Iridoviruses are large DNA viruses that cause diseases in fish, amphibians and insects. Singapore grouper iridovirus (SGIV) is isolated from cultured grouper and characterized as a ranavirus. ICP46 is defined to be a core gene of the family Iridoviridae and SGIV ICP46 was demonstrated to be an immediate-early (IE) gene associated with cell growth control and could contribute to virus replication in previous research.The transcription start site (TSS) and 5′-untranslated region (5′-UTR) of SGIV ICP46 were determined using 5′ RACE. The core promoter elements of ICP46s were analyzed by bioinformatics analysis. The core promoter region and the regulation model of SGIV ICP46 promoter were revealed by the construction of serially deleted promoter plasmids, transfections, drug treat and luciferase reporter assays. The identification of virion-associated transcriptional transactivator (VATT) that interact with SGIV ICP46 promoter and their binding site on promoter were performed by electrophoretic mobility shift assays (EMSA), DNA pull-down assays and mass spectrometry (MS).SGIV ICP46 was found to have short 5′-UTR and a presumptive downstream promoter element (DPE), AGACA, which locates at + 36 to + 39 nt downstream of the TSS. The core promoter region of SGIV ICP46 located from − 22 to + 42 nt relative to the TSS. VATTs were involved in the promoter activation of SGIV ICP46 and further identified to be VP12, VP39, VP57 and MCP. A 10-base DNA sequence “ATGGCTTTCG” between the TSS and presumptive DPE was determined to be the binding site of the VATTs.Our study showed that four VAATs  might bind with the SGIV ICP46 promoter and be involved in the promoter activation. Further, the binding site of the VATTs on promoter was a 10-base DNA sequence between the TSS and presumptive DPE. Iridoviruses are large DNA viruses that cause diseases in fish, amphibians, reptiles and insects, and result in significant economic and ecological losses , 2. SimiICP46 was firstly found to encode a very early protein of approximately 46 kDa in frog virus 3 (FV3), and have been identified as IE genes in FV3, SGIV and Bohle iridovirus (BIV) –13. By cIn the present study, we revealed the core region of SGIV ICP46 promoter, investigated the regulation model of SGIV ICP46 promoter associated with viral protein, identified the VATTs that interact with the SGIV ICP46 promoter, and determined the binding site of VATTs on SGIV ICP46 promoter. This study is important for the further study of the transcriptional regulation mechanism of SGIV ICP46, and contributes to better understanding of the iridovirus transcription, ultimately helps to develop novel control strategies for iridovirus diseases.Grouper spleen (GS) cells were cultured at 25 °C in Leibovitz’s L-15 medium containing 10% fetal bovine serum . Singapore grouper iridovirus  was propagated on GS cell monolayers as previously described , and SGIAccording to Huang’s method, UV-inactivated virus was treated by exposing SGIV suspension under a 30-W UV light source (10 cm distant) for 30 min at 4 °C . Heat-inTranscription start site (TSS) of SGIV ICP46 mRNA was determined using 5′ RACE System for Rapid Amplification of cDNA Ends  according to the manufacturer’s instructions. First strand cDNA was synthesized from 3 μg total RNA isolated at 12 h p.i. using a specific reverse primer . After RNAase treatment and cDNA purification, an oligo (dC) tail was added at the 5′ end. The resulting product was PCR amplified with a universal primer containing a poly-dG sequence and specific oligonucleotides  for the first and second PCR respectively. The amplified fragment was cloned into plasmid pMD18-T  and analyzed by automated sequencing .The TSS and 5′-UTR of SGIV ICP46 were performed bioinformatics analysis. According to the DNA sequences flanking the TSS (− 300 to + 200), the core promoter elements were analyzed for the TATA-box (TATA), CAAT-box ((C)CAAT) and downstream promoter element (DPE) (RGWYV (T)). Moreover, about 200 nucleotides (nt) flanking sequence of translation initiation site (TIS) from SGIV ICP46 and other 13 iridovirus ICP46s were chosen from the genome data of the NCBI blast server . Plasmids were purified using a Plasmid Endo-free Mini Kit  and the DNA concentrations were determined by spectrophotometry .5 cells per well in 500 μl of media and allowed to adhere overnight. All transient co-transfection experiments for luciferase reporter assays were carried out using 500 ng of each of the promoter reporter plasmids together with 50 ng of thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) and Lipofectamine 2000 according to the manufacturer’s instructions . At 24 h after transfection, cells were infected with SGIV at MOI of 2 and further incubated at 25 °C. Cells were harvested at 6 h p.i. for promoter analysis. Firefly and Renilla luciferase activities were measured in cell extracts using the dual luciferase reporter assay system  following the manufacturer’s instructions.GS cells were seeded in 24 well plates at a density of 2.5 × 10To clarify the regulation pattern of viral proteins on the activity of SGIV ICP46 promoter, GS cells were transfected with P-22, which containing the critical promoter regions, and then the transfected cells were either mock infected or infected with infectious SGIV, heat-inactivated SGIV and UV-inactivated SGIV, respectively. The GS cells transfected with pGL 4.17 were served as negative control. Finally, the luciferase reporter assays were carried out.To analysis whether the newly synthesized viral proteins can influence the activity of SGIV ICP46 promoter, GS cells were transfected with P-22 and then the transfected cells were pretreated with 50 μg/ml cycloheximide (CHX) or 100 μg/ml cytosine arabinofuranoside (AraC) for 1 h before and throughout the SGIV infection. CHX or AraC-pretreated cells were infected with SGIV at MOI of approximately 2, then harvested at 6 and 48 h p.i., respectively. The P-22 transfected GS cells infected with SGIV were collected at the same time points and served as negative control. The luciferase reporter assays were carried out following the manufacturer’s instructions.To confirm that the SGIV proteins may bind with the core region of SGIV ICP46 promoter, the electrophoretic mobility shift assay (EMSA) were carried out. According to the core promoter of SGIV ICP46, the 64 nt EMSA probe named as probe X was design and synthesis, the sequence of probe X is 5′-GCGCA AACTC GGAAC AAAAA TCGCC CTTGT GGAAG ATTTA AAAAT GGCTT TCGTC ACAGA CAAG − 3′. The whole protein extraction from GS cells (GS), SGIV-infected GS cells (SGIV+GS), CHX-pretreated and SGIV-infected GS cells (CHX + SGIV), AraC-pretreated and SGIV-infected GS cells (AraC+SGIV) and purified SGIV virion (SGIV) were obtained using the Cytoplasmic Protein/Nuclear Protein Extraction Kit  according to the manufacturer’s instructions. Then the EMSA were performed using Non-Radioactive EMSA Kits  following the manufacturer’s protocol. Briefly, the whole protein extraction (5 μg) from GS, SGIV+GS, CHX + SGIV, AraC+SGIV cells and purified SGIV virion were incubated with biotin-labeled probe X in reaction buffers for 20 min at room temperature. Incubated with biotin-labeled probe NFκB, the nuclear extracts with activated NFκB was used as positive control and nuclear extracts without activated NFκB was used as negative control. Protein-DNA complex was separated from free oligonucleotide on 5.5% nondenaturant polyacrylamide gels, and then transferred to positively charged nitrocellulose membranes  by wet trans-blot. After the transfer, the crosslink DNA on the membrane was immobilized in a UV linker . Then the binding-membrane was visualized with streptavidin-horseradish peroxidase followed by chemiluminescent detection.DNA pull-down experiments were performed using Pulldown Kits for Using Biotin-Probes  according to the manufacturer’s instructions. Briefly, the whole protein extraction (30–90 μg) from GS, SGIV+GS, CHX + SGIV, AraC+SGIV cells and purified SGIV virion were incubated with the biotin-labeled probe X (6–10 nmol) and non-specific competitors for 60 min at room temperature. Then the DNA/Protein binding mixture were bound to streptavidin-agarose beads by incubating 40 min at room temperature with gently shake, and washed three times to remove unbound DNA/Protein. Beads-bound proteins were dissociated by boiled for 5 min in a dissociation solution and separated by electrophoresis on a SDS-10% polyacrylamide gel. The differential protein bands were chosen and identified by NanoLC-ESI-MS/MS Analysis .The full length genes  encoding four VATTs of SGIV ICP46 promoter identified by DNA pull-down assays were cloned into eukaryotic vectors pcDNA3.1/His A  using corresponding primers  of SGIV ICP46 (about 40 nt) was included as well. The resulting plasmids were transfected to GS cells together with pRL-TK as a control plasmid to correct for variations in transfection efficiency. Luciferase reporter assays showed that the luciferase activity was markedly increased after transfected the promoter reporter plasmids P-3247, P-2180, P-1535, P-1159 and P-649 comparing with the negative control plasmid pGL4.17, and the luciferase level of these five constructs had no significantly difference  were used for luciferase activity assay. The results revealed that even the shortest deletion of − 22 to + 42 still had the transcriptional effects with regard to luciferase activity  from P-196, the levels of luciferase activity of P-∆196 markedly reduced in SGIV-infected GS cells Fig. c. This rThe heat-inactivated SGIV has denatured virion-associated proteins and replicable DNA, while the UV-inactivated SGIV has active virion-associated proteins and damaged DNA. Comparing with the mock-infected cells, luciferase activities were markedly increased after infected with the infectious SGIV and UV-inactivated SGIV in the P-22 transfected GS cells, while the luciferase activity of the P-22 transfected cells infected with heat-inactivated SGIV was similar with the mock-infected cells Fig. a. These To analysis whether the newly synthesized viral proteins can influence the activity of SGIV ICP46 promoter, P-22 transfected GS cells were treated with CHX and AraC, respectively. The luciferase reporter assay showed that CHX and Ara had no significant effect on the activity of SGIV ICP46 promoter . The protein extraction from GS, SGIV+GS, CHX + SGIV, AraC+SGIV cells and purified SGIV virion were used for EMSA analysis. The results show that all the protein extraction from GS, SGIV+GS, CHX + SGIV, AraC+SGIV cells and purified SGIV virion can combine with the probe X and form protein-DNA complexes. The band position of protein-DNA complexes from GS, SGIV+GS, CHX + SGIV and AraC+SGIV cells are basically the same, while the migration distance of protein-DNA complex from SGIV virion is further away from the sample holes Fig. . The resEpinephelus coioides were identified in the nine protein bands from SGIV+GS (data not shown), but no SGIV proteins were found might due to the low abundance. The MS data of the two DNA-binding protein bands  were constructed, and proved to be successfully expressed in GS cells by RT-PCR . Taken together, it can be concluded that the overlapped squence of probe 4 and probe 5, ATGGCTTTCG, is the binding site with VATTs on SGIV ICP46 promoter.Since the VATTs are involved in the activation of SGIV ICP46 promoter, the binding site with VATTs of the SGIV ICP46 promoter was defined. Probe X and probe 1–5 Table , which mThe TSS of SGIV ICP46 was at the G located 21 nt upstream of the TIS. It has been proved that the RNA polymerases preferentially initiate transcription with a purine residue , 21. Ourgene transcription by RNA polymerase II, the DPE functions cooperatively with the initiator (Inr) to bind to TFIID and directs accurate and efficient initiation of transcription in many TATA-less promoters [The location of typical TATA-box and CAAT-box is about − 30 and − 80 relative to the TSS respectively, while the core promoter elements analysis of SGIV ICP46 showed that no TATA-box and CAAT-box were found within 94 nt and 125 nt upstream of TSS, it seems that SGIV ICP46 promoter is a TATA-less and CAAT-less promoter. Meanwhile, a potential DPE was found at + 36 to + 39 nt downstream of the TSS. DPE is one of the core promoter element and plays an important role in the initiation of romoters .Bombyx mori nucleopolyhedrovirus (BmNPV) [gene transcription.The DPE is usually located about 30 nt downstream of the TSS and most commonly found in TATA-less promoters . It has  (BmNPV)  were allRanavirus, Lymphocystivirus and Megalocytivirus. Interestingly, Ranavirus, Lymphocystivirus and Megalocytivirus are exactly the three genera which can infect teleost fishes in family Iridoviridae. Seven fish pathogenic iridoviruses  were found to have presumptive DPE. Although belong to the same genus Ranavirus as SGIV and GIV, no DPE were found in FV3, STIV, ATV and TFV, which are infectious to amphibians and reptiles. It is uncertain whether the presumptive DPE of ICP46 from fish pathogenic iridoviruses is associated with the transcription characteristic in teleost fishes. The DPE as core promoter elements for ICP46s of fish infectious iridovirus still need to be verified and studied in further research.In addition, the core promoter elements analysis of 14 iridovirus ICP46s revealed that three of the five genera of Iridoviridae have potential DPE, they are Ranavirus and Lymphocystivirus. The function of VP12 is still unknown [The promoter analysis found that the core regions of SGIV ICP46 promoter include sequence 22 nt upstream and 42 nt downstream of the TSS. Previous research about FV3 ICP46 showed that the promoter was from 291 nt upstream to 195 nt downstream of TTS, and found that the FV3 ICP46 was overexpressed in the presence of CHX and inferred that certain early proteins have negative control over FV3 ICP46 tanscription . Differe unknown . Iridovi unknown . A homol unknown . Both SGCo-transfection and luciferase reporter assays showed that the transient expression of four VATTs alone or combined did not significantly affect the activity of the SGIV ICP46 promoter without SGIV infection. The virion protein composition of five iridoviruses have been determined, including SGIV , 36–38, The VATT binding site was defined by EMSA and reavealed that the 10-base DNA sequence “ATGGCTTTCG” between the TSS and the presumptive DPE is the binding site with VATTs. Transcription is a complicated process which involves the interactions of promoter elements with multiple transcription factors. The specific interactions rely not only on the specific sequence recognition but also on certain spatial arrangement of the factors in a complex . The stuIn conclusion, our present study found that the core region of SGIV ICP46 promoter locate from − 22 to + 42 nt relative to the TSS and probably is a TATA-less and DPE-containing promoter. This study also demonstrated that VATTs are required to initiate SGIV ICP46 transcription and the further molecular identities of VATTs are VP12, VP39, VP57 and MCP. Further examination revealed the binding site of VATTs interact with SGIV ICP46 promoter is ATGGCTTTCG between the TSS and presumptive DPE.


Redox homeostasis maintenance is essential to bring about cellular functions. Particularly, embryonic stem cells (ESCs) have high fidelity mechanisms for DNA repair, high activity of different antioxidant enzymes and low levels of oxidative stress. Although the expression and activity of antioxidant enzymes are reduced throughout the differentiation, the knowledge about the transcriptional regulation of genes involved in defense against oxidative stress is yet restricted. Since glutathione is a central component of a complex system involved in preserving cellular redox status, we aimed to study whether the expression of the glutathione reductase (Gsr) gene, which encodes an essential enzyme for cellular redox homeostasis, is modulated by the transcription factors critical for self-renewal and pluripotency of ESCs.We found that Gsr gene is expressed in ESCs during the pluripotent state and it was upregulated when these cells were induced to differentiate, concomitantly with Nanog decreased expression. Moreover, we found an increase in Gsr mRNA levels when Nanog was downregulated by a specific shRNA targeting this transcription factor in ESCs. Our results suggest that Nanog represses Gsr gene expression in ESCs, evidencing a role of this crucial pluripotency transcription factor in preservation of redox homeostasis in stem cells.The online version of this article (10.1186/s13104-019-4411-0) contains supplementary material, which is available to authorized users. Maintaining the homeostasis of redox state is essential for cellular functions. A complex system composed by different enzymes and low molecular weight compounds, such as antioxidant vitamins and glutathione, is involved in preserving cellular redox status. Within this network, glutathione reductase (Gsr) is one of the most important enzymes, since it catalyzes the reduction of glutathione disulfide to the thiol form of glutathione (GSH), maintaining the pool of reduced GSH. This molecule is the most abundant non-protein thiol in cells and has an essential role as a cellular redox buffer. It is co-substrate of other antioxidant enzymes which break down the reactive oxygen species (ROS), generated as by-products of cellular respiration.ROS play an important role as second messengers in various cellular functions such as proliferation, differentiation and apoptosis –5, henceEmbryonic stem cells (ESCs), which derive from the inner cell mass of blastocysts, possess several systems that secure genomic stability. This safeguard is essential since these cells physiologically originate all cell types of the organism, including the germ line. Thus, ESCs have high fidelity mechanisms involved in DNA repair, high activity of the multiple antioxidant enzymes and low levels of ROS with the consequent low mutation rate . Furthersod1 and sod2 genes, that encode for superoxide dismutases, are induced by the pluripotency transcription factors, which are essential for ESCs’ self-renewal and pluripotency [Some genes involved in the defense system against oxidative stress are modulated along ESCs differentiation , 10. Basipotency , 17.Although it has been reported that expression and activity of antioxidant enzymes are reduced throughout the differentiation , 6, the Gsr gene is modulated by the pluripotency transcription factors. We found that Glutathione reductase gene was expressed in undifferentiated ESCs and was upregulated when these cells were induced to differentiate, concomitantly with Nanog downregulation. In accordance with these results, we found that Gsr gene expression was induced in ESCs where Nanog gene was downregulated by transfection with shRNA vector targeting this transcription factor.The aim of this work was to study whether the expression of the Based on the growing evidence that antioxidant defense system is modulated when pluripotent stem cells are induced to differentiate, and that glutathione reductase is an important enzyme involved in the cellular response to oxidative stress, we decided to study Gsr gene modulation. In a previous work, we have in silico analyzed the upstream region of the coding sequence of diverse genes involved in antioxidant system, including Gsr and found the presence of multiple predicted binding sites for the transcription factors Oct4 and Nanog in a 5 kbp region upstream the transcription start site of this gene . In thisNext, to further investigate Gsr gene modulation we analyzed its expression pattern along a differentiation protocol to neural progenitor. We used the 46C ESC line for this approach, which is a reporter cell line that expresses GFP driven by Sox1 promoter, a specific marker of neuroectoderm . We veriGsr coding sequence, we decided to study whether Nanog modulates the endogenous Gsr gene expression by an shRNA approach. For this purpose, we downregulated this transcription factor’s mRNA levels using a shRNA targeting Nanog (shNanog). We transfected R1 ESCs with a vector encoding shNanog or targeting eGFP (shGFP) as control, and then analyzed Gsr mRNA levels by RT-qPCR. As outlined in Fig. Taking into account the induction of Gsr observed when Nanog was repressed along both differentiation protocols and the presence of six putative consensus sites for Nanog in the 3000 bp region upstream There is growing evidence showing that when pluripotent stem cells are induced to differentiate, antioxidant defense system is modulated , 19, 20.As aforementioned, we found that Gsr gene was expressed in pluripotent stem cells and increased throughout differentiation, showing an expression pattern opposed to Nanog’s when these cells were induced to differentiate by two distinct protocols. We have previously found by in silico analysis, multiple putative binding sites for Nanog in Gsr promoter region . SpecifiWe found that Gsr, which is critical for maintaining GSH levels and cellular redox status, is modulated by the stemness transcription factor Nanog evidencing a link between pluripotency transcription factors and redox homeostasis. Deep understanding of the antioxidant system in pluripotent stem cells and the relationship between ROS and the differentiation process is crucial for future applications of these promising cells.R1 ESC line (ATCC) were cultured and differentiated as previously described –34. 46C Gene expression was analyzed by RT-qPCR and immunofluorescence. A detailed description of the methodology, the antibodies and the sequence of the primers used is included in Additional file R1 ESCs were transfected in p60 plates with 3 µg pLKO.1-puro derived vectors (Sigma), expressing shRNA targeting Nanog  or eGFP (SHC005). Transfection, selection and mRNA analysis were carried out as previously described , 17. Fort test, except for Fig. Results were presented as mean ± Standard error mean (SEM). Statistical comparisons were performed using Student’s We attempted to evaluate Nanog effect on Gsr gene by ectopic expression in an heterologous system but we failed, presumably by the need of other factors missing in the system and/or different epigenetic landscape.Additional file 1: Figure S1. Region of Gsr genomic locus including Nanog binding peaks according to Chip-seq experiments downloaded from the Chip-Atlas Database (https://chip-atlas.org/). Sequence Read Archive Database identifiers are indicated in the figure.Additional file 2. Additional file contains Fig. S1 legend, Table S1 and additional methods.


During the phylotypic period, embryos from different genera show similar gene expression patterns, implying common regulatory mechanisms. Here we set out to identify enhancers involved in the initial events of cardiogenesis, which occurs during the phylotypic period. We isolate early cardiac progenitor cells from zebrafish embryos and characterize 3838 open chromatin regions specific to this cell population. Of these regions, 162 overlap with conserved non-coding elements (CNEs) that also map to open chromatin regions in human. Most of the zebrafish conserved open chromatin elements tested drive gene expression in the developing heart. Despite modest sequence identity, human orthologous open chromatin regions recapitulate the spatial temporal expression patterns of the zebrafish sequence, potentially providing a basis for phylotypic gene expression patterns. Genome-wide, we discover 5598 zebrafish-human conserved open chromatin regions, suggesting that a diverse repertoire of ancient enhancers is established prior to organogenesis and the phylotypic period. During early embryogenesis, critical cardiac specification events occur. Here the authors isolate cardiac progenitor cells from early zebrafish embryos and characterize accessible chromatin regions specific to this cell population, finding that many of these regions overlap with conserved non-coding elements that are ortholgous to accessible chromatin regions in human. The hourglass model is also supported by comparative transcriptomic studies that demonstrated that the most conserved gene expression patterns occur at the phylotypic stage5. The idea that conserved phylotypic gene expression is established through conserved enhancers is supported by several comparative epigenomic studies9. While most molecular studies of the phylotypic period have focused on whole embryos, recent evidence suggests that the exact developmental timing of maximal conservation varies in a tissue-specific manner8. We are only beginning to understand how conserved transcriptional programs for individual developmental lineages are set up prior to the phylotypic stage.The developmental hourglass model predicts a phylotypic stage during mid-embryogenesis when species within the same phylum display the greatest level of morphological similarities11. Crucial cardiac specification events occur during early embryogenesis15. For example, distinct subtypes of mouse cardiac progenitors emerge within the gastrula stage preceding the expression of the canonical cardiac progenitor marker Nkx2.5, long before any organ structure is formed14. However, how this potential early cardiac specification is controlled by enhancer elements, and the extent to which this process is evolutionarily conserved, is not known.The heart, derived from the cardiac mesoderm, is the first organ formed during embryogenesis. Heart development is orchestrated by conserved cardiac transcription factors (TFs) binding to cis-regulatory elements (CREs)21, computational predictions23, and in vivo validation in mouse and zebrafish embryos23. To date, the majority of in vivo heart enhancer discoveries and validation experiments were performed in embryos following establishment of the heart chamber structures or in adult hearts17. In vitro differentiation of embryonic stem cells (ESCs) into cardiac progenitors have also yielded insights into the early cardiac development and have enabled the discovery of cardiac enhancers21. However, more work is needed to identify the CREs that regulate the early differentiation of mesoderm progenitors to cardiac lineages in the context of the developing embryo.Heart enhancers have been extensively characterized in studies utilizing genome-wide profiling techniques including chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq)16. However, analysis of putative enhancers in mesoderm cells, derived from embryonic stem cells, show higher evolutionary constraint than the enhancers identified after organogenesis8. This suggests that the regulatory elements that establish the conserved cardiac transcriptional program may exist at the initial stages of heart development, presumptively during the time window from naive mesoderm to cardiac progenitors.Despite the highly conserved cardiac TFs necessary for heart development, heart enhancers identified at mouse E11.5 show limited phylogenetic conservation compared to brain enhancers identified at the same developmental stageNkx2.5. We generate a zebrafish GFP reporter line driven by a mouse Smarcd3 enhancer (Smarcd3-F6) that is active in early gastrulating mouse mesoderm12, in order to enrich for early zebrafish cardiac progenitors. We profile gene expression and the accessible chromatin landscape of GFP positive cells using single cell mRNA-seq, the assay for transposase-accessible chromatin using sequencing (ATAC-seq), lineage tracing, and RNA in-situ hybridization, both in wild-type embryos and following knockdown of the essential cardiac transcription factors Gata5/6. Results from these experiments indicate that we have purified a population of cells enriched for cardiac progenitors. Using direct and indirect DNA alignments25, we identify accessible chromatin regions shared between zebrafish and human. We find that these conserved accessible chromatin elements were highly associated with developmental transcription factors that are regulated by polycomb repressive complex 2 (PRC2). We confirm the cardiac activity and functional conservation of many anciently conserved open chromatin regions using in vivo reporter assays. In sum, our study identifies a set of conserved cardiac enhancers established prior to the phylotypic period, before the heart and other organ primordia appear, potentially providing a basis for common gene expression patterns across genera. Furthermore, we uncover ~ 6000 anciently conserved open chromatin regions that likely serve as enhancers for other cell lineages.Here, we set out to discover enhancers that are active in cardiac progenitor cells prior to the expression of the cardiac progenitor marker nkx2.5 expression, we tested a recently described early mouse cardiac enhancer, Smarcd3-F612 in our zebrafish model. Lineage tracing experiments demonstrated that this enhancer labeled cardiac progenitor cells prior to Nkx2.5 expression in mouse embryos12. We also found that the Smarcd3-F6 region was enriched for the active enhancer mark H3K27ac and contains a CRE co-bound by several conserved cardiac TFs  in mouse ESC differentiated cardiac precursors (CP) and cardiomyocytes (CM)21  transgenic line  along the embryonic margin   and Tg(nkx2.5:ZsYellow) expression indicated that the Smarcd3-F6 enhancer marked almost all cardiac mesoderm expressing nkx2.5 at early somite stages (13 hpf)  lines were generated to trace the fate of Smarcd3-F6 labeled cells. By crossing Tg(Smarcd3-F6:CreERT2) to a Tg(βactin2-loxP-dsRed-loxP-GFP) reporter line, we found that following 4-hydroxytamoxifen (4-HT) addition at 8 hpf, cells labeled by the Smarcd3-F6 enhancer contributed to heart formation  Fig. . Co-immupf) Fig. . Tg and unlabeled (GFP−) cells  and endoderm  markers showed significantly higher expression in Smarcd3-F6 labeled cells while ectoderm and axial mesoderm genes were relatively depleted  , mesodermal  and ectodermal  populations  in the GFP+ population and 153,777 in the GFP− population. After conducting differential analysis, we found most ATAC-seq peak regions  shared similar ATAC-seq signals in both GFP+ and GFP− populations. 5471 peaks showed significant quantitative differences , and 1633 peaks specifically increased in unlabeled cells (‘GFP− specific’)  and enhancers  that were previously identified from ChIP-seq experiments on whole zebrafish embryos of similar stages6. We found that 69% of accessible zebrafish regions did not overlap with active chromatin marks  and exonic regions  and enriched for introns  as compared to the genomic distribution of all ATAC-seq peaks  embryos. Supporting our motif enrichments, we found that Smarcd3-F6 enhancer activity requires Gata5 and Gata6 function  datasets, zCNEies Fig. . We assoP = 1.40e–46 using human CNEs; Chi-square test, two-sided)  Fig. . Previouely Fig. , our obs41. For ATAC-seq peaks overlapping CNEs, we asked if the orthologous human or mouse CNEs also contained accessible chromatin according to DNase I hypersensitivity sites (DHS) reported by ENCODE342  between species is a potentially powerful way to identify conserved regulatory function within alignable sequences for specific tissues or cell types342 Fig. . We refe342 Fig. . Overall25, we found that aCNEs were depleted for exonic regions  and were enriched for intronic regions  compared to all ATAC-seq peaks. We did not see an enrichment for promoter regions in aCNEs compared to all ATAC-seq peaks . While ATAC-seq peaks at promoters were more conserved than those not at promoters , promoter aCNEs did not show higher sequence constraint than non-promoter aCNEs  , stronger open chromatin signals  and slightly higher GC content  than the bulk zebrafish ATAC-seq peaks  when using all human DNase I sites as background. We next examined elements tested in the VISTA enhancer database44, which is the most comprehensive database of functionally validated enhancers . When we took a global view of all aCNEs from the human perspective, we found roughly 11% (958/8866) of human aCNEs were included in the VISTA Enhancer Browser. While less than 35% of the human regions (953/2787) profiled by VISTA were functionally validated as positive enhancers, two thirds of human aCNEs (640/958) tested were reported as positive enhancers, indicating a significant enrichment of active developmental enhancers amongst aCNEs .To gain insights into the cardiac enhancer activity of aCNEs, we compared the 281 GFP+ human aCNEs to putative heart enhancers predicated using curated epigenomic data46 , which only contains two sequences, enriched motifs were identified in all other categories. GATA, SMAD, RAR/RXR, ZNF263, and RREB1 motifs were found to be enriched in more than one enhancer category, suggesting that they may represent shared features of early cardiac enhancers , tbx20/TBX20 (aCNE20) and mef2cb/MEF2C . All of these zebrafish sequences drove robust and specific heart expression in stable transgenic lines   Fig. .Fig. 4AnWhen tested in vivo, the four orthologous human aCNE (HaCNE) sequences also drove GFP expression in the hearts of zebrafish transgenic lines, with 3 of them  demonstrating activities similar to that of their zebrafish orthologs Fig. . At 48 hgfp RNA in-situ to characterize the spatiotemporal dynamics of the aCNE activity. We found that both aCNE1 and aCNE20 pairs were active in cardiac lineages at early somite stages , 24 hpf, as well as 48 hpf  and compared their activities with that of the wild-type sequences  have at least one GATA motif and ~ 30% 52/162) have more than one GATA motif . Supporting the role of aCNEs as developmental enhancers, we observed that these regions were also highly enriched for genes regulated by PRC2 . To gain more insight into lineage-restricted functions of aCNEs, we compared the human aCNEs orthologous to our GFP+ specific and GFP− specific zebrafish ATAC-seq peaks. Although human aCNEs specific to the GFP+ and GFP− populations both showed significant enrichments for PRC2 target genes and homeobox transcription factors, only a minority of PRC2 regulated genes were associated with both GFP+ and GFP− aCNEs, indicating that these two sets of aCNEs may be involved in distinct developmental processes , with the top enriched factors largely overlapping  dataset which contains uniformly analyzed ChIP-seq binding profiles for 161 TFs in 91 cell types (Transcription Factor ChIP-seq Clusters V3 from ENCODE). We asked if any TF showed occupancy enrichment within aCNEs compared to randomly selected open chromatin regions  in the Roadmap Epigenomics Project48 Fig. . Support51, we compared aCNEs to three enhancer types defined in a recent study using mouse ESCs: poised , primed  and active 51. Compared to all open chromatin regions, aCNEs showed a five-fold enrichment for poised enhancers  with little enrichment for active  or primed  enhancers. We also observed a strong enrichment for poised enhancers that were identified in human ES cells 49.Given that lineage-specific activity and enrichment for PRC2 binding are well-established properties of poised enhancers in ESCs52. We found that nearly 90% (7846/8866) of human aCNEs fell into GRBs defined by using CNEs (70% identity over 50 bp) between human and chicken52, suggesting that aCNEs have been kept in a discrete set of syntenic regions during vertebrate evolution. Overall, the genomic and epigenomic properties of aCNEs suggest that many of them may serve as lineage-restricted enhancers that facilitate the expression of developmental genes.Conserved genomic regulatory blocks (GRBs), which are defined by cluster of CNEs, contain developmental genes, coincide the boundaries of topologically associating domains (TADs) and remain concordant between species of various evolutionary distances25 allowed us to identify more than 6000 zebrafish open chromatin regions that overlapped open chromatin regions in human or mouse genomes. Of these conserved accessible chromatin regions (aCNEs), 162 were unique to our cardiac progenitor-cell enriched population. While 27% (43/162) of the orthologous human regions have recently been predicted to be cardiac enhancers22, to our knowledge none of these 162 zebrafish open chromatin regions or their orthologous human or mouse sequences had been previously tested in vivo. We found that 16/18 pre-phylotypic aCNEs drove cardiac expression in stable zebrafish transgenic lines at 24 hpf, which is around the time considered to be the zebrafish phylotypic stage7, or later in development (48 hpf). Further experiments, such as ChIP-seq for individual transcription factors and posttranslational histone modifications will help understand the spatial and temporal dynamics activity of these aCNEs. Overall, our data support the existence of conserved cis-regulatory elements that are primed early in development prior to the establishment of the body plan and function during the phylotypic stage.The existence of a phylotypic period implies that the gene regulatory events that control phylotypic gene expression patterns are also likely to be evolutionarily constrained. In this study, we characterized a population of cells enriched for cardiac progenitors in early zebrafish embryos. By studying the open chromatin regions in this population, we uncovered a set of putative enhancers. Leveraging two resources that characterize non-coding elements conserved between zebrafish and humans55. This phenomenon is prevalent even within rodent56 or primate41 orders, let alone comparisons between species separated over large evolutionary distances, such as zebrafish and human (over 400 million years from the last common ancestor). Nonetheless, sequence comparisons between human and teleosts over great phylogenetic distance have discovered many functional non-coding elements active during development60 and CNEs are often found near genes encoding developmental regulators61. More direct evidence of conserved cis-regulatory events acting prior to the phylotypic period have come from profiling epigenetic changes around the phylotypic period. For example, a study using zebrafish embryos has shown that regions dynamically gaining epigenetic modification indicative of development enhancers (H3K27ac) at the gastrulation stage (8.5 hpf) are enriched for evolutionarily conserved DNA sequences6. Furthermore, differential DNA methylation changes observed during the phylotypic period in zebrafish, mouse, and frog were enriched for evolutionarily constrained DNA sequences and these DNA methylation changes were presumably guided by prior sequence-specific transcription binding events62. Our results identify a substantial number of aCNEs that are established in the early embryo, many of which drive tissue-specific gene expression patterns later in development.Many functional CREs do not share overt sequence similarities due to rapid turnover of the spacing between, and the sequences of, TF binding motifs63. Our results echo this finding, while at the same time highlighting a set of highly conserved heart enhancers. By comparing open chromatin within CNEs identified using approaches such as transitivity through a third species and ancestry reconstruction25, we discovered 60% more conserved open chromatin regions between zebrafish and human/mouse that would otherwise have been missed by using direct sequence alignment alone 52. We found that nearly 90% (7846/8866) of our human aCNEs fell into GRBs comprised of CNEs between human and chicken (70% identity over 50 bp)52. Overall the genomic and epigenomic properties of aCNEs suggest that many of them may serve as lineage-restricted enhancers that facilitate the expression of developmental genes.Attesting to their potential importance in regulating specific genes through long-range chromatin interactions, an established genomic property of CNEs is that they cluster into regions of conserved synteny, referred to as gene regulatory blocks (GRBs)66, new studies are beginning to demonstrate developmental anomalies68. Consistent with the existence of shadow enhancers, which can buffer the effects of individual enhancer loss71, two recent studies showed that the pairwise deletion of ultraconserved enhancers had an increased phenotypic impact71. While more work remains to be done to dissect the in vivo, spatial-temporal expression patterns and function of anciently conserved vertebrate enhancers, regions of deeply conserved open chromatin represent a solid foundation from which the regulation and evolution of the vertebrate body plan can be explored.We conclude that conserved open chromatin regions established prior to the phylotypic period, and shared over 450 million years of evolution, likely represent a set of ancient enhancers that contribute to diverse spatial and temporal gene expression patterns. Although the first deletions of ultraconserved non-coding elements did not reveal overt phenotypes72. All zebrafish lines used in the study were shown in Supplementary Method.Zebrafish were maintained and handled under the guidance and approval of the Canadian Council on Animal Care and the Hospital for Sick Children Laboratory Animal Services. Embryos were raised at 28.5 °C and developmentally staged by their morphologyp3E-CreERT2 were sub-cloned into pGEM Teasy vector  to make antisense probes . DIG-labeled in-situ probes were synthesized using DIG RNA Labeling kits (Roche). RNA in-situ hybridization was carried out using the following protocol73. Briefly, embryos of the right stages were fixed in 4% paraformaldehyde and then dehydrated in 100% MeOH. After rehydration, embryos were permeabilized and incubated with digoxigenin-labeled antisense RNA probe. RNA-probe hybrids were detected by an alkaline phosphatase-conjugated antibody  that catalyzed reaction on a chromogenic substrate. Stained embryos were cleared in BBA solution  and imaged under a Leica M205FA stereomicroscopeEGFP sequence from the ZED vector and Cre sequence from Tg (Smarcd3-F6: EGFP)hsc70 and Tg(nkx2.5: ZsYellow)fb7 crosses were fixed at 13 hpf in 4% paraformaldehyde at 4 °C overnight. After 3 × 5 min washes in PBS with 0.1% Triton, embryos were permeabilized in PBS with 0.5% Triton for 4 h at room temperature (RT). Embryos were then blocked in PBST (1% DMSO and 0.5% Triton X in PBS) with 5% Normal Goat Serum  for 1.5–2 h at RT before being incubated with primary antibodies  at 4 °C overnight. The Next day, embryos were washed for 3–4 h in PBS with 0.1% Triton at RT, with 6–8 changes of solution. Incubation of secondary antibodies  was carried out at 4 °C overnight, followed by the same washing procedure as that for primary antibodies. After staining, embryos were mounted in 1% (w/v) low melt agarose  and imaged under a Nikon A1R Si Point Scanning Confocal microscope.Embryos from Tg (Smarcd3-F6:CreERT2)hsc76 and Tg(βactin2:loxP-DsRed-STOP-loxP-EGFP)s928Tg crosses were dechorionated and incubated in 5 μM 4-OH-Tamoxifen for 12 h. The time of 4-HT addition was specified in Supplementary Fig. Embryos from Tg(Smarcd3-F6: EGFP)hsc70 embryos were dechorionated with pronase  and transferred to a 1.5 mL Eppendorf tube when they reached the bud stage. After incubation in 200 μl calcium-free Ringer solution  for 5 min, embryos were transferred into a 24-well plate with 500 μl TrypLE solution  for dissociation at room temperature. Embryos were gently homogenized every 5 min with P1000 tips. Dissociation was monitored under a dissection scope until most cells were in a single-cell suspension. The cell suspension was transferred into 200 μl ice-cold fetal bovine serum (FBS) to stop the reaction. Cells were centrifuged at 300 g for 3 min at 4 °C and washed with 500 μl ice-cold DMEM with 10% FBS before resuspended in 500 μl ice-cold DMEM with 1% FBS. Right before the Fluorescence-activated cell sorting (FACS), cells were filtered through a 40 μm strainer and DAPI was added at a concentration of 5 μg/ml to exclude dead cells.Around 100 FACS was performed on Beckman Coulter Mo Flo XDP or Mo Flo Astrios sorter, or Sony SH800S Cell Sorter with a 100 μm nozzle by the SickKids-UHN Flow and Mass Cytometry Facility. Cell-doublets and dead cells were excluded based on forward scatter, side scatter and DAPI channel. GFP+ and GFP− cells were sorted into 100% FBS and subjected to RNA-seq or ATAC-seq procedures immediately after sorting. Approximately 30,000–50,000 GFP+ cells and 100,000 GFP− cells were collected in one FACS run.Single-cell cDNA libraries were prepared using Fluidigm C1 system. After FACS, GFP+ cells were washed twice in DMEM with 3% FBS and filtered through a 40 μm cell strainer. Cells were adjusted to a concentration of 400–500 cells/μl and mixed with C1 suspension solution at a 5.2:4.8 ratio. 10 μl of the final cell mixture was loaded into a C1 medium or small Chip. Cell capture was examined under a microscope and only wells with a single cell captured were included in library construction. 3 ArrayControl™ RNA Spikes were added into the cell lysis mixture according to the Fluidigm C1 single-cell mRNA-seq protocol (PN 100–7168 Rev. B1). Cell lysis, Oligo-dT primer mediated reverse transcription, 21 cycles of PCR amplification and cDNA harvesting were performed as per manufacturer's instruction . We usually recovered 30–40 cells (30–40% capture efficiency) from one C1 Chip and 96 single-cell cDNA libraries were collected from three batches of experiments.For bulk mRNA-seq, RNA from 4000 GFP+ or GFP− cells were prepared using RNeasy Plus Micro Kit  and cDNA libraries were prepared following the Fluidigm tube control protocol (the same protocol as that for single-cell mRNA-seq except the input cell number is different). Three biological replicates were collected for both GFP+ and GFP− samples.For both single-cell and bulk mRNA-seq, final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit  and 120 bp pair-end sequenced on an Illumina HiSeq 2500 platform according to manufacturer’s instruction. Bulk RNA-seq libraries were sequenced to a depth of (18 ± 1.9) million reads and single-cell mRNA-seq libraries a depth of (3.0 ± 0.7) million reads.74. 30,000–50,000 cells obtained from FACS were used for nuclei prep. After tagmentation, transposed DNA fragments were amplified using the following PCR condition . Amplified libraries were purified twice with Agencourt Ampure XP beads  with a bead-to-sample ratio of 1.8:1. A Qubit fluorometer and Aglient Bioanalyzer were used to check library quality and concentration. Libraries were 50 bp single-end sequenced on Illumina HiSeq 2500 platform to a depth of (3.5–7.0) × 107 reads. Two biological replicates were collected for both GFP+ and GFP− cells.ATAC-seq was performed with minor modifications to the published protocolpDONOR221 vector  before they were cloned into E1b-Tol2-GFP-gw vector. 25 ng E1b-Tol2-GFP-gw plasmid carrying one aCNE and 150 ng Tol2 mRNA were injected into wild-type embryos at one-cell stage. F0 founder embryos were raised to 48–52 hpf before imaging and heart expression scoring. Candidate regions were considered as a heart enhancer if more than 30% of the injected embryos displayed GFP+ cells beating in the hearts, which was consistent with the criterion used in similar studies before75. 42–220 (average n = 86) injected embryos were analyzed for each candidate region. The genomic coordinates, lengths and nearby genes of candidate regions and primers used for cloning can be seen in the Supplementary Data Candidate regions containing the zebrafish ATAC-seq peaks (21 ZaCNEs) or human DHSs 4 HaCNEs) were amplified from genomic DNA and recombined into  HaCNEs wF0 embryos injected with enhancers that passed the 30% threshold were raised to screen for transgene germline carriers. Except enhancer ZaCNE18 for which only one carrier has been identified, 2–4 independent alleles have been identified for each enhancer (see Supplementary Data http://www.genomics.agilent.com/primerDesignProgram.jsp) .pDONOR221 vectors carrying the mutated aCNE1 enhancers were PCR amplified from the original pDONOR221 containing the wild-type aCNE1 sequences using the primers above. After amplification, 50 μl PCR mixture was incubated with 1 μl DpnI  at 37 °C to remove wild-type enhancer templates. DpnI was inactivated at 80 °C for 20 min before transformation. Plasmid clones with the correct GATA motif mutation were confirmed by Sanger sequencing and used as entry clones to make E1b-Tol2-GFP constructs containing mutated aCNE1 enhancers. Independent germline carriers have been identified for ZaCNE1_GATAMutated (n = 5) and HaCNE1_GATAMutated (n = 4) enhancers Two zebrafish CNE datasets were used for this analysis. First, Zebrafish CNEs (zCNEs) conserved with human or mouse identified from Hiller et al.25, which we referred to as garCNE. If the zebrafish coordinate of a garCNE does not overlap any zCNE records, we added it into our analysis and define it as indirectly aligned. Altogether, we collect 20,005 zebrafish CNEs conserved with human, with 10187 direct and 9818 indirect ones , and then add those CNEs on top of zCNE_mouse dataset in a similar way as what we did for human. At the end we obtained 18,827 zebrafish CNEs conserved with mouse, with 9429 direct and 9399 indirect ones  were used for identifying open regions anciently conserved between zebrafish and human or between zebrafish and mouse. Mouse DNase master peaks, which were provided as mm10 coordinates, were converted to mm9 coordinates using liftOver (-minMatch = 0.95). If a CNE that overlaps a zebrafish ATAC-seq peak by at least one base pair also overlaps a DNase I hypersensitivity site (DHS) from the DNase master lists by at least one base pair, then this orthologous ATAC-seq peak and DHS linked via CNEs are identified as accessible CNEs (aCNEs).Human or mouse DNase master peak lists from ENCODE3There are 1,199,722 non-overlapping regions in mouse DHSs master list after liftOver, 4,193,929 non-overlapping regions in human DHS master list (Supplementary Table We found analyses with the DHS master lists sometimes gave us a chain of DHSs that are conserved with the same ATAC-seq peaks. To avoid potential bias that may be introduced in analysis conducted by GREAT, we merged the DHSs that are within 200 bp and conserved with the same ATAC-seq peaks. We used merged DHSs for analyses in Fig. Comparative genomic analyses of aCNEs are shown in Supplementary InfoPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting Summary


Since the discovery of methylcytosine oxidase ten-eleven translocation (TET) proteins, we have witnessed an exponential increase in studies examining their roles in epigenetic regulation. TET family proteins catalyze the sequential oxidation of 5-methylcytosine (5mC) to oxidized methylcytosines including 5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine. TETs contribute to the regulation of lineage-specific gene expression via modulating DNA 5mC/5hmC balances at the proximal and distal regulatory elements of cell identity genes, and therefore enhance chromatin accessibility and gene transcription. Emerging evidence suggests that TET dioxygenases participate in the establishment and/or maintenance of hypomethylated bivalent domains at multiple differentiation-associated genes, and thus ensure developmental plasticity. Here, we review the current state of knowledge concerning TET family proteins, DNA hydroxymethylation, their distribution, and function in endoderm, mesoderm, and neuroectoderm specification. We will summarize the evidence pertaining to their crucial regulatory roles in lineage commitment and development. DNA methylation and the recently identified hydroxymethylation are essential epigenetic modifications in cells. DNA methylation is catalyzed by DNA cytosine-5-methyltransferases (DNMTs) via transferring a methyl group to the 5′ position of cytosine to form 5-methylcytosine (5mC). Heavily methylated DNA is often associated with repressed gene expression. In addition, DNA methylation can be actively demethylated by DNA methylcytosine dioxygenases, the ten-eleven translocation (TET) proteins, through oxidizing 5mC into 5-hydroxymethylcytosine (5hmC) and further to 5-formylcytosine (5fC) and then to 5-carboxylcytosine (5caC). Finally, 5fC and 5caC are removed by thymine DNA glycosylase (TDG) and cytosine is replaced by base excision repair (BER) –5. Succe2+) and α-ketoglutarate (α-KG) binding sites that are critical for TET catalytic activity [TET proteins are widely expressed in various organisms including human, mouse, Xenopus, and zebrafish , 10–14. activity , 18. TETactivity , 19, 20.activity . Recentlactivity , 23. TETactivity . DespiteThe three TET family members have distinct expression patterns among different cell types and are tightly regulated during development –26. TET1Since the discovery of TET family dioxygenases, studies examining the function of DNA hydroxymethylation have exponentially increased. Emerging evidence indicates that 5hmCs are enriched at low-to-intermediate CpG density regions of promoters and enhancers of developmental regulatory genes –42. SimiTet1, Tet2, and Tet3 triple knockout mouse ESCs, suggesting that these gene regulatory regions are the major targets of TETs [The establishment of cellular identities requires precise control of gene expression. Mounting evidence suggests that TET proteins work as epigenetic players to alter DNA methylation, histone modification, and chromatin accessibility, which regulate the transcription of key developmental genes. Strikingly, promoters, enhancers, and DNase I-hypersensitive sites accumulate significantly more 5mC in  of TETs , 48, 49.OCT4, are actively expressed in human ESCs and suppressed by promoter hypermethylation upon differentiation [TET induces promoter hypermethylation and aberrant gene expression in multiple lineage differentiation systems [NANOG promoter prevents DNA methylation [Xenopus, such as Pax6, Rx, and Ngn2, and sustains high levels of 5hmC at promoters [Tet2 and Tet3 downregulates P2rX7 expression along with reduction of 5hmC at the P2rX7 promoter during bone marrow mesenchymal stem cell differentiation [Accumulated evidence indicates that DNA methylation status at promoter regions influences gene transcription . Promotentiation . It has  systems , 51–53. hylation . In helphylation . TET1 anβ-N-acetylglucosamine transferase), which enhances methylation of histone H3 at lysine 4 by promoting the binding of a component of the H3K4 methyltransferase SET1/COMPASS complex to active promoters [FOXA2, GATA2, PAX6, and SOX17, by preventing their aberrant hypermethylation to ensure developmental competency [More interestingly, TET-mediated DNA demethylation has been suggested in association with the establishment and/or maintenance of bivalent promoters of developmental genes, in which H3K4me3 and H3K27me3 histone modifications take place simultaneously , 54. In romoters . It has mpetency . TogetheTet2 deletion in mESCs, Ren and colleagues have found that depletion of Tet2 leads to enhancer hypermethylation, accompanied by the loss of active enhancer mark H3K27ac and delayed the induction of Slit3, Lmo4, and Irx3 upon differentiation to a neural progenitor fate [Tet2 causes loss of 5hmC at enhancers and is accompanied by extensive elevation of DNA methylation, reduction of H3K27ac, and impaired gene expression during heart development [Tet2/3 double knockout pro-B cells restores chromatin accessibility at a genome-wide level as well as at the Igκ enhancer [In addition to facilitate promoter hypomethylation, TET proteins also play critical roles in regulating chromatin accessibility and enhancer architecture Fig. . 5hmC cotor fate . A similelopment . Re-exprenhancer . These denhancer .Igκ enhancer to facilitate DNA demethylation during early B cell maturation [In addition to chromatin accessibility, TETs may also contribute to enhancer priming. Prior to activation, the enhancer region is methylated and not accessible to general transcription factors. However, pioneer transcription factors, such as FOXA1, MEIS1, and PBX1, preferentially bind to oligonucleotide probes which contain methylated cytosines , 65. It turation . Furtherturation , 28, 63.turation , 69, indturation , 60. Theturation .+ T cells, TET1 and TET2 alter CTCF-dependent alternative pre-mRNA splicing through oxidation of 5mC to 5hmC and 5caC at corresponding intragenic CTCF-binding sites in the PTPRC (protein tyrosine phosphatase CD45) locus [Despite their plausible contribution to chromatin architecture, TETs may regulate gene transcription by interfering with RNA polymerization and RNA splicing. In CD45) locus , 72. Add5) locus , 47.Tet1/2/3 triple knockout mice are embryonic lethal with severe gastrulation defects during embryogenesis [TET2 skews spontaneous differentiation of human ESCs into neuroectoderm with the loss of mesoderm and endoderm [Growing evidence confirms that TET family proteins are important regulators for embryogenesis. ogenesis , 30, 48.ogenesis , 30. Furendoderm , while iendoderm . Thus, dendoderm , 73, 74.Tet1 knockout mice exhibit defects in neuron function including learning, memory consolidation, storage, and extinction [Npas4 [Tet1 knockout does not alter brain morphology. It has been revealed that compensatory upregulations of Tet2 and Tet3 were observed in the Tet1 knockout mouse brain [Ascl1, Brn2, and Ngn2 [The brain is one of the places in mammals with the most abundant 5hmC, suggesting that TET proteins and TET-mediated 5hmC might have significant impacts in neurogenesis. tinction , 75, 76.n [Npas4 , 78, Tetand Ngn2 . TherefoTET1/2/3 triple knockout human ESCs lose their ability to differentiate into PAX6+/SOX1+ neuroectoderm cells, which is mainly caused by aberrant hypermethylation at the PAX6 promoter [PAX6 bivalent promoter leads to the progressive oxidation of 5-methylcytosine. In the absence of 5hmC, de novo DNA methyltransferase DNMT3B anchors at PAX6 bivalent promoter P0 and induces promoter hypermethylation, which suppresses PAX6 induction and subsequent neuroectoderm differentiation. Targeted demethylation of the PAX6 P0 promoter by a catalytically inactive Cas9 (dCas9) fused with a TET1 catalytic domain partially restored PAX6 expression and rescued neuron differentiation defects [A recent study from Huangfu’s group has nicely demonstrated that promoter . In huma defects . These rTET2 mutations in CD34+ hematopoietic progenitor cells, the TET2-mutated CD34+ progenitors preferentially develop into myeloid instead of erythroid cells upon differentiation [Tet2 or double deletion of Tet2/3 or Tet1/2 alters T cell, B cell, NKT cell, red blood cell, or mast cell differentiation and maturation [Tet2 perturbs the induction of signature cytokine genes upon differentiation of CD4+ T cells toward helper T (Th) cells, which is associated with differential enrichment of 5hmC and p300 at the promoters of Ifng and Il17 [Tet1/Tet2 or Tet2/Tet3 double knockout mice contain less regulatory T (Treg) cells in the spleen. It has been further shown that Tet proteins can demethylate the conserved noncoding sequences (CNS) in Foxp3 loci to maintain the expression of Foxp3 [Foxp3 CNS functions as a super enhancer and docking site for chromatin organizer Satb1 binding [TET2 is ubiquitously expressed in multiple hematopoietic cells, and its mutation is frequently found in hematological malignancies , 80, 81.ntiation . Furtherturation , 83–85. and Il17 . In contof Foxp3 . One of  binding .Tet2 in undifferentiated C2C12 myoblasts significantly reduces the induction of myoblast differentiation-associated genes Myog and myoM with elevated methylation at their promoters [SRF, MYOCD, and MYH11, in human coronary artery smooth muscle cells [Tet2 knockdown deregulates a large number of genes associated with heart development and contraction. In particular, induction of the key cardiac gene Myh7 is suppressed upon cardiomyocyte differentiation in Tet2 knockdown cells, presumably due to the aberrant methylation at its enhancer [Moreover, TET dioxygenases have also been implicated to play crucial roles during cardiomyocytes and skeletal myoblast differentiation , 51, 88.enhancer .Axin2 and Lgr5 [HNF4A promoter P1 demethylation and activation upon hepatocyte differentiation [To date, functional analyses of TETs and 5hmC in the context of endodermal lineage specification are still limited. In the intestine, Tet1 is highly expressed in intestinal stem cells and positively regulates the expression of Wnt target genes such as and Lgr5 . In a montiation . They fontiation . Additiontiation . We deventiation , and furIn summary, TETs and TET-mediated 5hmC are dynamically redistributed in lineage descendants during development. A substantial number of studies have provided data supporting the role of TET proteins in the establishment of cell identity during differentiation. TET family members alter lineage specification as epigenetic modifiers through their dioxygenase activity to convert 5mC to oxidized methylcytosine. They modulate DNA methylation/hydroxymethylation balance at promoters and enhancers of cell fate-determining genes to further increase chromatin accessibility and facilitate gene transcription in lineage commitment. Strikingly, TET dioxygenases participate in the establishment and/or maintenance of bivalent domains of many differentiation-associated genes, and thusly ensure developmental plasticity. However, direct connections between TET dioxygenases and chromatin architecture are still not clear. Although 5hmC is enriched at distal regulatory elements, how the poised and active enhancer states are influenced by TETs remains elusive. It will be of great interest to identify whether oxidized methylcytosines can act as docking sites and recruit epigenetic readers to modulate histone modifications. Further studies aimed at unraveling the precise role of TET dioxygenases in the control of epigenetic machinery and gene regulation will contribute to the knowledge of how lineage specification is precisely regulated during development.


Translation is one of the final steps that regulate gene expression. The ribosome is the effector of translation through to its role in mRNA decoding and protein synthesis. Many mechanisms have been extensively described accounting for translational regulation. However it emerged only recently that ribosomes themselves could contribute to this regulation. Indeed, though it is well-known that the translational efficiency of the cell is linked to ribosome abundance, studies recently demonstrated that the composition of the ribosome could alter translation of specific mRNAs. Evidences suggest that according to the status, environment, development, or pathological conditions, cells produce different populations of ribosomes which differ in their ribosomal protein and/or RNA composition. Those observations gave rise to the concept of “specialized ribosomes”, which proposes that a unique ribosome composition determines the translational activity of this ribosome. The current review will present how technological advances have participated in the emergence of this concept, and to which extent the literature sustains this concept today. Eukaryotic gene expression follows many steps that are stringently controlled to adapt this expression to environmental challenges. Among these different steps, transcription and translation represent the first and last steps, respectively. While the role of transcriptional regulation in gene expression control is at present well-established and indisputable after extensive studies, that of translational regulation remains understudied and unclear. This could be due to the fact that analysis of translational regulation is relatively more complex than that of transcription. Transcription has been extensively analyzed thanks to genome-wide approaches since the development of transcriptomic analyses in the early 2000s and later on for chromatin availability and compaction. In contrast, such dedicated approaches were lacking for studying specifically translational step until recently. The impact of translational regulation on gene expression regulation was recently highlighted through several large-scale comparative proteomic and transcriptomic analyses in yeast , mouse  that unaTranslation results in the synthesis of proteins corresponding to specific messages that are contained within mRNAs. The regulation of translation is dependent on cell status, environment, development, and pathological conditions . TranslaTranslation initiation is the main rate-limiting stage of translation and comprises several steps . BrAll of these factors, i.e., eIFs, eEFs, and eTFs, which are part of the translational apparatus are well-characterized key trans-regulators of translation. To exert their functions they are subjected to post-translational modifications, mainly phosphorylation, following activation or inhibition of intra- and extracellular signaling cascades in response to stimuli occurring in physiological or pathological conditions. These modifications impact their activities and subsequently modify the global rate of translation by modulating initiation, elongation and termination rates. Many reviews have described in great details how and by which molecular mechanisms these trans-regulators modulate translational efficiency ,7,11. FuIn addition to these canonical translation factors regulating the three steps of translation, there is an increasing number of trans-regulators that do not belong to the core of the translational machinery, but that interact with it to modulate the translational efficiency of subsets of mRNAs. These additional trans-regulators of translation, including RNA binding proteins (RBPs) , internaA notion has been formulated recently that ribosomes themselves could be direct actors in the regulation of translation, in addition to the widely studied mechanisms of translational regulation cited above ,32,33. IThe human ribosome contains 80 ribosomal proteins (RPs) and 4 rRNAs  organized in two ribonucleo-proteic subunits, the SSU and the LSU. The SSU contains the 18S rRNA and 33 distinct RPs, and the LSU contains the 5S, the 5.8S, the 28S rRNAs and 47 distinct RPs . The ribRibosome biogenesis is a complex process involving more than 150 proteins, and approximately 70 small nucleolar RNAs (snoRNAs) for the synthesis, modification, assembly, and nucleus/cytoplasm shuttling of the 4 rRNAs and the 80 RPs . TheThe limiting step of ribosome biogenesis is the transcription of rDNA genes by the RNA polymerase I (RNA Pol I), which generates a polycistronic RNA precursor called the 47S pre-rRNA in humans. This precursor is sequentially processed mainly in the nucleoli, generating many rRNA processing intermediates, to form the pre-mature 5.8S, 18S and 28S rRNAs. They are then exported to the cytoplasm where they are submitted to a final maturation to produce the mature 5.8S, 18S and 28S rRNAs . The 5S Cells tightly regulate their quantity of ribosomes by controlling rDNA transcription. Hence, the amount of ribosomes can vary among different cell types and cell statuses. For instance, the circadian clock timely coordinates the transcription of rDNAs and mRNAs coding for RPs  and moduSeveral oncogenic pathways and oncogenes have been reported as key activators of ribosome biogenesis. The mammalian target of the rapamycin complex (mTOR) is a protein kinase which is activated by several stimuli, including nutrients, hormones and oncogenic signaling pathways. mTOR directly stimulates RNA Pol I and RNA Pol III by interacting with their promoters , and thuMyc has also been described as a regulator of ribosome biogenesis . Myc is In contrast, some tumor suppressor genes have been identified as inhibitors of ribosome biogenesis. The retinoblastoma protein 1 (RB1) gene was the first gene identified as a tumor suppressor since its mutational inactivation causes human cancer. The first cellular function described for protein pRb, and the most studied, is its pivotal role in the negative control of cell cycle. However, in addition, pRb represses RNA Pol I transcription through binding with the transcription factor UBF , and RNAThe protein p53, another extensively described protein exhibiting oncosuppressive activity is a multifunctional protein, first known for its activity as a transcriptional regulator. It recently emerged that p53 controls gene expression not only at the level of transcription, but also at the level of protein synthesis through the inhibition of ribosome biogenesis . IndBRCA1 is a tumor suppressor, the main function of which is to maintain genomic integrity via its critical role in DNA damage repair and its involvement in the control of a number of fundamental cellular processes such as cell cycle control, transcription, chromatin structure and apoptosis. However, in addition to its well-characterized functions, BRCA1 was recently identified as a translational regulator. Indeed, our team has shown that BRCA1 regulates protein synthesis through its interaction with PABP1 ,63. FurtInterestingly, in addition to the clear evidence exposed above showing that increased ribosome production affects translation, recent data revealed that, according to their status, environment, development, or pathological conditions, cells produce different types of ribosomes that differ in their RP composition  and chemical modifications of rRNAs. From these observations the concept of “specialized ribosomes” has emerged ,66, and For many years, the ribosome was considered to be a single structure made of 4 rRNAs and 80 RPs, each RP being present as a single copy, thus leading to the notion of a conserved RP stoichiometry. However, while the existence of such a stoichiometric ribosome is irrefutable, it has become increasingly evident that some ribosomes lack certain RPs. Hence, issues such as the abundance of these RP-defective ribosomes, their function, and the subsequent consequences of ribosome heterogeneity on cells have progressively been assessed over the past decade.Possibly, one of the first indications that different types of ribosomes existed was derived from the identification of numerous RP gene paralogs in yeast and plants. In 1995, analysis of RPL16 paralog gene expression patterns in Arabidopsis thaliana showed that the two paralogs were mutual exclusively expressed in different organs of the plant . In SaccThe evidence that various types of ribosomes may exist in humans came from a cohort of patients displaying genetic diseases collectively called ribosomopathies. Ribosomopathies are caused by haploinsufficiency of genes encoding key factors in ribosome biogenesis or RPs . For exaIn 2011, more than one decade after discovery of the first RP coding gene mutation, the first demonstration that a tissue-selective loss of expression of RPs could impact translation of a subset of mRNAs and drastically impact development stemmed from the analysis of the function of RPL38. Using a mouse model characterized by a 50% reduction in Rpl38 gene expression associated with tissue-specific patterning defects, Kondrashov et al. uncovered the important role of RPL38 in translational control by influencing only a subset of mRNAs, rather than affecting global translation. Indeed, a polysome profiling approach enabled this group to isolate and quantify mRNAs present in the polysome fraction and thus actively translated. A subset of 8 mRNAs among the 31 Hox mRNAs, genes crucial for the formation of the mammalian body plan, was found to be regulated in a RPL38-dependent manner ,74.Several studies confirmed that RP deficiency impacts translation of mRNA subsets. To identify the mRNAs that were translationally regulated upon depletion of RPS19, a microarray analysis of polysome-bound mRNAs in RPS19-depleted and non-depleted erythroblasts was performed. Upon RPS19 depletion, 130 genes were differentially recruited to polysomes . Using bStudies exploring the effect of RPs deficiency, including, but not limited to those exposed above, provide clues as to RPs deficiency implication in the translational regulation of subsets of mRNAs. However, to support the concept of “specialized ribosomes”, the fact that this translational reprogramming originated from ribosomes of heterogeneous compositions remained to be clearly demonstrated at that time.Thus, several proteomic-driven analyses explored the whole ribosome protein composition using purified ribosomes under various conditions to identify all RPs differentially expressed between those conditions. Hence, differential stoichiometry among RPs was associated with different translational statuses in murine embryonic stem cells (mESCs) . Indeed,Although those previous observations strongly supported the notion of a variation in RP expression then composition, technological improvements are ongoing in order to address directly and more accurately the possibility of a variability in RP stoichiometry. Recently, mass spectrometry was upgraded so as to provide data close to absolute quantification of proteins. This novel technology, called selected reaction monitoring (SRM)-based proteomics, uses the spiking of samples with known amounts of labeled peptides derived from RPs as a standard for absolute quantification. However, such approach being expensive, not all 80 RPs could be spiking for absolute quantification. Nevertheless, using this novel technology, absolute quantification of 15 RPs isolated from polysomes was assessed and 4 RPs were identified as being substoichiometric in mESC ribosomes, namely Rpl10A, Rpl38, Rps7, and Rps25, . Based oTo address the impact of ribosome heterogeneity on the translation of subsets of mRNAs, Rpl10A and Rps25 were chosen among the substoichiometric RPs of mESC in this study . Using tThis study therefore demonstrated in 2017 that heterogeneity in ribosome protein composition provides a given ribosome with a specificity for translating subsets of mRNAs, and thus strengthened the concept of “specialized ribosome”.Finally, a study published the same year reinforced this notion by demonstrating that ribosomes depleted in a given RP exhibit different translational activities compared to non-depleted ribosomes. Indeed, Ferretti et al. used a yeast system to assess whether RPS26, which is frequently mutated in DBA, influences the repertoire of translated mRNAs . A TAP tAlthough the initial proteomic studies to identify post-translational modifications of RPs date back to the years 2000 ,82, the The archetypal RP modification is undoubtedly RPS6 phosphorylation, which was associated with various physiological and pathological cellular contexts. Yet, despite a plethora of studies, whether ribosomes with phosphorylated RPS6 exhibit functions different from those harboring the unphosphorylated form remains debatable. Only one study suggests that RPS6 phosphorylation stimulates translation of a specific class of mRNAs containing a 5′-TOP sequence, in response to mTOR signaling, however it remains to be demonstrated that such alteration of translation results from phosphorylated RPS6-containing ribosome .Mutations in the leucine-rich repeat kinase 2 (LRRK2), which lead to Parkinson’s disease (PD), is another example. Martin et al. identified RPS15 as a substrate of the LRRK2 kinase . They shUntil recently, no systematic analysis of the effects of RP post-translational modifications on translation was conducted. A new approach, developed recently, called “polysome proteome profiling” (3P) combines sucrose gradient fractionation with stable isotope labeling using amino acids in cell culture (SILAC)-based quantification . This teAltogether, these studies demonstrate that post-translational modifications of RPs can regulate translation and extend previous findings that heterogeneous ribosomes preferentially translate specific subsets of mRNAs.rRNAs are central to translation, by directly supporting many of the key molecular interactions driving this process. This includes mRNA decoding , peptideRecently developed structural technologies, such as X-ray crystallography and cryo-EM, provided new insight into the specific organization of eukaryotic and human ribosomes ,93. For Hence, a few years ago, scientists intensified their investigations into whether rRNA modifications promote ribosome heterogeneity and impact ribosome activity, including ribosome-mediated regulation of translation as shown for RPs .Among the different types of chemical modifications, 2′-O-ribose-methylation (2′-O-Me) is the most abundant, with 106 sites mapped in human rRNA ,98. 2′-OAll ribosomes were first described as being methylated at each 2′-O-Me site, leading to the concept that 2′-O-Me was a constitutive rRNA modification in healthy proliferating eukaryotic cells ,101,102.To that end, the hybrid in vitro translation technique also called cell-free translation assay ,107, whiIt is well-known that 2′-O-Me is more densely localized within the functional domains of rRNA, notably the decoding center, the peptidyl transferase center (PTC) and intersubunit bridges , suggestThus, 2′-O-Me represents a source of ribosome population heterogeneity observed in different physio-pathological contexts, that preferentially affects several functional domains of ribosomes and impacts ribosomal activity (see review ).Another major rRNA chemical modification consists in the isomerization of uridine into pseudouridine (ψ) . ψ is thDKC1 is a ψ synthase that converts uridine residues into ψ in rRNA and snRNA. The role of DKC1 in rRNA pseudouridylation and its impact on ribosome function have been mainly approached using DKC1 functional inactivation models. X-linked dyskeratosis congenita (X-DC) is indeed a rare multisystemic inherited syndrome caused by mutations of the DKC1 gene, characterized by failure of proliferating tissues and associated with an increased risk of developing tumors .In 2006, a study unveiled that functional inactivation of DKC1 modifies translation, quantitatively and qualitatively. Indeed, in cells derived from DKC1-depleted mice or X-DC patients bearing DKC1 mutation, translation of mRNAs containing IRES elements, including those encoding the tumor suppressor p27 and the anti-apoptotic factors Bcl-xL and X-linked inhibitor of apoptosis protein (XIAP) was impaired . SeveralA thorough examination of the molecular mechanisms through which translation is deregulated upon DKC1 inactivation was conducted at the level of ribosomes. As such, ribosomes from DKC1 depleted human cells were purified in order to study whether DKC1 depletion impacts ribosome composition and function. By means of cell-free translation assay, the alterations in IRES-mediated translation, found in cells lacking DKC1, could definitely be ascribed to an intrinsic ribosomal defect .Analysis at the atomic level of ribosome structures indicate that these modifications participate in rRNA folding and are often located in the vicinity of sites involved in ribosome interaction with tRNA and mRNA ,95.Thus, from these data obtained through recent technologies, we can propose that, similarly to 2′-O-Me, pseudouridylation modulates the ribosome translational activity ,118,119.Finally, a few rRNA bases are also modified. In yeast, there are 12 modified bases on ribosomes. These modifications occur through the addition of one, or sometimes two methyl groups onto specific atoms, including at positions 1, 6, and 7 on the purine rings and positions 1, 3, and 5 on the pyrimidine rings . SpecifiBase modifications have been linked to development and disease. For example, point mutation of the EMG1 gene encoding the enzyme responsible for the m1acp3cψ1191 modification, causes Bowen–Conradi syndrome . DeletioIn yeast abolition of a single base modification, i.e., m1A645 located on the LSU, leads to a change in the quantity of a subset of 18 proteins . CompariThus base modification also contributes to modulating ribosomal activity, though whether these modifications are altered in physiological or pathological contexts remains to be studied in details.For several decades, the ribosome was mainly considered to play a constitutive role rather than a regulatory function in translation. This view was challenged by results showing that cells can produce different types of ribosomes, characterized by variable RP contents and rRNA chemical modifications. These findings led to formulating the concept of “specialized ribosomes” ,32, whicAn additional source of heterogeneous population of ribosomes may arise from the huge diversity of rDNA variants recently reported . Indeed,Interestingly, the concept of “specialized ribosomes” was challenged by the ribosome abundance concept ,129,130.The existence of ribosome heterogeneity adds numerous layers of subtlety when speaking about translational regulation. However, deciphering the fine-tuning of ribosome-mediated translational regulation requires in-depth scrutiny of ribosomes. The next challenges will be to identify and quantify each rRNA post-transcriptional modification, and to quantify each RP in one single cell and then in one single ribosome. Although innovative quantitative mass spectrometry approaches measuring the absolute abundance of RPs have proven their pertinence potential , furtherTranslation deregulation in human disease is a rapidly developing field of research, and some molecules are already in use to treat human diseases, including neurodevelopmental syndromes and cancer . In this