RNA polymerase III (Pol III) promoters, such as 7SK, U6, and H1, are widely used for the expression of small noncoding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a noncoding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter, we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly used H1 promoter in terms of not only activity and small promoter size but also concerning safety by exclusive expression of the desired therapeutic transcript (either pol II or pol III but not both). Type 3 RNA polymerase (Pol III) promoters, such as 7SK, U6, and H1, are popular for the expression of small noncoding RNAs because of their robust level of transcription in all cell types and defined transcription initiation and termination sites, such that a precise (therapeutic) transcript is made . These pWe previously reported that these Pol III promoters can also load Pol II and synthesize lengthy translation-competent mRNAs . These tA). The basal region is composed of a proximal sequence element (PSE) and a TATA box that are separated by a spacer. The DSE encodes an octamer motif and a Staf binding site (Staf). However, the relative position and exact nucleotide sequence of these elements differ between H1 and 7SK . To measure the promoter activities, the H1 variants were designed to express both a 41-nt small RNA (N41) by Pol III and a 1650-bp luciferase mRNA by Pol II . Equal molar amounts of the DNA constructs were transfected into human embryonic kidney 293T (HEK293T) cells, and cell extracts were prepared after 2 days. Pol II activity was quantified by luciferase assays, and Pol III activity was assessed by a Northern blot for the N41 transcript.A previous H1 promoter study showed that the important Pol III transcription elements are located within 100 bp (H1-100) immediately upstream of the transcription initiation site . RegardlFig. 1left panel of Fig. 1B, quantitation is shown in Fig. 1C). This result indicates that the sequence between position −124 and −74 is critical for Pol III activity. To map the critical elements in more detail, we introduced serial 5-bp truncations in H1-124. The constructs with deletions up to H1-99 did fully sustain Pol III transcriptional activity , but the smaller promoter variants either lost most (H1-94 and H1-87) or nearly all  Pol III activity. The 5-bp deletion going from H1-99 to H1-94 disrupts the octamer motif at position 97 to 90 , thus confirming the importance of this DSE enhancer for Pol III transcription. These results are also in line with the report that identified H1-100 as the minimal Pol III promoter . The first four deletion variants remain active, and the Pol II promoter activity is in fact somewhat increased such that H1-224 is 1.5- and 2-fold more active than H1-374 and H1-124, respectively. The Pol II activity drops to a level slightly below that of H1-374 for the subsequent deletions in H1-174 to H1-99. Deletion of the octamer in H1-94 and H1-89 nearly abrogated Pol II transcription, even to a greater extent than the concomitant drop in Pol III transcription. Full Pol II inactivity is scored for H1-84, H1-79, and H1-74. The overall Pol II/III activity profiles are thus quite similar, suggesting that Pol II and III complexes may use the same promoter elements and indicating that the octamer is critical for both activities. In summary, these initial results show that H1-99 (hereafter referred to as the H1-core) contains the minimal sequences required for both Pol II and III activity.Intriguingly, the Pol II activity profile based on luciferase assays for the set of truncated H1 promoters showed a similar trend as the Pol III activity C. The fiupper panel of Fig. 2A), but the latter lacks prominent Pol II activity. We reasoned that swapping of promoter elements from 7SK into H1 may transfer selective Pol III specificity onto the H1-core promoter.The H1-core promoter is particularly attractive for applications using viral vector systems that prefer a small Pol III expression cassette. However, its promiscuous Pol II activity is usually unwanted and can be problematic in the presence of a downstream RNA effector function  or an open reading frame that is translated into an unwanted and possibly immunogenic protein. We therefore set out to engineer a Pol III-specific H1-core promoter. There is architectural similarity between the H1 and 7SK promoters . We identified three hybrid constructs with significantly increased Pol III activity . M3, in which the PSE was replaced, showed a 4-fold increase, indicating that the 7SK PSE supports Pol III transcription to a greater extent than the H1 PSE. Intriguingly, this substitution did not affect the Pol II activity, suggesting that Pol II to III competition is not the underlying mechanism for the increased Pol III activity. Constructs M10 and M11 carry multiple 7SK elements  and enhance the Pol III activity to an even greater extent than M3 .We thus generated H1-core mutants by introducing individual or multiple 7SK elements  and subsequently analyzed the Pol II/III activity profile. We transfected these 11 mutant constructs (M1-11) into HEK293T cells and measured the Pol II and III activity by luciferase assays and Northern blotting, respectively. The mutants varied profoundly in N41 expression, which reflects the Pol III strength, compared with the parental H1-core promoter . Most mutants displayed reduced Pol II activity compared with H1-core, except for M3 that exhibited similar activity and M4 that showed increased activity. The most robust reduction in Pol II activity was apparent on combinatorial replacement of multiple elements (M5-7 and M9-11), with M11 showing the most dramatic reduction (11-fold), followed by M10 (8.5-fold). The M11 construct is the most Pol III-specific promoter, exhibiting the highest Pol III activity combined with a profoundly reduced Pol II activity. We note that M11, in which multiple H1 elements  were replaced, has more than 50% of its sequence derived from 7SK. It may thus be appropriate to call M11 a hybrid H1/7SK promoter, although this is a semantic issue. M11 characteristics are summarized in Pol II activity of the mutant constructs also varied considerably and did not seem to strictly follow the Pol III activity profile C. Most mA–C, quantification is shown in lower panels of each graph). We observed a very similar Pol III activity profile in these cell types: the H1-224 and H1-core promoters have similar Pol III activity, which is boosted in M10 and especially in M11. Perhaps remarkable, the M11 promoter is more active than the two parental H1-core and 7SK promoters.In HEK293T cells, M11 is the promoter of choice for Pol III applications because of its high Pol III activity and minimal Pol II activity. To allow applications in different fields, it is important that this hybrid promoter functions similarly in different cell types, each with a distinct pool of transcription factors. To test this, we compared the Pol III activity of M11 with H1-224, H1-core, M10, and two popular Pol III promoters (7SK and U6) in three cell lines of various origins. The DNA constructs were transfected into HEK293T, colon cancer cell lines (HCT116), and JC53-bl [clone 13] (TZM-b1) cells, and RNA was extracted after 2 days for Northern blot analysis (D), HCT116 (E), and TZM-bl cells (F). A similar activity profile was observed in all cell types. H1-224 and H1-core have high Pol II activity, which is significantly reduced for M10 and especially M11. Note that M11 harbors similarly low Pol II activity as the 7SK promoter  and measured luciferase activity after 2 days (A). The luciferase signal varied widely across the cell types, but a clear general trend was apparent for the H1 variants. We observed that H1-224 is roughly 2-fold more active than H1-core, in line with the results obtained in HEK293T cells (C). Both H1-224m and H1-corem showed enhanced Pol II activity compared with the respective parental promoters in most of the tested cell lines, albeit to various extents. Mutation of the TATA box enhanced Pol II activity to a greater extent in H1-corem than in H1-224m, but H1-corem remained weaker than H1-224 and H1-224m. Thus, H1-224m is the H1 variant with the highest Pol II activity, whereas H1-corem is the promoter of choice when the promoter size forms a restriction.Because of the unusual and robust Pol II activity of the small H1 promoter, we decided to study its potential as promoter of choice for mRNA/protein production. Construct H1-224 is clearly the most Pol II active, but the H1-core promoter, which is less than half the size, also shows decent activity. In thinking about further improvement of the Pol II activity, we reasoned that inactivation of the TATA box would not only knockout Pol III activity but also boost Pol II activity as we reported for H1-230 . We therr 2 days A. The lu3T cells C. Both Hm. The EF1 and CMV promoters direct the highest luciferase expression level across the cell lines and are also the largest. EF1-core is 5- to 35-fold less active than the full-length EF1 promoter but exhibits similar Pol II activity as PGK. SV40 early is the weakest promoter. Remarkably, the small H1-224m promoter is comparable in Pol II activity to EF1-core and PGK, thereby confirming that H1-224m is a bona fide Pol II promoter that sustains high level Pol II transcription.To get a better grasp of the absolute Pol II activity, we compared the four H1 variants to five classical Pol II promoters . The size of these promoters is listed in B). Lentiviral vectors containing promoter-GFP expression cassettes were constructed and transduced into six cell lines. The CMV construct was excluded because the same sequences are already present in the lentiviral vector backbone, which may cause unwanted recombination events during lentiviral vector production and subsequent transduction , indicating that H1-224m is the most active H1 variant across the cell lines tested. All four H1 variants exhibit greater activity than the SV40 promoter and are comparable in strength to the EF1-core and PGK promoters. In conclusion, the novel H1-224m and H1-corem promoters exhibit robust Pol II activity that is maintained in a chromosomal context, arguing that both promoters could become the promoter of choice for Pol II applications. H1-224m offers the advantage of potency, but H1-corem would be useful when the size of the transgene cassette is a concern, e.g., in viral vectors. H1-224m and H1-corem characteristics are summarized in As durable gene therapy applications will likely use transgene cassettes that become stably integrated into the human genome, we next assessed the activity of the novel H1 promoters in the chromosomal context B. Lentivsduction . We usedThe H1 promoter is a type 3 RNA Pol III promoter and is popular for the expression of small noncoding RNAs . It was The dual Pol II/III activity of the H1 promoter is an undesired feature in many applications. For example, in gene silencing applications where the H1 promoter is routinely used to express short shRNA transcripts, the silencing efficacy could be compromised as both Pol II and Pol III transcripts will fold the same hairpin structure that actually can refold to form an extended intermolecular base-pairing interaction between the two transcripts. In this way, the extended Pol II transcript can interfere with the folding and function of the short Pol III shRNA transcript. In addition, extended Pol II transcription initiated from the H1 promoter may run into downstream Pol II transcription units and cause head-to-head collision of the polymerase complexes or transcriptional interference , 10. FinAs dual promoter activity is generally undesired, we aimed to generate Pol II-and Pol III-specific H1 variants. Engineering of a Pol III-specific H1 promoter was achieved by replacing H1 promoter elements by those of 7SK, a popular type 3 Pol III promoter with high Pol III specificity . ReplaceOur results also provide insight into why the complete basal domain of 7SK is required for maximal Pol III activity and specificity. PSE-containing promoters come in two flavors: with or without a TATA box . PromoteChromatin immunoprecipitation sequencing experiments demonstrated that PSE-containing promoters without TATA almost exclusively recruit Pol II, whereas those with a TATA box recruit Pol III . The H1 TTG), which may explain their reduced Pol III activity.Unexplained remains the low Pol III activity of M8 and especially M9, in which the spacer or spacer and TATA box were replaced by 7SK elements, where one may have expected higher Pol III activity. Close inspection of the relevant sequences provides a possible solution. The H1 PSE that we replaced is TCACCATAAACGTGAAAT , whereasSome of the results may also suggest that the mechanism of Pol II/III specificity is even more complex. For example, in mutant M3, we have replaced the H1 PSE by that of 7SK and kept the H1 spacer length and TATA box. This mutant exhibits four times increased Pol III specificity over the parental H1 promoter by specifically increasing Pol III activity. Thus, the 7SK PSE element intrinsically sustains higher Pol III activity and specificity compared with the H1 PSE. Mutant M4 in which we replaced the H1 TATA box is also noteworthy as the 7SK TATA box enhances promoter activity, although not specificity, which may also contribute to the enhanced Pol III activity of M10 and M11.In conclusion, we propose that the complete basal 7SK region is required for maximal Pol III activity/specificity for at least three reasons: (1) the intrinsic high Pol III activity/specificity of the 7SK PSE; (2) the optimal 7SK spacer length for PIC binding to the PSE and TATA box; and (3) the strong 7SK TATA box that generally enhances Pol II and III activity.m and H1-224m. We previously used this TATA trick to render the H1 promoter strictly Pol II specific , but this usually results in a greatly reduced expression of the second transgene. It may thus be beneficial that the second transgene has its own promoter. When such size restrictions are apparent, a small and highly active Pol II promoter like H1-224m or H1-corem is highly recommended.The Pol II-specific H1 promoter may have some specific advantages over regular Pol II promoters for gene expression purposes. First, the Pol II-specific H1-224romoters . H1-corer family . The limm and H1-corem is independent of the cell lineage. This may have been expected as the physiological role of the H1 promoter is to drive the expression of the RNA component of RNase P, the enzyme involved in tRNA maturation in all cell types  encoding full-length H1-374 promoter-N41 sequence. This DNA fragment was ligated into the pGL3-basic vector using NheI and NcoI restriction enzyme sites by Gibson cloning, which constitutes the backbone construct pGL3-H1-374. To generate H1 constructs with 50-bp serial promoter truncations, the H1-truncation variants were PCR amplified and then ligated into pGL3-H1-374 on the NheI and XhoI sites. To generate H1-mutant constructs, gBlock gene fragments encoding the H1-mutant sequences  and S2 w6 cells in 2 ml medium were seeded per well in 6-well plate. For luciferase reporter assays, 1.5 × 105 cells in 0.5 ml medium were seeded per well in 24-well plate.HEK293T, C33A, HCT116, HeLa, TZM-bl, and Vero cells were grown as monolayer in DMEM (Invitrogen) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), streptomycin (100 U/ml), and minimal essential medium nonessential amino acids at 37 °C and 5% carbon dioxide. HCT116 is a human colon cancer cell line and TZM-bl is a HeLa cell derivative that was engineered by amphotropic retroviral transduction to express CD4, CCR5, and CXCR4. SupT1 cells were grown in advanced RPMI  supplemented with L-glutamine, 1% FCS, penicillin (30 U/ml), and streptomycin (30 μg/ml) at 37 °C and 5% carbon dioxide. Cells were trypsinized and seeded 1 day before transfection. For Northern blotting experiments, 1 × 1032P]-labeled decade RNA marker (Life Technologies) was used for size estimation. To check for equal sample loading, the gel was stained in 2 mg/ml of ethidium bromide for 20 min and visualized under UV light. The RNA in the gel was transferred to a positively charged nylon membrane (Boehringer Mannheim). Locked nucleic acid oligonucleotides (Pol47: 5'-ATTACTACTGCCCCTTCAC-3') were 5'-end labeled with [32P]-ATP  using kinaseMax kit (Ambion). The unincorporated nucleotides were removed by Sephadex G-25 spin columns (Amersham Biosciences). The membrane was incubated overnight with purified locked nucleic acid oligonucleotides in 10 ml ULTRAhyb hybridization buffer at 42 °C. The membrane was washed with low  and high  stringency buffers. The signals were captured by Typhoon FLA 9500  and quantitated using ImageJ software.Northern blotting for N41 detection was performed as described previously. Briefly, equimolar amounts of the DNA constructs  were transfected using Lipofectamine 2000 (Invitrogen). Total cellular RNA was extracted 2 days post-transfection using the mirVana miRNA isolation kit (Ambion), and 5 μg of total RNA with loading buffer was preheated for 5 min at 95 °C and then resolved in a 15% denaturing polyacrylamide gel . The γ[Equimolar amounts of H1-based constructs  and 2 ng of Renilla luciferase plasmid were cotransfected into cells in 24-well plates using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. At 48 h post-transfection, luciferase activity was measured with the dual-luciferase reporter assay kit , according to the manufacturer's protocol. The ratio of firefly to Renilla luciferase was calculated to represent the relative firefly luciferase activity. Three independent transfections were performed.Lentivirus was produced similarly as described previously . BrieflyPromoter activity from an integrated chromosomal position was measured 5 days after transduction with lentiviral vectors that express GFP, which was monitored by flow cytometry. Harvested cells were washed with fluorescence-activated cell sorting buffer (PBS with 2% FCS), and data acquisition was performed by an LSR Fortessa (BD Biosciences) interfaced with the fluorescence-activated cell sorting-Diva software system. The mean fluorescence intensity of GFP was analyzed by FlowJo version X software .All representative data are contained within the article.The authors declare that they have no conflicts of interest with the contents of this article.


NANOG is a core transcription factor (TF) in embryonic stem cells (ESCs) and primordial germ cells (PGCs). Regulation of the NANOG gene by TFs, epigenetic factors, and autoregulatory factors is well characterized in ESCs, and transcriptional regulation of NANOG is well established in these cells. Although NANOG plays a key role in germ cells, the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied. Therefore, we investigated the mechanism that regulates transcription of the chicken NANOG (cNANOG) gene in PGCs and ESCs.cNANOG by 5′-rapid amplification of cDNA ends PCR analysis. Then, we measured the promoter activity of various 5′ flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay. cNANOG expression required transcriptional regulatory elements, which were positively regulated by POU5F3 (OCT4) and SOX2 and negatively regulated by TP53 in PGCs. The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element (CCAAT/enhancer-binding protein (CEBP)-binding site) in ESCs. Furthermore, small interfering RNA-mediated knockdown demonstrated that POU5F3, SOX2, and CEBP played a role in cell type-specific transcription of cNANOG.We first identified the transcription start site of trans-regulatory elements control transcription of cNANOG in a cell type-specific manner. This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.We show for the first time that different  Gene transcription is mainly regulated by transcription factors (TFs) that bind to specific DNA sequences  located in the promoter regions of genes . Many TFNANOG, OCT4, and SOX2 control maintenance of pluripotency. Core TFs play an important role in establishing control of gene expression programs that define the identity of embryonic stem cells (ESCs) [NANOG gene is important for acquisition of pluripotency by ESCs and embryonic germ cells (EGCs) [NANOG that are required to maintain the self-renewal and pluripotency of ESCs [NANOG expression are Octamer- and Sox-binding elements present at the upstream of transcription start site (TSS) in its promoter region, and these elements are positively regulated by binding of OCT4 and SOX2 in ESCs [ZFP143 to the proximal region of the NANOG promoter regulates NANOG expression by modulating OCT4 binding [cis-regulatory elements of NANOG, including SP1/SP3-, SALL4-, and BRD4-binding sites, have been identified as positive regulators [P53-binding sites negatively regulate NANOG expression to induce differentiation of ESCs [NANOG expression plays a critical role in determining the fate of pluripotent cells.In mammals, core TFs such as s (ESCs) –16. In ps (EGCs) –19. Seve of ESCs –22. The  in ESCs , 23. Dir binding . In addigulators –27. On t of ESCs . TherefoNANOG, POU5F3, and SOX2, and their expression controls transcription of germness-related genes in these cells [NANOG plays an essential role during early germ cell development as a key TF required for the formation of PGCs and maintenance of early germ cells [NANOG-deficient PGCs reportedly undergo apoptotic death [NANOG regulates PGC-specific epigenetic programming and global histone methylation [NANOG is evolutionarily conserved in mammals and most of the lower vertebrate species, including chicken. In particular, NANOG orthologs from chicken, zebrafish, and axolotl are highly conserved [NANOG is crucial to maintain pluripotency and self-renewal of chicken ESCs [NANOG is expressed during chicken intrauterine embryonic development and is exclusively expressed in PGCs from Hamburger and Hamilton stage 5 (HH5) to HH8. Therefore, NANOG is also important to maintain pluripotency and cell proliferation in chicken intrauterine embryos and PGCs [PGCs express several pluripotency-related TFs such as se cells , 29. NANrm cells , 31. NANic death . It was hylation , 34. NANonserved –37. Simiken ESCs . NANOG iand PGCs , 38.NANOG in chicken PGCs, the molecular mechanism that regulates its transcription in these cells has not been fully clarified. This study investigated enhancers and suppressors of the proximal promoter region of the chicken NANOG (cNANOG) gene in PGCs and ESCs. Furthermore, we investigated transcriptional control of cNANOG expression via trans-regulatory elements and TFs, which are important for its cell type-specific expression.Despite the exclusive expression of cis- and trans-regulatory elements that are important for modulating transcription of the NANOG gene in chicken PGCs using the dual luciferase assay and transcriptome analysis. The management of White Leghorn (WL) chickens was approved by the Institute of Laboratory Animal Resources, Seoul National University, Korea (SNU-190401-1-1). The chickens were housed according to standard procedures at the University Animal Farm, Seoul National University, Korea.This study investigated the cNANOG gene (Gene ID: 100272166), 5′-RACE PCR was performed using a GeneRacer Kit  following the manufacturer’s instructions. Gene Racer RNA Oligo-ligated mRNA was reverse-transcribed into cDNA. Single-stranded cDNA served as the template in nested 5′-RACE PCR using the GeneRacer 5′ Primer and reverse gene-specific primers (GSPs). The cNANOG reverse GSP was 5′-GTC TGC AGT AGG GCT AGT GGC AGA GTC T-3′. The RACE products were identified by DNA sequencing analysis. To confirm the quality of adapter-ligated RNA, 5′-RACE PCR was performed with a chicken β-actin reverse GSP, which was 872 bp in size and contained 828 bp of β-actin and 44 bp of the GeneRacer RNA Oligo.To determine the TSS of the cNANOG gene was amplified using genomic DNA extracted from adult chicken blood and inserted into the pGEM-T Easy vector . Primer sets were used to clone differently sized fragments of the cNANOG promoter (Table cNANOG gene were inserted between the KpnI and XhoI sites of the pNL1.2 vector (Promega).To construct NanoLuc luciferase expression vectors, the 5′ flanking region of the er Table . Then, dcNANOG promoter activity. Prepared cells were seeded in a 96-well plate and co-transfected with the pGL4.53 firefly luciferase (Fluc) and pNL1.2 (NlucP/cNANOG RE) NanoLuc luciferase (Nluc) plasmids using Lipofectamine 2000 (Invitrogen). After transfection for 24 h, cells were lysed with lysis buffer containing Fluc substrate. Fluc signals were then quenched, followed by reaction with Nluc substrate. Signals in arbitrary units (AU) of Nluc and Fluc were measured using a luminometer . Promoter activities were calculated by determining the ratio of Nluc/Fluc signals in AU. pNL1.2, an empty vector, was used as a negative control. All reporter assays were repeated at least three times.The Nano-Glo Dual Reporter Assay System (Promega) was used to assess L-glutamine, 1× nonessential amino acids, β-mercaptoethanol, 10 mmol/L sodium pyruvate, 1× antibiotic-antimycotic , and 10 ng/mL human basic fibroblast growth factor (MilliporeSigma). PGCs were sub-cultured onto mitomycin-inactivated mouse embryonic fibroblasts at an interval of 5–6 d via gentle pipetting.WL PGCs were maintained and sub-passaged in KnockOut DMEM  supplemented with 20% fetal bovine serum , 2% chicken serum , 1× nucleosides (MilliporeSigma), 2 mmol/L Chicken ESCs were generously provided by Dr. Bertrand Pain (INSERM-INRAE). These cells were maintained and sub-passaged as previously described . Briefly2 and 60–70% relative humidity.Chicken DF-1 cells  and chicken embryonic fibroblasts (CEFs) were cultured as negative controls. Chicken DF-1 cells were maintained and sub-passaged in DMEM (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1× ABAM (Thermo Fisher-Invitrogen). CEFs were derived from 6-day-old WL embryos and maintained in DMEM (Hyclone) supplemented with 10% fetal bovine serum (Hyclone) and 1× ABAM (Thermo Fisher-Invitrogen). All chicken cells  were cultured in an incubator at 37 °C under an atmosphere of 5% COwww.genomatix.de/) using TRANSFAC matrices , and PROMO 3.0, which uses TRANSFAC version 8.3 .TF-binding sites were predicted by MatInspector, a Genomatix program (http://http://www.ambion.com) (Table CEBP) genes, including CEBPA, CEBPB, CEBPD, CEBPG, and CEBPZ, and TP53 using Lipofectamine 2000 (Invitrogen). After siRNA transfection for 24 h, the knockdown efficiencies of the predicted TFs and the effects on cNANOG gene transcription were measured by quantitative reverse-transcription PCR (RT-qPCR).siRNAs targeting predicted TFs were designed using siRNA Target Finder (m) Table . CommercGAPDH) expression as an endogenous control.Total RNA was extracted from test samples using TRIzol reagent  in accordance with the manufacturer’s protocol and reverse-transcribed using the Superscript III First-Strand Synthesis System (Invitrogen). The PCR mixture contained 2 μL of PCR buffer, 1 μL of 20× EvaGreen qPCR dye , 0.4 μL of 10 mmol/L dNTP mixture, and 10 pmol each of gene-specific forward and reverse primers Table . RT-qPCRP < 0.05 indicated statistical significance.Statistical analysis was performed using GraphPad Prism . Significant differences between groups were determined by a one-way analysis of variance with Bonferroni’s multiple comparison test and the unpaired t-test. A value of cNANOG gene, we first determined the TSS of this gene by 5′-RACE PCR analysis. A 470 bp PCR product was obtained using a reverse GSP that targeted exon 2 of the cNANOG gene . Several TF-binding sites, including AIRE-, NFY-, CMYB-, ISL1-, E2F-, and OSNT -binding sites, were identified in this region  significantly reduced relative luciferase activity in PGCs. Moreover, relative luciferase activity was reduced significantly more by mutation of the SOX2-binding site alone than by mutation of the POU5F3-binding site alone in PGCs   Fig. a. Among cription , 40, 41.cNANOG promoter via deletion of the 5′ upstream region. Among the four constructs, the − 407/+ 70 bp, − 377/+ 70 bp, and − 312/+ 70 bp fragments exhibited significantly reduced cNANOG promoter activity in ESCs  to identify important TF-binding sites that maintain the basal activity of the  bp Fig. c. To exaent Fig. d. Mutatiion Fig. e. Taken CEBP genes  was significantly higher in ESCs than in other cells. By contrast, expression of POU5F3 and SOX2/3 was significantly higher in PGCs and ESCs than in DF-1 cells and CEFs. Expression of POU5F3 and SOX3 did not differ between PGCs and ESCs, while SOX2 was significantly upregulated in PGCs. Additionally, expression of TP53 was significantly higher in PGCs than in other cells  containing wild type binding sites, whereas, knockdown of TP53 is significantly increased the activity of the cNANOG promoter − 250/+ 70 bp fragment in PGCs (Fig. CEBPA, CEBPB, CEBPD, CEBPG, and CEBPZ in ESCs dramatically reduced the activity of the cNANOG promoter − 442/+ 70 bp fragment containing wild type CEBP binding site (Fig. cNANOG by directly interacting with its promoter in a cell type-specific manner.We further examined whether these TFs affect the transcription of ion Fig. a. KnockdSCs Fig. b–f. We aSCs Fig. . KnockdoGCs Fig. a and b. ite Fig. c. These NANOG is important to maintain pluripotency in mammalian pluripotent cells during embryonic development [NANOG was also important for maintaining the pluripotency in PGCs and ESCs [NANOG gene in chicken PGCs and ESCs remain unclear. In this regard, we characterized the structure of cNANOG and analyzed its promoter activity in chicken PGCs and ESCs.The homeodomain TF elopment . Therefoelopment , 22, 23.elopment  and mainelopment . It has elopment . In chicand ESCs , 38, 44.cNANOG under the control of the proximal regulatory region located within 130 bp upstream of the TSS in PGCs. Furthermore, we identified the regulatory region of cNANOG located within 442 bp upstream of the TSS in ESCs. Moreover, we showed that TP53 suppresses cNANOG transcription in PGCs. These results suggest that the cNANOG promoter functions in a cell type-specific manner. Similarly, Yeom et al. reported that the mouse Oct4 gene contains two separate regulatory elements [Oct4 gene is regulated in a stage-specific manner. Our findings indicate which elements are critical for gene expression in PGCs. This is the first report of a transcriptional regulatory factors of NANOG that is differentially active in a cell type-specific manner in chicken.We successfully transcribed elements . The disNANOG gene. Most of the positive regulation of NANOG transcription has been discovered in the proximal region, which encompasses OCT3/4 and SOX2 in mouse ESCs. This region is strongly conserved in various mammalian species [NANOG. Therefore, OCT3/4 and SOX2 play an important role in regulation of the NANOG gene promoter in mammalian ESCs [POU5F3, SOX2/3, KLF2, and SALL4 are highly expressed in chicken ES cells and PGCs [POU5F3- and SOX2-binding sites in the proximal region significantly reduced cNANOG promoter activity in PGCs. Although the DNA sequences of POU5F3 and SOX2, which are recognized by mouse core pluripotency factors, are not well conserved in chicken, POU5F3 and SOX2 are key regulators of cNANOG transcription. Further investigation by the electrophoretic mobility shift assay and chromatin immunoprecipitation sequencing is required to determine the core TFs in chicken PGCs.Many researchers have studied mammalian ESCs to determine which core factors regulate the  species , 23. Mutand PGCs . Accordiand PGCs . In the Drosophila melanogaster, TP53 is reportedly involved in elimination of excess PGCs during PGC development [TP53 binds to the NANOG promoter and suppresses NANOG expression for maintenance of genome stability in ESCs [TP53-binding site negatively controlled NANOG transcription in chicken PGCs. Therefore, we propose that TP53 plays important roles in the regulation of NANOG transcription to maintain genome stability in PGCs.Programmed death of PGCs is essential to remove abnormal, misplaced, and excess cells during PGC development and this is important to establish the next generation. In elopment  and, mouelopment . In addi in ESCs . InteresCEBPB interacts with p300 to modulate histone acetylation [p300 is a co-activator that binds to NANOG for maintenance of pluripotency in ESCs [CEBPA, CEBPB, CEBPD, CEBPG, and CEBPZ were significantly upregulated in chicken ESCs. In addition, knockdown of these TFs dramatically decreased transcription of cNANOG in chicken ESCs. These results suggest that CEBP in chicken ESCs participate in regulation of cNANOG transcription by directly interacting with putative binding sites in the cNANOG promoter.tylation , and p30 in ESCs . In our NANOG is conserved in mammals, although DNA sequences of regulation factors differ between chicken and mammals. Typically, mammalian PGCs can be induced by cell signaling [Nanog is key regulator of PGCs-like cells independent of BMP4 and Wnt signals by activating the expression of germ cell-specific TFs [VASA and DAZL in germ plasm [NANOG in chicken PGCs has been investigated by our group to understand the molecular mechanisms involved in the specification of germ cells [NANOG in chicken germ cell specification is still unclear. In this study, we shown that chicken NANOG has differential regulatory roles in PGCs and ESCs, even though cNANOG promoter region sharing the common transcription factor binding sites. These finding provided insights into germ cell and stem cell-specific transcriptional regulatory mechanisms.As described above, transcription regulation of cignaling . Interesific TFs . On the rm plasm , 54. Recrm cells . HowevercNANOG gene differs between PGCs and ESCs. We showed that the cNANOG gene is positively regulated by POU5F3 and SOX2 and negatively regulated by TP53 in PGCs, while it is positively regulated by CEBP in ESCs. Collectively, these findings aid understanding of transcriptional regulation of the cNANOG gene in PGCs and ESCs (Fig. This study demonstrated that the proximal regulatory region of the SCs Fig. .Fig. 9A


Human embryogenesis and the development of its most unique product, the human brain, are believed to be precisely regulated by factors adopted during human evolution that differentiate us from other species. Nevertheless, increasing evidence shows an unthinkable “alien” factor may have contributed to the process. Pervasive horizontal gene transfer between species mediated by retroviruses is such a defining factor of evolution . RetroviENVV1, due to a one nucleotide insertion leading to a frame shift. Both ENVV1 and ENVV2 show placenta-specific expression in humans and a baboon species [Although mostly silenced in somatic cells, increased activities of retroviral and other transposable elements have been observed during embrogenesis and in pluripotent stem cells. These retro elements include LINE-1 –8, the o species . HERV-R  species .HERV transcripts are increased during cell transformation and in human pluripotent stem cells . We compin vivo, competes with sycytin-1 for its receptor (ASCT-2), and inhibits sycytin-1 mediated trophoblast cell-cell fusion [The mechanisms of retro elements influencing embryonic development are just being realized. The LTRs function in a stage-specific manner by activating transcription, altering protein-coding sequences, producing noncoding RNAs, and even supporting the evolution of new protein-coding genes, resulting in mRNAs, lncRNAs, or proteins with regulatory roles (46). In the 2 cell-stage, many gene’ transcripts are initiated from ERV LTRs, such as MERVL (47). Some ERV families contain preserved splice sites that join the ERV segment with non-ERV exons in their genomic vicinity (48). The envelope protein of HERV-W is expressed in mammals as syncytin. It has been adopted by human species as an essential cellular protein for trophoblast and placental development ,17,18. Al fusion . HERV-H l fusion  and playl fusion . HERV-H l fusion . Along tl fusion .gag, pro, pol, and env genes [env containing plasmid, the neuronal induction process was inhibited, as indicated by the cells’ morphology and lower nestin expression. Nestin is a marker of neural stem cells but is not expressed in pluripotent stem cells. On the contrary, inhibition of HML-2 env in iPSCs facilitated induction of neural stem cells and eventually neuronal differentiation [Recently acquired HERV-K subfamily HML-2 has nearly full-length viral sequences with open reading frames for nv genes . HML-2 anv genes , mesenchnv genes  and certnv genes . We founnv genes . When HMntiation .LINE-1s are abundant retrotransposons that comprise approximately 20% of mammalian genomes. Activation of LINE-1 occurs mainly in early embryonic development and during hippocampal neurogenesis . Active There are many mechanisms that regulate HERV expression throughout development and in differentiated cells. Transcriptional activity of HERVs is regulated by binding of epigenetic modifiers and transcription factors to the long terminal repeats (LTRs) that flank the 5’ and 3’ ends of the viral genome. Many HERVs exist as only solo LTRs, most likely due to homologous recombination between the 5’ and 3’ LTRs that results in deletion of the internal coding sequence . TranscrAberrant expression of endogenous retroviral genes at an inappropriate embryonic stage or in tissues where their transcription is normally suppressed is strongly associated with development of disease. Therefore, control of HERV expression by epigenetic regulation is a crucial part of tissue homeostasis. DNA (CpG) methylation performed by DNA methyltransferase 1 (DNMT1) is an important mechanism of retroelement silencing, particularly for the most intact and transcriptionally active member, HERV-K/HML-2; the LTRs of HERVs are often hypermethylated in healthy tissues, and loss of CpG methylation results in increased HML-2 transcription that may contribute to oncogenesis ,35. In aIt has been hypothesized that nucleosomal positioning plays a role in HERV transcription by a mechanism observed in latent HIV infections. In a transcriptionally inactive HIV promoter, there is a nucleosome positioned immediately downstream of the transcription start site, which prevents access to transcriptional machinery. Binding of specific transcription factors to enhancers in the LTR or differential methylation or acetylation of the histones within the nucleosome induces repositioning of the nucleosome to allow for transcription . HERV-K/PRODH gene exhibits tissue-specific enhancer activity with maximal expression in hippocampus. The enhancer activity is regulated by methylation and involves the binding of SOX2. PRODH is the mitochondrial proline dehydrogenase that regulates proline catabolism. PRODH is critical for normal CNS function and has been associated with schizophrenia [Recent work in breast cancer cell lines has revealed a role for the progesterone-response element and the octamer-binding transcription factor 4 binding sites in some HML-2 LTRs . An isofophrenia .env resulted in high levels of rpS6 and LPCAT1 expression, further implying that HML-2 incorporation in the human genome caused the increased activity of mTOR in human stem cells and may have played a role in human brain evolution [Retroviral envelope proteins are known to facilitate cell-to-cell adhesion and in some cases cause cell fusion. For example, HERV-W syncytin leads to fusion of trophoblasts to form the placenta ,45. Intevolution .The neuronal gene Arc/Arg3.1 plays an essential role in the consolidation of synaptic plasticity and long-term memory  and the In the presence of RNA, purified Arc protein spontaneously assembles into oligomeric, virus-like capsid structures . Arc proThe RNA of LINE-1 is localized to the nucleus in embryonic stem cells and pre-implantation embryos. DMNT-1 is responsible for the high CpG methylation of LINE-1 lineages that are younger than 12.5 million years, corresponding to hominoid-specific elements, including many that are human-specific. Deletion of DMNT-1 resulted in hypomethylation of DNA and chromatin remodeling and increased activation of these younger LINE-1s, which are responsible for transcriptional enhancement of many protein-coding genes involved in neuronal functions and psychiatric disorders . LINE-1 LINE-1 functions as a nuclear RNA scaffold, which recruits Nucleolin and Kap1/Trim28 to repress Dux, the master activator of a transcriptional program specific to the 2-cell embryo. In humans, there is a strong association between the establishment of accessible chromatin and embryonic genome activation. A large proportion of the early activated genes and HERVs are bound by DUX4 and become accessible as early as the 2- to 4-cell stages .Many transcription factors important to neural development regulate, or are regulated by, LINE-1. For example, one LINE-1 element contains overlapping Sox2 and T-cell factor/lymphoid enhancer factor (TCF/LEF)-binding sites (Sox/LEF), which make up a transcriptional site regulated by Wnt and β-catenin signals. Wnt3a ligand and β-catenin increased acetylated histone H3 levels in the LINE-1 genomic region, inducing the active chromatin state, causing an increase in the amount of LINE-1 ORF2 mRNA in neuronal stem cells. Wnt3a ligand and β-catenin signaling may also affect nearby genes such as DCX and neurogulin-4, which are important in regulating neural development . The SOXWe have presented evidence from recent literature that retroviral elements incorporated in the human genome have played important roles during human evolution. Although most of the insertions have been silenced, a few of the retroviral elements such as HERVs and LINE-1s can still be active in human embryogenesis and play important functions in human development. The fine regulation of HERV-K and LINE-1 are especially important in human brain development. HERV-K activation is a key event that regulates mTOR activation, which differentiates human beings from other primates. Additionally, the increased LINE-1 activity during neural genesis mediates adult neuron diversity in the brain.The regulation of the retroviral elements is influenced by a general mechanism regulating DNA methylation status, which is lower in certain stages of embryogenesis. There are also regulatory mechanisms specific to species, tissues, even cell types leading to activation of individual retroviral elements. It is notable that there are also interactions among retroviral elements, which may be either positive or negative associations. Our knowledge of the role of retroviral elements in brain development and disease is still rudimentary but this is a fertile and promising area of investigation that can provide novel insights into disease pathogenesis and identify new targets for treatment.


Timely and accurate RNA synthesis depends on accessory proteins that instruct RNA polymerase (RNAP) where and when to start and stop transcription. Among thousands of transcription factors, NusG/Spt5 stand out as the only universally conserved family of regulators. These proteins interact with RNAP to promote uninterrupted RNA synthesis and with diverse cellular partners to couple transcription to RNA processing, modification or translation, or to trigger premature termination of aberrant transcription. NusG homologs are present in all cells that utilize bacterial-type RNAP, from endosymbionts to plants, underscoring their ancient and essential function. Yet, in stark contrast to other core RNAP components, NusG family is actively evolving: horizontal gene transfer and sub-functionalization drive emergence of NusG paralogs, such as bacterial LoaP, RfaH, and UpxY. These specialized regulators activate a few (or just one) operons required for expression of antibiotics, capsules, secretion systems, toxins, and other niche-specific macromolecules. Despite their common origin and binding site on the RNAP, NusG homologs differ in their target selection, interacting partners and effects on RNA synthesis. Even among housekeeping NusGs from diverse bacteria, some factors promote pause-free transcription while others slow the RNAP down. Here, we discuss structure, function, and evolution of NusG proteins, focusing on unique mechanisms that determine their effects on gene expression and enable bacterial adaptation to diverse ecological niches. Escherichia coli and Bacillus subtilis are only 50% identical. Differences in cellular transcriptional machinery are thought to reflect unique regulatory constraints imposed by diverse habitats. In support of this notion, even basal general transcription factors that assist RNAP during each step of the transcription cycle are not conserved between kingdoms. The sole exception to this trend is a transcription elongation factor NusG  carry out the first step of gene expression, transcription of a DNA template into an RNA copy. Reflecting their common evolutionary origin in the last universal common ancestor (LUCA) and the basic mechanism of RNA synthesis, RNAPs share an overall architecture and structural elements that play key roles in the assembly of transcription complexes, substrate selection and catalysis, interactions with nucleic acids, etc. ,b. Howevtor NusG .N-utilization substance) proteins have been identified genetically based on their requirement for the coliphage λ development  composed of RNAP and NusABEG proteins  bind to an evolutionary conserved site on the largest RNAP subunit . The NusE. coli NusG discovery by the data in support of its role as a termination-promoting factor. NusG is essential in wild-type E. coli  and a C-terminal domain with a 27-residue long Kyrpides-Ouzounis-Woese (KOW) motif common among RNA-binding proteins  domain  domain ; in meta) domain . Apart f) domain .All NGN domains make very similar contacts to two conserved RNAP elements , the claIn addition, some NGNs make sequence-specific contacts to the non-template DNA strand in the transcription bubble of the transcription elongation complex . The NGN binding site on the TEC is structurally analogous to binding sites of transcription initiation factors in promoter complexes; e.g., bacterial σ factors recognize non-template DNA sequences and an adjacent region on the β’ CH during promoter-dependent initiation . ConsequE. coli NusG, interactions between the KOW domain and Rho facilitate termination  element for loading onto the nascent RNA and subsequent TEC dissociation, Rho has recently emerged as a global multi-functional regulator  protein, a prototypical xenogeneic silencer, to limit unwanted gene expression. In E. coli, Rho and H-NS co-localize on the chromosome  from Rho is conferred by multicomponent TACs. Recently solved cryo-EM structures of these TACs  and raise a possibility that an alternative mechanism of transcription noise silencing operates in these species.In wild-type  strains  and confsubtilis , in whicsubtilis , early ssubtilis , and mossubtilis . In contess RNAs , Rho-depelements . TogetheE. coli NusG is bridging the RNAP and the ribosome  comprise a set of alternative transcription elongation factors that compete for the transcribing RNAP, an analogy further strengthened by their recruitment to the same site on RNAP  which are present in leader regions of several operons silenced by NusG and Rho  provides additional time for RfaH, which is present in few copies/cell, to find its target; and (ii) presents the ops bases in a small hairpin, with a conserved T residue flipped out for specific recognition by RfaH   by RfaH . This isant NusG , RfaH biant NusG , essentisequence .ops signal in the first place? Unlike NusG, in which the RNAP-binding site on the NGN domain is exposed, this site is blocked by the KOW domain in free RfaH folds into a β-barrel when expressed alone, separated from the NGN domain upon proteolytic cleavage of the linker, or as a result of interface-destabilizing substitutions . Deuteroparation . The α→βparation .E. coli RfaH is the only known transformer protein. However, it is possible that other KOW domains are capable of transformation. In particular, an amazingly broad repertoire of known cellular targets of eukaryotic NusG homologs  are adjacent to their target operons  are enriched in genes involved in cell envelope biogenesis, with glycosyltransferases, nucleoside-diphosphate-sugar epimerases, and exopolysaccharide biosynthesis enzymes being the most common  type 1 secretion system (T1SS), which is found in uropathogenic  strains . HlyA is strains . RfaH, ay operon . Inactivnfection . The capcapacity .K. pneumoniae is an urgent public health threat and a leading cause of pneumonia in hospitalized patients  required for infection in vertebrate hosts. Among them, SPI4 plays a role in the initial interaction with the intestinal epithelium and possibly contributes to long-term persistence (S. enterica RfaH is required for the expression of SPI4, which encodes a T1SS and its adhesin substrate (S. enterica serovar Typhimurium lacking rfaH are efficient as vaccines against salmonellosis and induce strong serum immune responses (Antibiotic-resistant patients . Functio strains . Vibrio eveloped . Loss ofan serum . Expressmination . S. entethe rise . Salmonesistence . S. enteubstrate , as wellubstrate . Mutantsesponses . Given tesponses , other NrfaH, prevented cefotaxime-induced up-regulation of traF and decreased the conjugative transfer of the resistance plasmid (Antibiotic resistance determinants are frequently encoded on conjugative plasmids and can be rapidly transferred between bacteria . RfaH ac plasmid .Vibrio, Yersinia, Salmonella, and Klebsiella bind to the E. coli TEC in vitro and complement the E. coli rfaH gene deletion (E. coli and K. pneumoniae RfaH to RNAP (RfaH proteins from deletion . Small m to RNAP  may haveNusG homologs comprise the only universally conserved family of transcription factors, which includes housekeeping regulators and their specialized paralogs . DespiteThis regulatory plasticity depends on dynamic interactions of the NGN and KOW domains with each other, RNAP, single and double-stranded nucleic acids, and many auxiliary cellular proteins. While bound to the TEC through contacts mediated by highly conserved residues within RNAP and NGN, NusG homologs employ divergent residues in their NGN and KOW domains to enact a range of responses demanded by specific cellular circumstances. Some NusG paralogs augment their regulatory prowess by undergoing an unprecedented and reversible refolding of an entire KOW domain, during which the protein turns inside out. The presence of NusG in all free-living organisms, sometimes in several copies, confirms its unique place in gene expression control, from LUCA to present life forms.BW prepared all original figures and wrote the first draft. IA revised and expanded the manuscript. Both authors prepared figures and edited the draft while preparing a revised manuscript.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.


Mono-ubiquitylation of histone H2B (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. It remained unclear whether Cdk9 and H2Bub1 cooperate to regulate the expression of individual genes. Here, we show that Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of ∼10% of fission yeast genes, with each perturbation affecting largely distinct subsets; ablation of both pathways de-represses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both modifications are enriched, and directly proportional to each other, in coding regions, and decrease abruptly around the cleavage and polyadenylation signal (CPS). Cdk9-dependence of antisense suppression at specific genes correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Genetic interactions link Cdk9, H2Bub1 and the histone deacetylase Clr6-CII, while combined Cdk9 inhibition and H2Bub1 loss impair Clr6-CII recruitment to chromatin and lead to decreased occupancy and increased acetylation of histones within gene coding regions. These results uncover novel interactions between co-transcriptional histone modification pathways, which link regulation of RNAPII transcription elongation to suppression of aberrant initiation. The elongation phase of transcription by RNA polymerase II (RNAPII) is subject to stringent regulation . Among tSaccharomyces cerevisiae Bre1 (Mono-ubiquitylation of histone H2B (H2Bub1) occurs in concert with RNAPII elongation, and is catalyzed by the E2 ubiquitin conjugating enzyme Rad6 and E3 ubiquitin ligases related to iae Bre1 . H2Bub1 iae Bre1 ,8—but aliae Bre1 ,10, for iae Bre1 . The meciae Bre1 ,9,11,14.Schizosaccharomyces pombe, Cdk9 activity is needed to overcome an early checkpoint control in transcription and allow RNAPII to attain maximal rates of elongation  and Set1 act in parallel pathways downstream of H2Bub1 , an essential CDK with multiple functions and targets . In metaongation . Near thongation . Cdk9 woongation . Rtf1 anongation ,23; an aongation . In S. pS. cerevisiae, Clr6-CII in S. pombe, Sin3B in human cells) and suppressing aberrant transcription initiation (Factors that control elongation by RNAPII influence the structure of transcribed chromatin and play key roles in suppressing the aberrant initiation of transcription (sense or antisense) from within gene coding regions. These mechanisms involve cooperation between elongation factors and proteins that modify chromatin structure, including histone chaperones, ATP-dependent chromatin remodelers, and histone-modifying enzymes . For exaitiation . Althougitiation ,32, crosTo uncover gene-regulatory mechanisms dependent on Cdk9 and H2Bub1, we analyzed (i) steady-state RNA levels upon loss of H2Bub1, inhibition of Cdk9, or both perturbations, by strand-specific RNA sequencing (RNA-seq); and (ii) distributions of H2Bub1 and pSpt5 on chromatin in unperturbed cells, by chromatin immunoprecipitation and sequencing (ChIP-seq). The most prevalent changes in RNA abundance due to loss of either Cdk9 activity or H2Bub1 were increases in antisense transcripts derived from gene coding regions. The genes at which Cdk9 inhibition alone induced antisense transcription were enriched for ones that have higher-than-average H2Bub1 occupancy and exhibit promoter–proximal RNAPII pausing . Genes tcph1Δ::kanMX4) was derived from an h+/h+ diploid (purchased from Bioneer) after transformation with plasmid pON177 to induce sporulation  of strains JTB362 (WT), JTB425 (ascdk9), JTB97 (htb1-K119R), and JTB508 (as htb1-K119Rcdk9) were grown to OD600 of 0.2 before treatment with DMSO or 20 μM 3-MB-PP1 for 1 h. Total RNA was extracted by a hot phenol method  and gene-specific primers listed in act1+ sense transcript in each strain.For strand-specific RT-qPCR to measure levels of S. pombe cell cultures were grown in YES to OD600 of 0.3–0.6, treated with DMSO or 20 μM 3-MB-PP1 for 20 min, and crosslinked with 1% formaldehyde for 15 min at room temperature. To terminate crosslinking, 2.5 M glycine was added to a final concentration of 125 mM for 5 min. Cells were pelleted by centrifugation, washed twice with 10 mL cold TBS , frozen on dry ice and stored at –80°C. Cell pellets from 50 ml cultures were resuspended in 0.4 ml FA lysis buffer  and lysed in a mini-bead beater (Biospec products) in the presence of glass beads  at 4°C for 2 min. Lysates were centrifuged at 16,100 × gav for 15 min at 4°C. Pellets containing chromatin were resuspended in 1 ml FA Lysis buffer and transferred to 15 ml polycarbonate tubes. Lysates were sonicated for 20 min at 4°C  using a waterbath sonicator (Diagenode Bioruptor), transferred to new 1.5-ml tubes and centrifuged at 16,100 × gav for 5 min at 4°C. 100 μl of supernatant was kept as ‘input’ and the remainder (∼900 μl) was subjected to immunoprecipitation with antibodies against histone H3 (Abcam 1791), K9,14-acetylated histone H3 (Millipore 06-599), K36 trimethylated histone H3 (Abcam 9050), FLAG epitope , or control IgG. Lysates prepared in parallel from S. cerevisiae strains BY4743 or a derivative expressing Rpb1-FLAG were spiked in to each IP (10% of lysate volume) for normalization. After incubation at 4°C with mixing for ≥4 h, immune complexes were recovered using 15 μl protein A/G agarose beads (Thermo Fisher) and incubated for an additional hour. Beads were washed sequentially with 0.5 ml FA Lysis + 0.1% SDS, FA Lysis + 0.1% SDS + 500 mM NaCl, LiCl buffer , and TE . Each wash was for 4 min with mixing at room temperature. Immune complexes were eluted in 100 μl elution buffer  at 65°C for 20 min. Beads were washed with 150 μl TE + 0.67% SDS, which was combined with the eluate. 150 μl TE + 0.67% SDS was also added to the input samples, and both IP and input samples were incubated at 65°C overnight to reverse protein–DNA crosslinks. DNA was purified by phenol/chloroform extraction as described previously . Aligned sequences of each biological replicate were fed into MACS2  to call peaks from alignment results. Generated ‘bedgraph treatment’ files were concatenated  to combine replicates of each sample, converted into bigwig using ‘Wig/BedGraph-to-bigWig converter’  and processed using computeMatrix  in DeepTools to prepare data for plotting heatmaps and/ or profiles of given regions. Heatmap and Metagene plots were generated using ‘plotHeatmap’  and ‘plotProfile’  tools, respectively. The pSpt5: Spt5 and H2Bub1: H2B signal ratios were calculated using bigwigCompare .ChIP-seq analysis was carried out in a strain expressing FLAG-tagged histone H2B (strain MS265) using an H2Bub1-specific antibody (Active Motif) and anti-FLAG antibody (Sigma). Total Spt5 and phospho-Spt5 were immunoprecipitated from a Myc-tagged Spt5 strain (strain CS111) using anti-Myc antibody (EMD Millipore) and a previously described pSpt5-specific antibody , respectpst2-myc using anti-myc antibody (BioLegend # 626802) and 8WG16 (BioLegend # 664912), which recognizes the Rpb1 carboxy-terminal domain (CTD), respectively. Lysates from S. cerevisiae strain BY4743 expressing TFIIB-myc were spiked in to IPs  for normalization, which was applied after alignment of FASTQ files to both S. cerevisiae and S. pombe genomes.ChIP-seq of Clr6-CII and RNAPII was performed in strains expressing as) mutation (ascdk9) that confers sensitivity to inhibition by the bulky adenine analog 3-MB-PP1 (htb1-K119R or 3) both mutations  an analog-sensitive  htb1utations . Cdk9 inutations , consistutations . We alsoS. pombe genes but increased antisense transcript levels of ∼10% of genes , but not for the other classes of affected transcripts , as confirmed by specific RT-PCR  (spt5(T1A)7–did not increase antisense transcription of +cdc2 or erg32+ in an +htb1 strain, but it produced levels of antisense transcripts similar to those caused by Cdk9 inhibition in an htb1-K119R background 7 strains remained sensitive to Cdk9 inhibition; treatment of as htb1-K119R spt5(T1Acdk9)7 cells with 3-MB-PP1 produced the highest levels of antisense transcripts detected at either gene. Therefore, whereas constitutive loss of pSpt5 is not sufficient to derepress +cdc2 or +erg32antisense transcription in H2Bub1-proficient cells, it sensitizes cells to antisense induction by H2Bub1 loss, and to combined H2Bub1 loss and Cdk9 inhibition. This suggests that Cdk9 inhibition might derepress antisense transcription by more than one mechanism, possibly by affecting multiple Cdk9 substrates.We next sought to implicate Spt5, a known target of Cdk9, in antisense suppression. The majority of Spt5 phosphorylation by Cdk9 occurs within a carboxy-terminal domain (CTD) comprising 18 repeats of a nonapeptide motif (consensus: T6G7S8K9) . Within 6G7S8K9) , and thi6G7S8K9) . We anal6G7S8K9) . A Thr→Ad Figure , suggestS. pombe genes based on pSpt5:Spt5 ratio sorted them by H2Bub1:H2B ratio as well  was enriched for functions involved in ribonucleoprotein complex biogenesis, RNA processing and ribosome biogenesis , exo2+ or dcr1+ did not increase either antisense transcript, indicating that they are not regulated post-transcriptionally. These results point to regulators of chromatin organization—Clr6-CII, Set2 and Hrp3—as candidate effectors of antisense suppression by Cdk9 and H2Bub1.Previous studies in fission yeast identified two classes of factors involved in regulation of antisense transcription: (i) those that work by modifying chromatin, such as Clr6-CII ,45 and , setg factor ) , indicating genetic epistasis  antisense levels, whereas inhibition of Cdk9 in combination with either hrp3Δ or set2Δ led to effects on antisense transcription that varied, i.e., were either additive or epistatic, at different genes , we measured s Figure . The strs Figure . A similtructure ,49 . Sims Figure . Taken t+cph1—encoding a subunit of the Clr6-CII HDAC complex—was epistatic with both htb1-K119R and ascdk9 at all four loci. Therefore, to ask if H2Bub1 loss or Cdk9 inactivation affected Clr6-CII recruitment to transcribed chromatin, we performed parallel ChIP-seq analyses of RNAPII and the Clr6-CII subunit Pst2 in pst2-myc strains. Metagene analysis revealed that Cdk9 inhibition caused a shift of RNAPII density toward the 5′ ends of gene coding regions and enhanced RNAPII occupancy throughout gene bodies by ∼3-fold  indicated a requirement for Cdk9 activity for Set2 recruitment at both genes. However, there were no effects on H3K36me levels , likely due to slow turnover of this histone mark  revealed a slight increase in H3 occupancy in the latter. In contrast, 3-MB-PP1 treatment of the as htb1-K119Rcdk9 strain led to significantly decreased H3 crosslinking (2–3-fold) at both +cdc2 and +erg32, relative to the DMSO-treated cells and 3-MB-PP1-treated ascdk9 cells. This indicates redundant roles of Cdk9 and H2Bub1 in maintaining nucleosome occupancy at these genes. The effect on nucleosome occupancy was less pronounced at two subtelomeric genes and at an intergenic region near cen3, suggesting that it was driven by RNAPII transcription , as is evident in single-gene tracks  allele, suggests that Cdk9-mediated suppression of cryptic initiation occurs through at least one pathway that does not depend on Spt5 phosphorylation at Thr1 within its CTD repeats. The lack of an effect due to 7spt5(T1A) alone, moreover, implies that those mechanisms can compensate for loss of pSpt5 in H2Bub1-proficient cells.Induced degradation of Spt5 has been shown to promote antisense transcription in S. pombe . Here, win vivo, although the human ortholog of one Clr6-CII subunit, MRG15, interacts with H2Bub1 in vitro -seq analyses in htb1-K119R cells demonstrated that nucleosome stability was generally compromised within gene coding regions  .The processed sequencing files from RNA-seq are available on NCBI BioProject ID:382240. ChIP-seq files have been submitted to the NCBI Gene Expression Omnibus (GEO; gkaa474_Supplemental_FilesClick here for additional data file.


Drosophila melanogaster embryos to extend the catalog of in vivo translated small ORFs, and to reveal the translational regulation of both small and canonical ORFs from mRNAs across embryogenesis.Ribosomal profiling has revealed the translation of thousands of sequences outside annotated protein-coding genes, including small open reading frames of less than 100 codons, and the translational regulation of many genes. Here we present an improved version of Poly-Ribo-Seq and apply it to Drosophila, footprints mapping to codons in a phased pattern, the hallmark of productive translation. We propose a simple binomial probability metric to ascertain translation probability. Our results also reveal reproducible ribosomal binding apparently not resulting in productive translation. This non-productive ribosomal binding seems to be especially prevalent amongst upstream short ORFs located in the 5′ mRNA leaders, and amongst canonical ORFs during the activation of the zygotic translatome at the maternal-to zygotic transition.We obtain highly correlated samples across five embryonic stages, with nearly 500 million putative ribosomal footprints mapped to mRNAs, and compare them to existing Ribo-Seq and proteomic data. Our analysis reveals, for the first time in We suggest that this non-productive ribosomal binding might be due to cis-regulatory ribosomal binding and to defective ribosomal scanning of ORFs outside periods of productive translation. Our results are compatible with the main function of upstream short ORFs being to buffer the translation of canonical canonical ORFs; and show that, in general, small ORFs in mRNAs display markers compatible with an evolutionary transitory state towards full coding function.Supplementary information accompanies this paper at 10.1186/s13059-020-02011-5. Ribosomal profiling, the genome-wide sequencing of ribosome-protected RNA fragments (Ribo-Seq), has been increasing our understanding of a crucial event in all living genomes: protein translation , 2. RiboDrosophila [Drosophila embryogenesis is a highly coordinated and complex process that is completed in a time span of just 24 h [Drosophila embryogenesis has not yet been revealed.At the same time, the comparison of RNA-Seq and Ribo-Seq for the same biological samples allows the transcriptome-wide study of an important but often forgotten regulatory process in genome function: the regulation of translation. Typically, expression of an mRNA transcript is equated with the automatic translation of any encoded canonical ORF (of more than 100 codons). However, translation is not automatic, but is regulated, often with great relevance for organismal function, both under normal and altered conditions –10. Tranosophila –14. Drosust 24 h . During ust 24 h , 17. Aftust 24 h , 18–20. Drosophila melanogaster life-cycle has detected less than 40% of all unique canonical proteins [Ribo-Seq results regarding non-canonical and regulated translation have been the subject of debate. While it has become accepted that both processes may occur more extensively than previously thought, there is no consensus on the actual fraction of smORFs and non-canonical ORFs whose translation is shown by Ribo-Seq –25. The proteins . This nuproteins . Howeverproteins , 2. A biproteins , 27. We proteins .Drosophila melanogaster embryogenesis. We have both improved our experimental Poly-Ribo-Seq and the subsequent data analysis pipeline, to obtain unprecedented levels of Ribo-Seq efficiency (reads mapped to ORFs) and quality, including codon framing as the hallmark of productive, biologically meaningful translation. Thus, we can ascertain translation and its regulation in vivo and across development for both canonical and non-canonical ORFs. We detect the translation of thousands of non-annotated ORFs and identify hundreds of mRNAs whose translation is highly regulated during embryogenesis. However, our results also reveal reproducible ribosomal binding not resulting in productive translation. This non-productive ribosomal binding seems to be especially prevalent amongst upstream short ORFs located in the 5′ mRNA leaders, and amongst canonical ORFs during the activation of the zygotic translatome at the maternal to zygotic transition. We suggest that this type of ribosomal binding might be due to either cis-regulatory ribosomal activity, or to defective ribosomal scanning of ORFs outside periods of productive translation.Here we present an in vivo Poly-Ribo-Seq study covering a time-course of Drosophila S2 cell line was an excellent tool for the development of the technique. However, the S2 cell line is derived from just one type of tissue (macrophage-like) from late stage Drosophila melanogaster embryos [Drosophila translation. Only 60% of both canonical genes and uORFs, and only about a third of annotated smORFs  appear or sCDSs . Therefosis Fig. a. The orsis Fig. ”, and wesis Fig. , purity sis Fig. b, and reDrosophila embryogenesis into three temporal 8-h windows: early , mid (8–16 h AEL), and late embryogenesis (16–24 h AEL) , mid 8–6 h AEL, Drosophila embryogenesis at a genomic scale. We studied several classes of ORFs as defined in Couso and Patraquim [We collected two biological replicas  from each embryonic stage, extracting simultaneously total RNA and ribosomal footprints. Sequencing and analysis of these samples was used to determine transcription and translation levels, which in turn were used to reveal the extent and quality of translational regulation during atraquim , altogetr = 0.9) of RPKMs  for each ORF amongst the two replicas  and Ribo-Seq (RPKMFP) samples. Only reads of 26 to 36 nucleotides (nt) were counted for RPKMFP, as this length distribution corresponded to 97% of the reads obtained in all cases and is within the size range previously described for ribosomal FPs [tarsal-less, where the FPs mapped to the ORFs experimentally proven to be translated [As the Ribo-Seq protocol typically results in a composite library with a majority of ribosomal rRNA reads, and a minority of ribosomal footprints (FPs), we sequenced an exhaustive amount, totalling 1.2 billion Ribo-Seq reads, yielding some 345 million ribosomal footprints (FPs) mapped to the genome . To determine translation, we use two combined filters. First, a quantitative filter: a minimal amount of reproducible ribosomal binding must be detected to indicate signal above error and background; this “ribosomal bound” state was achieved by obtaining RPKMFP>1 in both T and B replicas in a given stage. Second, a qualitative filter, the nature of that binding: the ribosomal movement across an ORF during translation generates tri-nucleotide phasing of ribosomal protected footprints, or framing  following n independent trials (number of reads mapping to the ORF) follows a classical binomial distribution, which can be consulted using widely available tables [p < 0.01  whose translation has been determined experimentally [SclA and SclB) achieve RPKMFP above 1 in both T and B replicas during mid and late embryogenesis. Most of its FP reads mapped to frame “2”, but SclB only achieved a binomial p value below 0.01 during late embryogenesis. Thus, our data indicated that SclA is ribosomal-bound and translated at mid and late embryogenesis, but SclB is only productively translated during late embryogenesis, when scl function in regulating muscle contraction would have its maximum requirement  but only the likelihood that the observed ribosomal binding leads to productive ORF translation. This binomial framing metric also ensures that the translation detected belongs to a given ORF, not to another putative ORF overlapping it.Here, we have opted for a simpler approach that is easy to extend to past and future datasets. We treat framing as a simple coin-toss problem, which offers clear statistical probabilities, even with a low number of reads. As each footprint is the result of an independent RNA-ribosome molecular interaction, each resulting sequencing read has in principle a 1/3 probability of aligning to the right frame in an ORF, and 2/3 of aligning to the wrong frame , whereas the percentage of actually translated ORFs  was 91% of Canonical, 70% of short CDSs, and 11% of transcribed uORFs  with a replica of their stage (either ours or modENCODE), than with samples of other stages , and second with samples from adjacent time periods  showed the highest correlation with our RPKMFP samples from early embryogenesis (0–8 h AEL) (Spearman’s r ≈ 0.8)  Fig. . Our bio.8) Fig. ”. A tota.8) Fig. b. HoweveDrosophila embryo-derived S2 cell cultures [We also compared our embryo FP data with cultures , which icultures , adding FP values , especially between 1<RPKMFP<1024, indicating that RPKMFP can be a good proxy of protein-production by canonical ORF translation at moderate to high levels. However, 40% of canonical ORFs detected as transcribed by RNA-Seq by us and modENCODE during embryogenesis are not detected by proteomics  is much higher than for those not detected (13.80), indicating that proteomics detection needs higher levels of protein translation, as noticed previously [Hox genes except the more widely expressed Ubx and abd-A. This “proteomics detectability” issue is not attributable to trypsination treatments, since we observed that 99% of canonical and 92% of non-canonical ORFs could produce K- and R-cleaved peptides of 7 to 24 amino acids (AA) suitable for mass-spec (MS) detection. Hence, other yet unknown factors must influence the proteomics detection of proteins. In addition, small peptides of less than 50 amino acids suffer from enhanced degradation [FP of those detected is 172.1 . This TE analysis revealed both on-off translation changes, and modulations of sustained translation, from one embryonic stage to the next. The average TE varied to some extent across development , whereas 33% were translated only at specific stages of development Fig. a. Stage ent Fig. b, but inZ-ratio analysis, we observed in total 367 canonical ORFs with significantly changed translational efficiency between the maternal and the early zygotic translatomes  and 136 upregulated, showing RNA expression patterns preferentially registered as “maternal” or “ubiquitous” at this stage , but we observed 369 ORFs translated in S2 cells but not in late embryos, whereas 8021 ORFs appeared translated in late embryos but not in S2 cells  might rios Fig. d. In priios Fig. ”. Interer = 0.95, Additional file FP>1 in two replicates, Fig. Drosophila melanogaster might not have a peptide-productive role  in both early-to-mid (694 ORFs) and mid-to-late embryogenesis  located downstream in their transcripts . This reto-mid 69 ORFs andto-mid 69 ORFs andto-mid 69 ORFs andPreviously, a negative correlation between the number of uORFs within a 5′ leader and the TE of the main ORF was found in zebrafish genome-wide studies  and acroZ-ratios (≥ |1.5|) and vice versa  is similar to recent estimates [In our previous study , we estiges Fig. f, and diges Fig. b and frages Fig. C. The pestimates , but thestimates . Yet, thstimates , 56, 67,stimates , 66, 68)stimates ) and passtimates , 71, verstimates , and humstimates , where aA mildly beneficial buffering role could explain the ubiquity of uORFs in otherwise canonical mRNAs. The minority of uORFs showing peptide-productive translation could be a tolerable by-product of this buffering role, which would otherwise be mostly mediated by the much more prevalent uORFs with non-productive ribosomal-binding-only. Interestingly, we observe subtly different Kozak scores, conservation levels, and amino acid usage for translated uORFs, which seem to fit into a stepped continuum ranging from transcribed-only uORFs to canonical ORFs. It is tempting to speculate that, just as ribosomal-bound-only in canonical ORFs may be a developmental transitory state during the maternal to zygotic translation, ribosome-bound-only uORFs may be an evolutionary transitory state poised for a transition to full coding function. In this way, the 5′ leaders of canonical genes would act as “proto-gene nurseries” offering a tolerant environment for random ORF creation  and hence a TE Z-ratio significantly increased. This sampling issue can contrive an artefactual increase of expression and translation (RPKM and TE) of hemotin and similar cell-specific transcripts in cell lines [Despite the improvements to our Poly-Ribo-Seq protocol, and the large number of reads that we have collected, it is possible that not all embryonic translation has been detected in our samples. Lowly abundant or transient mRNAs may not be adequately detected in our 8-h developmental windows, or may be drowned out by noise and not be able to achieve RPKM > 1. This could be especially true of mRNAs expressed in a few cells of the embryo, a situation that must arise often towards the end of embryogenesis, and could be prevalent in differentiated tissues. Our macrophage-like S2 cell line data allows for an approximation to this issue, and the problem of integrating and comparing Ribo-Seq and genomic data from whole embryos and cell lines. Direct comparison between late embryos and S2 cells creates a sampling issue. The embryonic counterparts of S2 cells could be only a few cells amongst many embryonic cell types and some 30,000 cells overall , 75. Thua embryo . Yet ourll lines  when comll lines , 79), will lines .Drosophila melanogaster. Translation levels appear more reproducible than transcription, yet highly correlated with it, together yielding highly constant expression of canonical ORFs and sCDSs, and higher temporal specificity of uORFs. However, 11–19% ORFs show specific regulation of their translation, including “poising” at the maternal to zygotic transition. Finally, a large pool of near 7000 uORFs also show ribosomal binding without achieving either productive translation or a significant regulatory input on downstream ORFs. These and other data suggest that, in general, 5′ leaders with uORFs act as “translation buffers” at the gene level (helping downstream ORFs to stabilize translation levels) and as “proto-gene nurseries” at the genomic level (providing a favorable environment for random ORF creation).The likelihood of translation can be determined in Ribo-Seq data by comparing the frequency of reads in frame against a simple binomial probability. Using this metric and a standard RPKM>1 filter for ribosomal binding, almost 16,000 canonical ORFs, more than 300 sCDSs, and more than 1200 uORFs appear translated during the embryonic development of g to pellet the monosomes. The 50 nt fragmented mRNAs and 28–34-nt footprints were isolated from 10% denaturing acrylamide gels and subsequently T4 PNK treated for library preparation.The RNA-Seq and Poly-Ribo-Seq experiments were conducted as previously described in Aspden et al.  with theThe NEBNext Small RNA Library Prep Set for Illumina  was used following the manufacturers’ instructions with small modifications as follows. After the 3′ adapter ligation step, ribosome footprints were rRNA depleted using biotinylated DNA fragments and oligos from rRNAs and going through two consecutive rounds of subtractive hybridization (except for the T 0-8 h sample having only one round) using MyOne Streptavidin C1 Dynabeads (Invitrogen). After ethanol precipitation, samples were processed as NEB guidelines. Libraries were isolated from 10% non-denaturing gels by size selection and their quality and quantity measure by DNA high sensitivity chips (Agilent) with Bioanalyzer and Qubit.D. melanogaster reference genome (Release 6.13) using HISAT2 with default options. Due to the very low frequency of multimapping reads, we retained all genome-aligned reads for further analyses.Ribosomal footprints were filtered  and clipped for adapters using the FASTX-Toolkit. The resulting reads were aligned to a FASTA file containing rRNA, tRNA, snoRNA, and snRNA from Flybase Release 6.13 annotations using Bowtie, discarding the successful alignments and collecting all unaligned reads. Unaligned reads were then mapped to the FlyBase getorf program. Next, all ORFs with fully redundant CDS coordinates were identified, and duplicates were discarded within each class . Additionally, we discarded uORFs overlapping with any annotated CDS, or fully included within a longer, in-frame uORF, but kept uORFs overlapping in different frames. uORFs in dicistronic transcripts were then added to the uORF set, based on a full overlap with annotated ORFs contained within the 5′ leader of an immediately downstream annotated CDS.All annotated coding sequences (CDSs) in FlyBase, r6.13 were retrieved and divided into groups of either longer than or shorter than 303 nucleotides (100 AA). CDSs longer than the cut off were assigned to the canonical ORF category. CDSs shorter or equal to 303 nt but arising from the same locus as canonicals  were diFP) was measured by counting Ribo-Seq reads of 26 to 36 nt overlapping each ORF using the R package Rsamtols (absolute ribosome density), and scaling this number by ORF length and the total number of genome-aligned reads [RNA) was ascertained using the same ORF coordinates and applying the same method on RNA-Seq measurements. Correlation analyses across replicates were measured by Spearman’s rho on ORF RPKM values.Relative ribosome density (RPKM+). The main read length and its most overrepresented frame in each sample was then used to evaluate the framing of each ORF in each Ribo-Seq sample. For each ORF, we then compared successful framed reads  with total mapped reads using a binomial test, implemented by the cumulative pbinom function in R to measure the probability of obtaining the observed framing (or better), when compared to the expectation by random (1 in 3 probability). This was implemented using the expression pbinom. For binomial tests of ORF translation, different biological replicates (if available) were merged into a single one. Although the precision of the binomial probability method improves with the amount of evidence  were first aligned to an artificially constructed transcriptome set, consisting of all ORFs analyzed in this study as well as their surrounding regions − 18 nucleotides upstream and + 15 nucleotides downstream, thus allowing for the alignment of full ribosomal reads spanning the START and STOP codons. The resulting alignments were analyzed using the R package RiboSeqR to extract the global framing patterns across ribosome footprint lengths for three pools of RPFs: all pooled embryonic RiboSeq samples, S2 cell samples, and pool of mature oocyte and activated eggs RPFs  as well as the corresponding RNA-Seq experiments (SRR1039764 and SRR1039765), all deposited under accession number GSE52799. The activated egg and oocyte reads were merged to obtain Ribo-Seq and RNA-Seq “unfertilized egg” samples, which were analyzed employing the same pipeline as our own data. In the case of S2-cell data, we merged our previously published Ribo-Seq  and RNA-Seq datasets (SRR1548660-SRR1548661) [We obtained previously published  fastq fi1548661)  and addeDrosophila melanogaster from [We compared the mass spectrometry-based developmental proteome of ter from  with ourZ-ratio calculations of TE variation as per Cheadle et al. [Z-score of each ORF in each sample:Translation efficiency (TE) per ORF was calculated as the fraction of ribosome-density (Poly-Ribo-Seq RPKM) over the total mRNA read density (RNA-Seq RPKM). To detect significant events of translational regulation, we performed e et al. . First, Z-ratios across contiguous developmental stages, e.g., early and mid embryogenesis, were calculated for each ORF:Second, Third, the cut-off of ≥ |1.5| was used to identify biologically significant differences , 48 betwDrosophila melanogaster genes with expression information and expressed as a p value for each significantly enriched tissue, after the Benjamini-Hochberg multiple-comparisons correction.We used the unique gene identifiers (FBgn) for genes identified as translationally regulated across stages see “” to calcThe pre-computed phyloP nucleotide scores for 27-way insect multiple sequence alignments were downloaded from UCSC as bigWig files. We then used the UCSC bigWigAverageOverBed package to compare phyloP scores with BED files containing genome-coordinates for each ORF in our set, obtaining average phyloP scores per ORF.aa_composition.pl, [We used a previously published own script  to calcbona fide Kozak contexts with which to score individual ORFs in all classes. The final score per ORF was then obtained by adding the individual position-specific values for all observed nucleotides.For all canonical ORFs, we extracted the nucleotide composition around—but excluding—the annotated START codon . For each position, we then calculated the log odds ratio between observed and background nucleotide frequencies. This provided a scoring table of position-specific nucleotide frequencies from Additional file 1. Patraquim_Supplemental_Information_Additional File 1.pdf: Supplementary Figures and Tables.Additional file 2. Patraquim_canonical_ORFs_Additional_File_2.csv: transcriptional, ribosome-binding, framing and translational regulation analysis results for canonical ORFs.Additional file 3. Patraquim_shortCDSs_Additional_File_3.csv: transcriptional, ribosome-binding, framing and translational regulation analysis results for shortCDSs.Additional file 4. Patraquim_uORFs_Additional_File_4.csv: transcriptional, ribosome-binding, framing and translational regulation analysis results for uORFs.Additional file 5. Review history.