogens, coronaviruses such as SARS-CoV and MERS-CoV have evolved genetic functions that delay and/or antagonize pathogen recognition as well as ISG effector functions. SARS-CoV encodes several proteins that modulate innate immune signaling through the antagonism of the induction of interferon. As mentioned above, several nsps that are encoded by ORF1 (ORF1a/b), like nsp1, nsp3 papain-like protease, nsp14 and nsp16 antagonize various sensing or signaling programs and NFκβ, or function to cap viral messenger RNAs (mRNAs) and evade interferon-induced protein with tetratricopeptide repeats (IFIT) 1-3 ISGs ,23,24,25Epigenetic regulation bridges genotype and phenotype by changing the function of the gene locus without changing the sequence of the underlying DNA. Over the last decade, research efforts have revealed a dynamic range of epigenetic factors that shape and regulate chromatin status, leading to changes in host gene expression patterns, and therefore to alterations in phenotypes. Epigenetic modifications are significant in regulating cellular mechanisms and pathways during embryonic development, in memory function, in immunity and in disease ,33. WhilOver the last 20 years, the development of many biochemical and in particular high-throughput approaches have revolutionized our understanding of chromatin biology and function. Chromatin biology is now at the point where studies can be performed that use its tools to discover and validate new players and pathways in epigenetics and their role in a variety of biological disciplines, including developmental biology, oncology and infectious diseases, like bacterial and viral infections.The human genome project (HGP) was officially completed in 2003, providing the research community with a detailed map of the genetic organization and structure of the human genome as well as the epigenome . AnotherThe genetic information in eukaryotic cells is encoded in the chromosomes and mitochondrial DNA. Chromosomes exist in deoxyribonucleoprotein complexes called chromatin. Chromatin is found in two variations: the euchromatin and the heterochromatin, which were originally distinguished cytogenetically by Giemsa staining procedures. Darker staining heterochromatin indicates tightly packaged protein and nucleic acid complexes found at centromers and telomers. These contain mostly inactive satellite DNA as opposed to the lighter-stained loosely-packed euchromatin, which defines genome regions under active transcription and gene expression . Chromatin organization is complex and composed of a specialized set of proteins—the histones (H)—that organize the DNA into the nucleosome. The nucleosome is composed of a tightly-packed histone octamer consisting of the core histones H3, H4, H2A, and H2B with roughly 165 base pairs of DNA wrapped around it, much like beads on a string. This structure maintains stability and most importantly, protein and transcription factor accessibility to the DNA genome, allowing the chromatin to guarantee tight packaging of the genomic DNA, accurate replication, and distribution into the daughter cells during cell division, as well as transcriptional regulation of gene expression .The histone’s N-termini, the so-called histone tails, extend from the globular protein unit and as such are targets for post-translational modifications. At this time, several chemical modifications have been identified and characterized: lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, and lysine ubiquitination and sumoylation. These modifications are found on H2A, H2B, H3, and H4 histone subunits ,49. In 2The last decade has shown that this regulatory instance of nucleosomes and chromatin structure on the genome has emerged as a critically important determinant of cellular transcription, replication, and differentiation state.The histone tails of the nucleosome are subject to post-translational modulations. These modulations are covalently attached to the tails and include methylation of arginine residues, and methylation, acetylation, ubiquitination, phosphorylation, and sumoylation of serines (S) and threonines (T) A. Over the last decade, many modifications have been associated with active or non-active transcription. Modifications like the acetylation (ac) of H3 and H4, as well as the di- or tri-methylation (me) of H3 on lysine 4 (H3K4), are associated with an active transcription state. On the other hand, methylation of H3K9 and H3K27 are now associated with transcriptional repression of the particular gene . Based oLikewise, many modifications are almost uniquely associated with gene organization components, like promoters, TSSs, enhancers or gene bodies, slicing sites, and transcriptional end sites (TES). These histone marks help to organize the chromatin by modulating accessibility, thereby defining regulatory regions and elements, like promoters, enhancers and insulators, within the genome . Some of the histone mark distributions are uniquely associated with the transcription rates of particular genes. As mentioned above, main histone marks for regulating the TSS are H3K4me3 and H3K27me3; both modifications are exclusively found at the TSS and in the appropriate promoter region of the particular gene . H3K4me3Despite these differences, both modifications perform a crucial function in bivalent or ‘poised’ promoters . A promoThe state of the chromatin is modulated by a large number of proteins which can be seen as ‘writers’, ‘readers’, and ‘erasers’ . ‘WriterThe recognition of histone modifications is mediated by ‘readers’; proteins which are tightly regulated by phosphorylation and dephosphorylation through signaling pathways, recruitment and binding of co-factors, like transcription factors and adaptor proteins . OverallBased on the theory of the histone code, the same histone mark can have very different physiological outcomes depending on the location in the chromatin, the neighboring modifications, and the combination of modifications . In otheThis circumstance influences strongly the nature of the modification-binding proteins, the so-called readers and the following down-stream processes. This way, a rather small set of separate histone modifications results in a broad range of different outcomes for the cell. Overall, it is not surprising that these chromatin-modifying enzymes play an important role in maintaining chromatin structure and dynamics. It is important to note, however, that chromatin marks can be easily reversed. As a consequence, they can rapidly respond to external stimuli, thereby regulating the accessibility of the underlying DNA to the transcriptional machinery and ensuring the correct association of expressed genes in the appropriate situation ,63. In all mammalian cells, DNA methylation takes place post DNA replication. It occurs at the 5′ position of the cytosine ring within CpG nucleotides by adding a methyl group to create 5-methylcytosine (5mC). The modification is mediated by a family of enzymes, the DNA methyltransferases (DNMTs) B. DNMT3aThe presence of 5mC CGIs plays a critical role in regulation of gene expression. More than 60% of coding genes contain CGIs in promoter-associated regions. These CGIs are generally unmethylated, and therefore easily accessible to transcription factors and other chromatin-associated proteins for the expression of most housekeeping genes and other regulated genes . HoweverStarting in the early 2000s, the field of non-coding RNAs (ncRNAs) evolved from its historic origins as “junk RNA” and quickly expanded into its own field of research. Based on their function and their genetic origin ncRNAs can be divided into long non-coding RNAs (lncRNAs) and small non-coding RNAs (sncRNAs), based on whether each RNA is greater than or less than 200 bp in length . For manA number of high throughput technologies have been developed to study the epigenetic landscape and epigenetic modifications genome-wide and on sequence-specific levels . As a broad strategy to identify modifications of histones and the localization of histone marks across the genome, FAIRE was developed and has been applied to understand the chromatin status of target cells and of DNA viruses under different conditions of infection . FAIRE iThis technique is used to determine whether a given protein binds to, or is localized to, a specific DNA sequence in vivo. Cross-linked chromatin is sheared and the DNA-binding protein of interest is precipitated by using a protein-specific antibody. The bound DNA is then isolated by reverting the cross-linking and can be analyzed by utilizing microarrays (ChIP-on-chip), next-generation sequencing (ChIP-Seq), and quantitative PCR (ChIP-PCR) ,80. The The combination of the ChIP technology with next-generation sequencing allows and improves the characterization of binding sites for transcription factors and other DNA-binding proteins and the identification and characterization of DNA sequence motifs across the entire genome. The advancement in high resolution is crucial in profiling nucleosome positioning, the systematic cataloguing of histone modification patterns, and the establishment of precise histone modification maps throughout the entire genome ,80,82. To quantify the global distribution of active and inactive states of chromatin across the genome, several methods and technologies have been developed to measure the methylation status across the genomic DNA . RecentlHpaII/MspI, which are blocked or not blocked by CpG methylation. After treatment of the total DNA with the enzymes the distribution and extent of DNA methylation can be analyzed by quantitative PCR targeting regions of interest. However, the enzymatic approach is prone to bias based on the sequence specificity of the utilized restriction enzymes. This limits the analysis to certain sequence 3 motifs, which can be unevenly distributed across the whole genome [There are other complementary approaches to study the genome-wide methylation status of chromatin based on methylation arrays and methylation-sensitive and methylation-insensitive restriction enzymes. The methylation array technology is based on the Infinium MethylationEPIC technology (formerly the Infinium Human Methylation 450 array) which allows low sample input and fast read-out but has the disadvantage of not covering all annotated genes and shows bias which is based on the array technology. Another approach uses restriction enzymes, like e genome . Based on functional and spatial patterns, the immune system is broadly divided into two broad arms: the innate immune system and the adaptive immune system. Both systems include a wide range of cell types that communicate via direct cell–cell interactions or by the secretion of mediators such as interleukins, cytokines, and chemokines. The innate immune system not only regulates cell intrinsic defense programs in response to microbial attack but also has a critical role in activating and shaping the adaptive immune response. The innate immune system accomplishes this by being able to generate and drive a transcriptional response that is both cell- and stimulus-specific. Based on these mechanisms, the signal-specific induced response guarantees initiation of the appropriate innate and adaptive immune responses that have the greatest potential to successfully control a particular pathogen ,88,89. Much of the innate immune response is regulated by membrane-bound and intracellular PAMPs, like TLRs, RIG-I, MDA-5 and cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING) and other sensors that detect invading pathogens ,91. ThesA basic feature of innate immune cells is the ability to start a transcriptional response program that is specific to the stimulus, and then mounting a signal with a high degree of cell type and stimulus specificity ,97,98.➔Recruitment of transcription factors/machinery;➔Prevention of unwanted expression of potent mediators; and➔Repression or activation of secondary gene programs ,99.Recent studies have involved epigenetic factors in every aspect of activation and shaping innate and adaptive immune responses. Major contributions are the:The main players of the innate immune system are primary response genes like IFN and tumor necrosis factor (TNF), which are rapidly induced and whose promoters show the characteristics of a poised promoter. Often, the promoters of these genes also contain CpG islands which are resistant to epigenetic modifications like DNA methylation and histone tail modification. These common modifications can be found at promoters of highly active transcribed genes, which also show high levels of RNA Pol II occupancy . To the Two cell types of the innate immune system, dendritic cells and macrophages, are the primary sensors of ‘danger’ signals. Once these cells are activated, it is especially important that their signals are both cell-specific and stimulus-specific to ensure the initiation of a temporal and spatial response. These cell-specific signals can be mediated through cell–cell contact or by secretion of IFN and TNF. Thus, the ability of their epigenome to change within minutes after a stimulus is not just essential for initiating a rapid antiviral host response but is also essential to ensure a persistent and specific defense response. This way, epigenetic mechanisms are responsible for the priming and the memory of these responses and for guaranteeing a functional and highly regulated host response beyond the initial activation wave.Besides the histone marks for poised promoters H3K4me3 and H3K27me3, an important role as a major player in regulating the activation of IFN has been described for H3K9me2. Fang et al. correlated the levels of H3K9me2 modification with the level of interferon expression in vitro. H3K9me2 is a repressive histone mark that contributes to DNA methylation and heterochromatin formation and thereby prohibits histone tail acetylation by recruiting the transcriptional repressor of the heterochromatin protein 1 family . HoweverOn the other hand, H3K4me3 is a histone modification exclusively found at active promoters and is therefore often enriched in promoter regions regulating TLRs. A recent study has shown that 60 min after lipopolysaccharide (LPS) stimulation of macrophages and dendritic cells, the overall histone acetylation and the binding of polymerase II (Pol II) at the specific promoters was tremendously increased, demonstrating an efficient and specific induction of the innate immune response by epigenetic control mechanisms . Interferons are important mediators of an antiviral state and initiators of pathogen-driven immune response by the inactivation of ISGs ,105. TheMarazzi et al. have demonstrated that the highly pathogenic H3N2 influenza A virus inhibits the initiation of the host innate immune response in part by interfering with the epigenetic control of gene expression. Using histone mimicry, it has been proposed that the carboxyterminus of the H3N2 nonstructural protein NS1 shares homologue sequences with the aminoterminus of the histone H3 tail . EssentiPreviously, we compared ISG profiles of pathogenic influenza viruses and coronaviruses in Calu3 cells, a human airway epithelial cell line, by using a transcriptomics and proteomics dataset . The infIn our laboratory, by using ChIP-PCR approaches, we could determine differential occupancy of histone marks at the promoters of ISG genes. We showed that the promoter regions of ISG genes contained more histones with active marks of H3K4me than the repressive H3K27me3 mark, therefore favoring open chromatin and promoting active transcription and ISG expression during H1N1-2009 and SARS-CoV infection. In contrast, infection of Calu3 with the highly pathogenic HPAI and MERS-CoV resulted in increased levels of H3K27me3 and decreased levels of H3K4me3 occupancy at the promoter regions of subsets of specific ISGs, which were not induced, demonstrating that these viruses have developed antagonistic mechanisms to specifically target the IFN arm of the innate immunity . We have expanded this dataset by using a genome-wide ChIP-Seq approach. This allows us to choose any set of cellular effector molecules and to study their histone modification profile during infection. While histone mimicry has been identified for H3N2 virus, the responsible binding motif is not contained in NS1 protein encoded by H5N1-VN1203, although the NS1 protein may contribute to this phenotype . This suCalu3 cells are a continuous human airway epithelial cell line that can be differentiated in ciliated cells and are commonly used to study respiratory cell function under different physiological stresses and conditions . By utilHost–pathogen relationships are plastic and dynamic. In particular, selective pressure put on the pathogen by the host increases the degree of plasticity and adaptability of the pathogen. On the other hand, the host phenotype is altered by the pathogen and the pathogen’s virulence, which leads to a co-adaptation that influences pathogen and host equally. For a successful viral life cycle, viruses have co-evolved with the host, which often means that the virus has to adjust to the host’s immune system, resulting in distinct mechanisms to repress or evade the host’s immune response. Co-evolution with the host may well have expanded the capabilities of epigenetic regulation, adding additional mechanisms and sources of dynamic reversible phenotype variation . The evoSeveral complementary approaches can be combined to study, define, and map the epigenetic landscape during coronavirus infection and other highly pathogenic human viruses . CombiniRecent advancements through basic research in the understanding of the mechanisms involved in viral chromatin modification in lytic viruses have opened a new window into previously unknown mechanisms of viral antagonism and host–virus interactions, including genetic factors that influence both protective or pathogenic host responses. The subsequent identification and improved understanding of these mechanisms open new avenues for the development of antiviral drugs by illuminating new targets for specific inhibitors. Established model systems for latent and persistent viruses such as human immunodeficiency virus 1 (HIV-1) and herpesviruses have already demonstrated modulation of the viral infection by chromatin ,120. As


Understanding the mechanisms that regulate cell type-specific transcriptional programs requires developing a lexicon of their genomic regulatory elements. We developed a lineage-selective method to map transcriptional enhancers, regulatory genomic regions that activate transcription, in mice. Since most tissue-specific enhancers are bound by the transcriptional co-activator Ep300, we used Cre-directed, lineage-specific Ep300 biotinylation and pulldown on immobilized streptavidin followed by next generation sequencing of co-precipitated DNA to identify lineage-specific enhancers. By driving this system with lineage-specific Cre transgenes, we mapped enhancers active in embryonic endothelial cells/blood or skeletal muscle. Analysis of these enhancers identified new transcription factor heterodimer motifs that likely regulate transcription in these lineages. Furthermore, we identified candidate enhancers that regulate adult heart- or lung- specific endothelial cell specialization. Our strategy for tissue-specific protein biotinylation opens new avenues for studying lineage-specific protein-DNA and protein-protein interactions.DOI:http://dx.doi.org/10.7554/eLife.22039.001 The diverse cell types of a multicellular organism share the same genome but express distinct gene expression programs. In mammals, precise cell-type specific regulation of gene expression depends on transcriptional enhancers, non-coding regions of the genome required to activate expression of their target genes . EnhanceEp300 are marginal for robust ChIP-seq, particularly from tissues, leading to low reproducibility, variation between antibody lots, and inefficient enhancer identification  has been used to map potential enhancer. DNase hypersensitivity , H3K27acfication .Mammalian tissues are composed of multiple cell types, each with their own lineage-specific transcriptional enhancers. Thus defining lineage-specific enhancers from mammalian tissues requires developing strategies that overcome the cellular heterogeneity of mammalian tissues, particularly when the lineage of interest comprises a small fraction of the cells in the tissue. Past efforts to surmount this challenge have taken the strategy of purifying nuclei from the cell type of interest using a lineage-specific tag. For instance, nuclei labeled by lineage-specific expression of a fluorescent protein have been purified by FACS . This meEp300 in which the protein is labeled by the bio peptide sequence  and skeletal muscle lineage enhancers active during embryonic development. Extending the approach to adult organs, we defined adult EC enhancers, including enhancers associated with distinct EC gene expression programs in heart compared to lung. Analysis of motifs enriched in EC or skeletal muscle lineage enhancers predicted novel transcription factor motif signatures that govern EC gene expression.Here, we report an approach to identify murine enhancers active in a specific lineage within a tissue. We developed a knock-in allele of sequence . Cre rectope tag , allows Ep300 allele, fbEp300, in which FLAG and bio epitopes  from fb/fb; Rosa26BirA/BirAEp300 mice. We then performed Ep300fbbiotin-mediated chromatin precipitation followed by sequencing (bioChiP-seq), in which high affinity biotin-streptavidin interaction is used to pull down Ep300 and its associated chromatin  embryos in biological duplicate  were common between tissues  functional terms relevant to the respective tissue . We compared the genome-wide signal of Ep300seq data  was lessseq data  comparedectively , suggestfb bioChiP-seq data. The VISTA Enhancer database  of these regions overlapped the regions previously reported to be bound by Ep300 using antibody ChIP-seq.We analyzed the prediction of active enhancers by our Ep300database  containsRecently human and mouse Ep300 and H3K27ac ChIP-seq data from fetal and adult heart were combined to yield a ‘compendium’ of heart enhancers, with the strength of ChIP-seq signal used to provide an ‘enhancer score’ ranging from 0 to 1 that correlated with the likelihood of regions covered in the VISTA database to show heart activity . We compfb regions common to both replicates. In comparison, only 6/40 (15%) intersected the regions detected previously by Ep300 antibody ChIP-seq. Few heart enhancers were found in the regions unique to Ep300 antibody ChIP-seq (11/185 VISTA and 2/40 non-VISTA), compared to regions unique to Ep300fb bioChIP-seq (36/185 VISTA and 20/40 non-VISTA). We conclude that Ep300fb ChIP-seq predicts heart enhancers with sensitivity that is superior to antibody-mediated Ep300 ChIP-seq.Ep300 antibody ChIP-seq was one of the criteria used to select some of the test regions in the VISTA Enhancer database; as an independent test free of this potentially confounding effect, we searched the literature for other heart enhancers that were confirmed using the transient transgenic assay. We identified 40 additional heart enhancers. Of these, 24 (60%) intersected the Ep300Other chromatin features have been used to predict transcriptional enhancers. We compared the accuracy of Ep300 bioChiP-seq to other chromatin features for heart enhancer prediction. To map accessible chromatin, we performed ATAC-seq (assay for transposable-accessible chromatin followed by sequencing) on E12.5 cardiomyocytes. E12.5 heart ChIP-seq data for modified histones  were obtained from publicly available datasets . Using a machine learning approach and the VISTA enhancer database as the gold standard, we evaluated the accuracy of each of these chromatin features, compared to Ep300 bioChiP-seq, for predictive heart enhancers . This anfb requires co-expression of the biotinylating enzyme BirA. We reasoned that Ep300fb bioChIP-seq could be targeted to a Cre-labeled lineage by making BirA expression Cre-dependent. Therefore, we established fsBirARosa26, in which BirA expression is contingent upon Cre excision of a floxed-stop (fs) cassette (fsBirARosa26 expression of BirA was Cre-dependent (fb biotinylation (Tek regulatory elements (Tg(Tek-cre)1Ywa/J; also known as Tie2Cre), BirA was expressed in endothelial and blood lineages  (tm3(cre)Sor/J    , Myf5tm3lineage) , or Tg(Equitous) . For theChIP-seq . Viewingenhancer , as wellTie2Cre or Myf5Cre) Ep300fb signal was at least 1.5 times the ubiquitous Cre (EIIaCre) Ep300fb signal (Tie2Cre (Ep300-T-fb) and 1292 regions with enriched signal in Myf5Cre (Ep300-M-fb), compared to 17382 regions with Ep300fb occupancy detected with ubiquitous biotinylation  had been described previously  of regions identified by lineage-selective, Cre-directed bioChIP-seq have appropriate and reproducible in vivo activity.To further evaluate the enhancer activity of Ep300-T-fb regions, we searched the literature and the VISTA Enhancer database  for genoeviously  . Clustering and selection of representative non-redundant motifs left 40 motifs that were enriched in either Ep300-T-fb or Ep300-M-fb, or both . Many clTie2Cre-marked blood and EC lineages .The database used for our motif search included 315 heterodimer motifs that were recently discovered through high throughput sequencing of DNA concurrently bound by two different transcription factors . This alTie2Cre-enriched motifs within the central 200 bp of Ep300-T-fb regions. Whereas randomly selected 12 bp sequences from these regions exhibited a distribution of scores heavily weighted towards low conservation values, sequences matching Tie2Cre-enriched motifs showed a bimodal distribution consisting of highly conserved and poorly conserved sequences , and transcriptional activity was measured by luciferase assay, normalized to the enhancerless promoter-luciferase construct . The welenhancer  robustlyTo further assess and compare the transcriptional activity of these heterodimer motifs, we cloned upstream of luciferase 3x repeated motifs into a consistent DNA context that had minimal endogenous enhancer activity . The ETSTogether, unbiased discovery of cell type specific enhancers coupled with motif analysis identified novel transcription factor signatures that are likely important for gene expression programs of blood, vasculature, and skeletal muscle.ECs in different adult organs have distinct gene expression programs that underlie organ-specific EC functions . For exafb bioChIP-seq would allow us to identify enhancers linked to organ-specific EC gene expression. For these experiments, we used Tg(Cdh5-cre/ERT2)1Rha   heart and lungs from fb/+; VEcad-CreERT2+; Rosa26fsBirA/+Ep300 mice and performed bioChiP-seq. Triplicate repeat experiments were highly reproducible   to driven in ECs ; unlike oducible . Inspect tissues . Interes disease . On the richment .10.7554/fb bioChIP-seq results, regions bound by Ep300fb in either heart (14251) or lung (22174) were rank-ordered by the heart to lung Ep300fb signal ratio  had higher median mRNA transcript levels than lung  had higher median mRNA transcript levels in lung than heart. We then focused on genes with selective expression in heart or lung. More decile 1 Ep300fb regions were associated with genes with heart-selective expression than lung-selective expression, and more decile 10 Ep300fb regions were associated with genes with lung-selective expression  were significantly associated with terms relevant to coronary artery disease, such as ‘coronary heart disease', 'arteriosclerosis’, and ’myocardial infarction’, whereas genes neighboring decile 10 regions  were less enriched. Conversely, genes neighboring decile 10 regions were selectively associated with hypertension and cerebral artery disease.We analyzed the GO terms that are over-represented for genes neighboring Ep300elopment . AnalysiTo gain insights into transcriptional regulators that preferentially drive heart or lung EC enhancers, we searched for motifs with differential enrichment in decile one compared to decile 10. Using decile one regions as the foreground sequences and decile 10 regions as the background sequences, we detected significant enrichment of the TCF motif in regions preferentially occupied by Ep300 in heart ECs , consistbio epitope tag. Furthermore, we show that combining this strategy with the Ep300fb knockin allele permits efficient identification of cell type-specific enhancers. The bio epitope has been knocked into a number of different transcription factor loci (Rbpj), 025980 (Ep300), 025983 (Mef2c), 025978 (Nkx2-5), 025979 (Srf), and 025977 (Zfpm2)), and these alleles can be combined with Cre-activated BirA to permit lineage-specific mapping of transcription factor binding sites. Cell type-specific protein biotinylation will also be useful for mapping protein-protein interactions in specific cell lineages.Here we show that Cre-mediated, tissue-specific activation of BirA permits high affinity bioChiP-seq of factors with a tor loci  and JackUsing this technique, we identified thousands of candidate skeletal muscle and EC enhancers and showed that many of these candidate enhancers are likely to be functional. Furthermore, we showed that the technique can identify tissue-specific enhancers in postnatal tissues, and identified novel candidate enhancers that regulate organ-specific endothelial gene expression. These enhancer regions will be a valuable resource for future studies of transcriptional regulation in these systems.Large scale identification of tissue-specific enhancers will facilitate decoding the mechanisms responsible for cell type-specific gene expression in development and disease. Here. By analyzing these candidate regulatory regions, we revealed novel transcriptional regulatory motifs that likely participate in skeletal muscle development, angiogenesis, and organ specific EC gene expression. Our recovery of a previously described FOX-ETS heterodimer binding site in EC enhancers  validateAdditional novel heterodimer motifs, such as ETS:EBox, ETS:HOMEO, and ETS:Tbox, were enriched in EC enhancers from both developing and adult ECs. This suggests that these motifs, and potentially the protein heterodimers that were reported to bind them , are impA limitation of our current protocol is the need for several million cells to obtain robust ChIP-seq signal. Optimization of chromatin pulldown and purification through application of streamlined protocols or microfluidics , combinefbEp300 mice were generated by homologous recombination in embryonic stem cells. Targeted ESCs were used to generate a mouse line, which was bred to homozygosity. This line has been donated to Jackson labs (Jax 025980). The fsBirARosa26 allele was derived from the previously described fsTRAPRosa26 mouse . AP mouse   and PECAM1  antibodies and imaged by confocal microscopy (Olympus FV1000).For immunostaining, cryosections were stained with HA  and PECAM1  antibodies and imaged by confocal microscopy (Olympus FV1000).fb/fb;Rosa26BirA/BirAEp300 female mice were hormonally primed and mated overnight with eight weeks old fb/fb;Rosa26BirA/BirAEp300 male mice. Uteri were collected on 3.5 dpc and embryos were flushed out under a microscope. After removal of zona pellucida by treating the embryos with Tyrode’s solution , the embryos were cultured in ESC medium , supplemented with 50 µM PD98059  for 7–10 days. The outgrowth was dissociated with trypsin, and the cells were cultured in ESC medium in 24 well plates to obtain colonies, which were then clonally expanded. Five male ESC lines were retained for further experiments. Pluripotency of these ESC lines was confirmed by immunostaining for pluripotency markers Oct4, Sox2, and SSEA, and they were negative for mycoplasma.ESCs were derived as described previously . Five wefb/fb;Rosa26BirA/BirAEp300 ESC lines were cultured in 150 mm dishes to 70–80% confluence. Crosslinking was performed by adding formaldehyde to 1% and incubating at room temperature for 15 min. Chromatin was fragmented using a microtip sonicator (QSONICA Q700). The chromatin from 3 ESC lines was pooled for replicate one and from the other 2 ESC lines for replicate 2. Ep300fb and bound chromatin were pulled down by incubation with streptavidin beads (Life Technologies #11206D).The five Candidate motif sequences were synthesized as oligonucleotides  and clonImmunoblotting was performed using standard protocols and the following primary antibodies: GAPDH, Fitzgerald, 10 R-G109A ; Ep300, Millipore, 05257 (1:2000 dilution); BirA, Abcam, ab14002 (1:1000 dilution).bioChIP-seq was performed as described previously , with mifb/fb;Rosa26BirA/BirAEp300 males. Cells were dissociated in a 2 mL glass dounce homogenizer  and then cross-linked in 1% formaldehyde-containing PBS for 15 min at room temperature. Glycine was added to final concentration of 125 mM to quench formaldehyde. Chromatin isolation was performed as previously described (E12.5 embryonic forebrain and ventricle apex tissues were isolated from Swiss Webster (Charles River) females crossed to escribed  30 forebescribed . For H3KmTmGRosa26 females crossed to fb/fb;Rosa26fsBirA/BriA;Tie2CreEp300 males. Tie2Cre positive embryos were picked under a fluorescence microscope for further experiments. Crosslinking and sonication were performed as described above. We used 15 Tie2Cre positive embryos for each bioChIP replicate. We used 750 µL Dynabeads M-280 Streptavidin beads for Ep300fb pull-down; this amount was determined by empiric titration.E11.5 embryos were isolated from pregnant mTmGRosa26 females crossed to fb/fb;Rosa26fsBirA/BriA;Myf5CreEp300 males. Cre positive embryos were picked under a fluorescence microscope for further experiments. five embryos were used for each bioChIP replicate and incubated with 750 µl Dynabeads M-280 Streptavidin beads for Ep300fb pull-down.E13.5 embryos were isolated from pregnant fb/fb;Rosa26fsBirA/mTmG;VEcad-CreERT2Ep300 pups were given two consecutive intragastic injections of 50 µl tamoxifen (2 mg/ml in sunflower seed oil) on postnatal day P1 and P2 to induce the activity of Cre. The lungs and heart apexes were collected when the mice were eight weeks old. Cross-linking and sonication were performed as described above. Six mice were used for each replicate. We used 600 µl (heart) or 1.5 mL (lung) streptavidin beads for the bioChIP.Libraries were constructed using a ChIP-seq library preparation kit (KAPA Biosystems KK8500). 50 ng of sonicated chromatin without pull-down was used as input.Sequencing (50 nt single end) was performed on an Illumina HiSeq 2500. Reads were aligned to mm9 using Bowtie2  using deTo associate genomic regions to genes, we used Homer’s AnnotatePeaks  to selecE12.5 heart ventricles were dissociated into single cell-suspensions using the Neonatal Heart Dissociation Kit (Miltenyi Biotec #130-098-373) with minor modifications from the manufacturer’s protocol. Embryonic tissue samples were incubated twice with enzyme dissociation mixes at 37°C for 15 min with gentle agitation by tube inversions between incubations. Cell mixtures were gently filtered through a 70 μm cell strainer and centrifuged at 300 x g, 5 min, 4°C. Red blood cells were lysed with 10X Red Blood Cell Lysis Solution (Miltenyi #130-094-183) and myocytes were isolated using the Neonatal Cardiomyocyte Isolation Kit (Miltenyi Biotec #130-100-825). 75,000 isolated cardiomyocytes were used for each ATAC-Seq experiment. Libraries were prepped as previously described .Translating ribosome RNA purification (TRAP) and RNA-seq were performed as described . The expGene Ontology analysis was performed using GREAT . ResultsHomer  was usedfsTrap/+;Tie2Cre embryos were isolated from Swiss Webster strain pregnant females crossed to Rosa26fsTrap/Trap;Tie2Cre males. TRAP RNA from 50 embryos was pooled for RNA-seq. The polyadenylated RNA was purified by binding to oligo (dT) magnetic beads . RNA-seq libraries were prepared with ScriptSeq v2 kit  according to the manufacturer’s instructions. RNA-seq reads were aligned with TopHat (Translating ribosome RNA purification (TRAP) was performed as described . E10.5 Rh TopHat  and exprh TopHat . Adult oEnhancer regions in the VISTA database were used as the golden standard. Each chromatin feature that was analyzed was from E12.5 heart. Data sources are listed below under ‘Data Sources’. For each chromatin factor, average read intensity in each VISTA regions with or without heart enhancer activity was calculated and used as the starting point for machine learning. We used the weighted KNN method as the classifier. The parameters used were: 10 nearest neighbors, Euclidean distance, and squared inverse weight. The classifier was trained and tested using 10-fold cross validation. ROC (Receiver operating characteristic) curve was used to evaluate the predictive accuracy of each factor, as measured by the area under the ROC curve (AUC).The phastCons score for the multiple alignments of 30 vertebrates to mouse mm9 genome was used as the conservation score . SequencHeart and endothelial cell enhancers were identified by searching PubMed for ‘heart enhancer' or ’endothelial cell enhancer’, respectively, and then manually curating references for mouse or human enhancers with appropriate activity in murine transient transgenic assays.fb/fb;Rosa26BirA/BirAEp300 ESCs; (2) FB_Ep300_bioChIP from E12.5 fb/+;Rosa26BirA/+Ep300 forebrains; (3) FB_H3K27ac_ChIP from E12.5 fb/+;Rosa26BirA/+Ep300 forebrains; (4) He_Ep300_bioChIP from E12.5 fb/+;Rosa26BirA/+Ep300 heart apex; (5) He_H3K27ac_ChIP from fb/+;Rosa26BirA/+E12.5 Ep300 heart apex; (6) EIIaCre_Ep300_bioChIP from E11.5 fb/+;Rosa26BirA/+Ep300 whole embryos; Myf5Cre_Ep300_bioChIP from E13.5 fb/+;Rosa26fsBirA/+;Myf5CreEp300 whole embryos; Tie2Cre_Ep300_bioChIP from E11.5 fb/+;Rosa26fsBirA/+;Tie2Cre+Ep300 whole embryos; Tie2Cre-TRAP and Tie2Cre-Input from E10.5 fsTrap/+;Tie2Cre+Rosa26 whole embryos; and ATAC-seq from wild-type E12.5 ventricular cardiomyocytes. These data are available via the Gene Expression Omnibus (accession number GSE88789) or the Cardiovascular Development Consortium server .Sequencing data generated for this study are as follows: (1) ES_Ep300_bioChIP from The following public data sources were used for this study: ES_Ep300_ChIP, GSE36027; ES_Ep300_input, GSE36027; E12.5_Histone_input, GSE82850, E12.5_H3K27ac, GSE82449; E12.5_H3K27me3, GSE82448; E12.5_H3K36me3, GSE82970; E12.5_H3K4me1, GSE82697; E12.5_H3K4me2, GSE82667; E12.5_H3K4me3, GSE82882; E12.5_H3K9ac, GSE83056; E12.5_H3K9me3, GSE82787. Antibody Ep300 ChIP-seq data on forebrain  and hearhttps://b2b.hci.utah.edu/gnomex/; login as guest; instructions for reviewer access are provided in an supplementary file).Sequencing data generated for this study are available via the Gene Expression Omnibus (accession number GSE88789) or the Cardiovascular Development Consortium server ; Joshua D Wythe (Reviewer #2).Thank you for submitting your article "Mapping cell type-specific transcriptional enhancers using high affinity, lineage-specific p300 bioChIP-seq" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:We appreciate the novel in vivo biotinylation strategy, via generation of a flag-bio tagged allele of endogenous p300 in combination with a published R26-lsl-birA line and multiple tissue-specific Cre drivers, which demonstrated that p300 occupancy predicts lineage-specific transcription. Validation of the novel p300 biotinylation system in vivo through rigorous criteria demonstrate the utility of this reagent in predicting enhancer usage. We believe this is a high quality manuscript that will be of interest to the general community. However, there are a number of points that should be addressed, which are detailed below. Most importantly, please address the issue of how "neighboring genes" were determined and also describe the metrics by which enhancers were determined be positive in transgenic embryos.Essential revisions:1) The authors state, after performing the TRAP-seq, that they compared the expression in ECs of "neighboring genes". However, we could not find within the manuscript any bioinformatic criteria explaining how they decided what a neighboring gene was. The genomic criteria  for suggesting a relationship of p300 occupancy and translating ribosomal-bound message should be clearly stated . Additionally, they should discuss many p300-T-fb enriched regions were near genes that weren't actively translating in the EC sample.2) Overall, the criteria of 2 embryos (out of at least 5 PCR positive embryos) leading to a call as a "positive" enhancer lacks any interpretation or comment on the strength of the enhancer, as some of their elements  appear very weak and others appear somewhat non-specific . Additionally, scoring an enhancer as positive when only 3 out of 22 showed EC activity seems arbitrary . The transgenic results are somewhat puzzling, as the Sox7 enhancer  appears  Essential revisions:1) The authors state, after performing the TRAP-seq, that they compared the expression in ECs of "neighboring genes". However, we could not find within the manuscript any bioinformatic criteria explaining how they decided what a neighboring gene was. The genomic criteria  for suggesting a relationship of p300 occupancy and translating ribosomal-bound message should be clearly stated . Additionally, they should discuss many p300-T-fb enriched regions were near genes that weren't actively translating in the EC sample.The definition we used was the TSS closest to the p300 peak, without a maximal distance. This definition has been widely used, for example Blow et al., Nature Genetics, 2010. In the revision we also repeated the comparison with a series of different maximal distance thresholds and showed that the overall conclusion is not affected by the specific maximal threshold used .As suggested, we also compared expression of p300-associated genes to those without a p300 peak. We found that p300-associated genes were more highly expressed.Finally, we calculated the fraction of genes with and without associated p300 that were not detected within actively translating transcripts. We found that a higher fraction of p300-associated genes were detectably expressed. However, not all p300-associated genes were expressed, and not all expressed genes were p300-associated. This likely reflects multiple mechanisms of transcriptional activation, as well as imperfect rules used to associate genes to p300 regions.2) Overall, the criteria of 2 embryos (out of at least 5 PCR positive embryos) leading to a call as a "positive" enhancer lacks any interpretation or comment on the strength of the enhancer, as some of their elements  appear very weak and others appear somewhat non-specific . Additionally, scoring an enhancer as positive when only 3 out of 22 showed EC activity seems arbitrary . The transgenic results are somewhat puzzling, as the Sox7 enhancer  appears We agree that scoring regions as positive or negatively overly simplifies the activity, which also includes spatial pattern, specificity, and strength. For this reason, we provide the raw data on the regions, as both whole embryo images and sections. However, scoring regions as positive or negative simplifies summarizing the activity of many regions, and comparison to prior studies.We adopted the definition of an active enhancer that has been used by the Pennacchio and Visel groups. In their series of studies, an active enhancer was defined as one that drove expression in the target tissue . In other words, a “heart enhancer” is an enhancer that drives expression in the heart, without regard to its specificity for heart. The Pennacchio/Visel definition of a positive enhancer is one that shows expected activity in three or more independent embryos. This demonstrates reproducibility. In our original manuscript, we used a criteria of two or more independent embryos, but in the revision we adhered to the definition of 3 or more for calculation of the validation rate. We retained the description of the results for the additional two enhancers with reproducible activity in two but not 3 embryos as these we feel it likely that this activity is biologically meaningful. We rewrote the text to reflect these changes and to remove the statement that 55% activity is consistent with previously reported validation rates.For Sox7, the enhancer had activity in the endocardium of the outflow tract, not the atria. This is accurately reflected in the tissue sections.