Tpsb2 and Tpsg1 genes, which reside at the 3′-end of the tryptase locus, is significantly decreased by the reduction of the GATA transcription factors GATA1 or GATA2. Chromatin immunoprecipitation assays have shown that the GATA factors bind at multiple regions within the locus, including 1.0 and 72.8 kb upstream of the Tpsb2 gene, and that GATA1 and GATA2 facilitate each other’s DNA binding activity to these regions. Deletion of the −72.8 kb region by genome editing significantly reduced the Tpsb2 and Tpsg1 mRNA levels in MEDMC-BRC6 cells. Furthermore, binding of CTCF and the cohesin subunit Rad21 was found upstream of the −72.8 kb region and was significantly reduced in the absence of GATA1. These results suggest that mouse tryptase gene expression is coordinately regulated by GATA1 and GATA2 in BMMCs. Mast cell tryptases have crucial roles in allergic and inflammatory diseases. The mouse tryptase genes represent a cluster of loci on chromosome 16p3.3. While their functional studies have been extensively performed, transcriptional regulation of tryptase genes is poorly understood. In this study, we examined the molecular basis of the tryptase gene expression in bone marrow-derived mast cells (BMMCs) of C57BL/6 mice and in MEDMC-BRC6 mast cells. The expression of the  Mast cell tryptases are expressed abundantly and are the major component in secretory granules. In humans, elevated levels of tryptase in serum are associated with anaphylaxis  and systTPSG1, TPSB2, TPSAB1 and TPSD1. TPSG1 encodes γ-tryptase, the only membrane-anchored member of the family. In humans, there are three soluble tryptases—α-, β-  and δ-tryptase—that are transcribed from three genes, TPSB2, TPSAB1 and TPSD1. The βII and βIII isoforms are transcribed from the TPSB2 gene, whereas the α and βI isoforms are transcribed from the TPSAB1 gene. In mice, the transcripts from the Tpsg1, Tpsb2 and Tpsab1 genes are mTMT, mMCP6 and mMCP7, respectively. The mTMT is membrane-anchored, whereas mMCP6 and mMCP7 are soluble tryptases.The genes encoding mast cell tryptase form a cluster of loci on chromosome 16p3.3 and 17A3.3 in humans and mice, respectively. Human tryptase loci contain four genes expressed in mast cells: TPSAB1 and TPSB2 genes, and the expression of these genes is polymorphic [TPSD1 gene has been found. Although the overall structure and number of tryptase genes have been well conserved in mammals [TPSAB1 gene have been identified, and an increased copy number of the TPSAB1 gene leads to an elevated basal serum tryptase concentration, which is associated with multisystem disorders in humans [A strong linkage disequilibrium has been demonstrated between the ymorphic ,6. In mi mammals , genomic mammals ,8,9,10. Tpsb2 [Tpsab1 [Tpsg1 [Tpsb2 and Tpsg1 [Tpsab1 activation by MITF was mediated by the activation of c-Jun [Tpsb2 activation, polyomavirus enhancer binding protein 2 (PEBP2) physically interacts with MITF and synergistically activates the Tpsb2 gene transcription [However, while genetic and functional studies have been extensively performed, transcriptional regulation of tryptase genes is less well defined . A basicTpsb2 ,13, Tpsa [Tpsab1  and Tpsg1 [Tpsg1  genes. Wnd Tpsg1 ,15, the of c-Jun . Regardicription .Tpsb2 and Tpsg1 [Tpsb2 and Tpsg1 was also observed [In addition to MITF, we previously reported that the GATA transcription factors GATA1 and GATA2 are also involved in the tryptase gene regulation ,18. We sobserved . Furtherobserved . HoweverIn the present study, we investigated how GATA1 and GATA2 regulate tryptase gene expression in BMMCs. Because region A resides at the 5′-end of the locus, we hypothesized that the genes on this locus might be coordinately regulated by the GATA factors.Cpa3 gene were the most abundant, followed by those from the Cma1 and Tpsb2 genes. To precisely evaluate the contribution of GATA1 and GATA2 to mast cell protease gene expression, siRNA targeting either GATA1 or GATA2 was introduced into BMMCs, and the mRNA levels of mast cell protease genes were assessed by reverse transcription quantitative polymerase chain reaction (qRT-PCR). The introduction of GATA1 and GATA2 siRNAs led to a significant reduction in the corresponding GATA factor expression at both the mRNA and protein levels at 24 h after transfection A,B. In oCpa3 or protease genes located on chromosome 14 . In contrast, consistent with our previous findings for GATA1 knockout BMMCs [Tpsb2 and Tpsg1) was significantly reduced in the GATA1 knockdown cells  within a region of less than 80 kb. We evaluated the mRNA levels of these genes by qRT-PCR in BMMCs and quantified the relative expression level to that of KIT  that are known to be associated with active enhancers ,21. As st of KIT B. The Tpart site A. Regionart site A.Gata2 gene were amplified as positive and negative controls for GATA factor binding, respectively [Gata2 −3.9 kb region. GATA1 binding was observed at similar regions, although the percent input values were smaller than that of the Gata2 −3.9 kb region. The DNA binding of both GATA1 and GATA2 was weak or undetectable at the −67.9, −34.0 and +2.6 kb regions. Although additional peaks of GATA2 binding were observed at −84.3, −20.7 and −13.5 kb in the ChIP-seq data , but not upstream (Ube2i), genes. Alternatively, although this region is bound by the GATA factors, this region may not have a significant role in gene regulation. Contrary to our hypothesis, the mRNA level of the 5′-neighboring A2 siRNA D. In conA2 siRNA D. These T2Rosa26CreER gene and their gene recombination is induced by the 4-OHT treatment in BMMCs [Kit −114 kb region was examined as a positive locus for GATA factor binding [To further examine the DNA binding specificity of GATA1 and GATA2 to the −72.8 and −1.1 kb regions, we utilized BMMCs prepared from conditional knockout mice of GATA1 (G1KO) and GATA2 (G2KO) and examined whether or not GATA1 and GATA2 affect each other’s DNA binding activity to these regions. These mice express in BMMCs ,18. We pin BMMCs . In cont binding . The GATTaken together, these results indicate that GATA1 and GATA2 affect each other’s binding activity to the −72.8 and −1.1 kb regions. In addition, GATA1 might have a critical role in GATA2 binding to the distal −72.8 kb region, whereas GATA2 might be required for GATA1 binding to the proximal −1.1 kb region.Tpsb2 and Tpsg1 gene expression, this region was deleted using CRISPR/Cas9-mediated genome editing in MEDMC-BRC6 (BRC6) murine mast cells [Tpsb2 and Tpsg1 were significantly decreased in the GATA1 or GATA2 knockdown cells  that were used for the qRT-PCR analysis  were found to be expressed in the BMMCs of C57BL/6 mice. These genes reside next to each other in the 3′-end of the locus. To our surprise, deletion of the −72.8 kb region that resides in the 5′-end of the locus severely affected the expression of Tpsb2 and Tpsg1 genes. The −72.8 kb region contains seven classical GATA recognition sequences, (A/T)GATAA, and two of them match the chromatin occupancy sequence for GATA1, (C/G)(A/T)GATAA(G/A/C)(G/A/C), that was reported in previous ChIP-seq studies [Cacna1h gene but not the 5′-neighboring Ube2i gene. Similar results were also obtained in BMMCs treated with either GATA1 or GATA2 siRNA. The expression of the Ube2i gene encoding the SUMO-conjugating enzyme Ubc9 is not mast cell-specific, and its transcript is detected in a variety of tissues [In the present study, we investigated the regulation of mouse tryptase gene expression by GATA factors. Our data suggest that the genes encoding mast cell tryptase on mouse chromosome 17A3.3 are coordinately regulated by GATA factors. Within the mouse tryptase gene locus spanning approximately 75 kb, only two genes  assay and related Hi-C technique reveal that the three-dimensional structure of chromosomes is dynamically regulated, depending on the cell type, stage of development and extracellular condition ,31. EukaTpsb2 and Tpsg1, GATA1 might be required for the GATA2-mediated activation of the −72.8 kb region. Notably, ablation of GATA1, but not GATA2, affected the CTCF and Rad21 binding to the −81.3 and −75.9 kb regions. Thus, it is possible that GATA1 facilitates the recruitment of CTCF and cohesin to the 5′-region of the tryptase locus and thereby promotes the formation of active chromatin structure. Interestingly, GATA2 ablation attenuated the GATA1 binding to the proximal −1.1 kb region, whereas the binding to the −72.8 kb was not affected. Thus, GATA2 might have a dominant role in activating the Tpsb2 and Tpsg1 promoters. Our data indicate that GATA1 plays specific roles that cannot be compensated for by GATA2 in the regulation of mast cell tryptase genes. The ChIP analyses showed that GATA1 ablation significantly reduced the GATA2 binding to the distal −72.8 kb region. Given that deletion of the −72.8 kb region affects the expression of In erythroid and megakaryocytic cells, the genome-wide DNA binding of GATA1 and GATA2 has been comprehensively studied by ChIP-seq analyses ,28,36,37An earlier study showed that the expression of tryptase genes in vivo is restricted to a particular cell type or tissue, and each individual gene has a different expression profile . These dGata1 (Gata1flox/y) and Gata2 (Gata2flox/flox) were kindly provided by S. Philipsen  and S. A. Camper , respectively [T2) were kindly provided by Anton Berns, The Netherlands Cancer Institute. The Gata1 and Gata2 knockout phenotype was examined in BMMCs prepared from hemizygous (Gata1−/y) and homozygous (Gata2−/−) male mice expressing CreERT2. The study was approved by the Animal Experiment Committee of the Takasaki University of Health and Welfare . The animal experiments were carried out in accordance with The Guidelines on Animal Experiments in Takasaki University of Health and Welfare, Japanese Government Animal Protection and Management Law (No.105) and Japanese Government Notification on Feeding and Safekeeping of Animals (No.88). The mice were maintained in an animal facility of Takasaki University of Health and Welfare in accordance with institutional guidelines. Conditional knockout mice of ectively ,44. The l-glutamine . Bone marrow mononuclear cells isolated from the femurs and tibiae were cultured for two weeks in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and penicillin-streptomycin in the presence of 10 ng/mL recombinant murine IL-3  and subsequently cultured for two weeks with IL-3 and 10 ng/mL recombinant murine stem cell factor (Peprotech). The 293T cells (Thermo Fisher Scientific) were cultured in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. MEDMC-BRC6 cells  were pur6 cells) were transfected with 200 pmol of siRNA and 2.5 µg of plasmid DNA by electroporation, and the cells expressing DsRed were sorted using a FACSJazz cell sorter  at 24 h after transfection. In the plasmid transfection experiments shown in The siRNA duplexes for mouse GATA1 and GATA2 were purchased from Invitrogen. The control siRNA was purchased from Sigma. In the knockdown experiments, BMMCs (2.0 × 106 cells) were transfected with 200 pmol of siRNA by electroporation using an Amaxa Nucleofector . Cells were harvested 24 h after transfection and used for the analyses. In the co-expression experiments shown in Polr2a) gene. The primer sequences used for PCR are shown in Total RNA was isolated from cells using a NucleoSpin RNA II . The reverse transcription reactions were performed using a ReverTra Ace qPCR RT Kit  according to the manufacturer’s instructions. Quantitative RT-PCR (qRT-PCR) was performed using the Go Taq qPCR Master Mix  and an Mx3000P real-time PCR system , as described previously . The resThe whole lysates were prepared as previously described (ref). The samples were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the Western blot analyses were performed as described previously  using anA ChIP assay was performed as in a prior report  using anhttps://crispr.dbcls.jp). The sgRNA target sequences were synthesized as DNA oligonucleotides, and a pair of annealed oligonucleotides was inserted into either PX458 or PX459 using the golden gate method. To prepare a targeting vector for homologous recombination, the 5′ and 3′ homology arms were amplified by PCR. The resulting 5′ (1.1 kb) and 3′ (1 kb) arms and the PGK-gb2-neo template  were cloned into a pBluescript KS vector (Agilent Technologies). BRC6 cells were co-transfected with 2 µg each of PX458 and PX459 vectors harboring the 5′ and 3′ gRNAs, respectively, and 1 µg of the targeting plasmid by electroporation using NEPA21 . At 24 h after electroporation, the cells were cultured with 1.0 µg/mL of puromycin for another 24 h, and subsequently, the GFP-positive cells were sorted using a FACSJazz cell sorter into 96-well plates. At 24 h after sorting, G418 was added at a concentration of 1.5 mg/mL, and the cells were cultured for 10 days. The genomic deletion was examined by PCR using genomic DNA isolated with PBND buffer . For homozygous deletion mutants, deletion of the −72.8 kb region was further confirmed by DNA sequencing. The sgRNA target sequences and PCR primers used for the arms are shown in To delete the −72.8 kb region using the CRISPR/Cas9 system, we used two Cas9 expression plasmids: pSpCas9(BB)-2A-GFP (PX458) and pSpCas9(BB)-2A-Puro (PX459) V2.0. These plasmids were a gift from Feng Zhang (Addgene plasmid #48139 and #48138) [The previously described ChIP-seq data sets (GSE48086 and GSE97253) ,21 were t-test. Data are presented as the means ± standard deviation. For all of the analyses, statistical significance was defined as a p-value < 0.05.Comparisons between two groups were made using Student’s


Multicellular organisms require precise gene regulation during ontogeny, and epigenetic modifications, such as DNA methylation and histone modification, facilitate this precise regulation. The conservative reprogramming patterns of DNA methylation in vertebrates have been well described. However, knowledge of how histone modifications are passed on from gametes to early embryos is limited, and whether histone modification reprogramming is conserved is not clear.We profiled H3K4me3/H3K27me3 modifications in gametes and early embryos in zebrafish and found that the patterns in gene promoter regions have been largely set to either co-occupied or active states in gametes and then passed on to early embryos. Co-occupied states are partially maintained, while active states are largely restored to nearly match the sperm’s pattern prior to zygotic genome activation (ZGA). However, repressive H3K27me3 modifications in promoter regions are largely discarded in early embryos. Prior to ZGA, patterns of genes that initialize ZGA are converted to nonrepressive states to coordinate gene expression. Moreover, promoter peaks that mark stage-specific genes are hypermethylated, and histone modifications in these regions are erased independently of DNA methylation reprogramming. Furthermore, comparative analysis revealed that the functions of co-occupied and active genes passed on from gametes are conserved in vertebrates. Gene age preferences by co-occupied and active histone modifications are also confirmed in vertebrates.Our data provide fundamental resources for understanding H3K4me3/H3K27me3 modifications in early zebrafish embryos. The data also reveal that the reprogramming progress of histone modifications is conserved in vertebrates and coordinates with gene expression during ZGA.The online version of this article (10.1186/s12864-019-5611-7) contains supplementary material, which is available to authorized users. Multicellular organisms have evolved numerous cell types that provide sophisticated biological functions and thus require delicate gene regulation mechanisms. Epigenetic modifications such as DNA methylation and histone alterations play vital roles in cell-type decisions. Histones are found in a wide range of eukaryotic organisms, and modifications of histones, such as methylation, acetylation and phosphorylation, are crucial epigenetic marks informing gene activities and chromatin states. These modifications are found around the transcription start site (TSS) of genes in diverse states, in the gene body and in the heterochromatin regions, and they even serve as a signature of DNA damage . Of the The reprograming dynamics of DNA methylation 5mC) during early embryo development have been well illustrated in zebrafish , 4 and mmC duringThe most challenging part of profiling histone modifications in early embryos is that chromatin immunoprecipitation (ChIP) requires large amounts of experimental materials, and early stage embryos are not affordable. Particularly for zebrafish, there is abundant yolk protein that may interfere with immunoprecipitation. Consequently, previously published studies largely focused on stages around ZGA (zygotic genome activation) where a relatively large number of cells are available. Hints from currently available results have indicated that in zebrafish, the competition between histone and gene transcription factors for binding to DNA regulates the onset of ZGA  and histWe profiled genome-wide H3K4me3 and H3K27me3 occupancy in zebrafish gametes and early embryos  with ChIP-seq. For each stage, data from two replicates  primarily according to their states in gametes Fig. a. To ourhha Fig. c, sox2 . Sperm-specific co-occupied genes progressively lost their co-occupancy after the first zygotic cleavage, leaving 15% (770 genes) of them remaining in co-occupied states across all of the investigated stages. Notably, for those sperm-specific co-occupied genes that lost their co-occupancy, they tend to be converted to active states if they are in an active state in oocytes; otherwise they are converted to repressive states and then lose their repressive markers eventually. As a result of the above reprogramming, the final state in the early embryo is not similar to either the sperm or the oocyte, but instead shares features of both gametes. However, in mouse, based on the currently available data, the reprogramming procedure is straightforward, namely, H3K27me3 enrichments are removed from the corresponding co-occupied gene group after the first cell cleavage.Genes in different modification states undergo distinct reprogramming procedures. The co-occupied gene group (group A) undergoes extensive reprogramming progress in zebrafish compared to that group in mouse. For these genes, less than 10% (543 genes) of co-occupied genes are shared in sperm and oocyte, i.e., germline co-occupied genes. However, less than half of these germline co-occupied genes’ states are kept stable across the investigated stages, such as fbxl18; Additional file blf). Among these genes, gamete-shared genes, e.g., gfm1 , histone modifications are partially erased and then reprogrammed nearly but not completely to match the sperm’s pattern upon ZGA, similar to DNA methylation. In our data, we observed that H3K4me3 modifications of more than half of genes are first erased until the 16-cell stage and then restored around the 1 K-cell stage  and oocyte-originated H3K27me3-marked genes, e.g., f4a Fig. c, which Histone modifications could be indicators of gene expression activities. In our data, we found that gene expression and the gene histone modification intensity of H3K4me3 in promoter regions are globally positively correlated Fig. a. Moreovpou5f3, the soxB gene family and nanog, were examined, and we found that histone modification co-occupancy on pou5f3, sox19b and sox2  and protein domain enrichment analysis for each gene group in zebrafish and mouse to see if there are conservations in gene functions . We asked whether there are preferences in gene age in the context of histone modification states, as new genes evolved to render novel functions during species evolution. To address this question, a phylostratigraphic approach  was usedFor the co-occupied gene group in zebrafish Fig. c, the prFor the active gene groups, genes originating in primitive unicellular organisms (phylostratum 1) and eukaryotic organisms (phylostratum 2) were significantly enriched. The proportion of genes that diverged from holozoa organism onward (phylostrata 5–14) are significantly lower in zebrafish . In our study, we profiled histone modifications of early embryos in zebrafish and revealed that there is extensive reprogramming of histone modifications. Compared with the reprogramming process in mouse, we found that although there are differences in histone modification reprogramming, there are conserved principles followed in both zebrafish and mouse. Our data suggested that the reprogramming of histone modifications is likely independent of reprogramming DNA methylation. Histone modification patterns passed on from gametes are reprogrammed according to their associated genes’ functions, thereby facilitating maternal-to-zygotic gene expression transitions and embryogenesis.Wild-type zebrafish line TU, maintained in our own laboratory, was used in this study. Zebrafish were raised for three to twelve months under standard conditions  before experimental materials were collected.Adult male fish were first anesthetized in system water containing 0.016% Tricaine. Testes were dissected from adult male fish as described  and placApproximately 5 min after spawning three washes with system water, unfertilized oocytes were collected by squeezing the abdomen of females. The squeezed females were transferred to separate tanks for recovery. Embryos were collected for 10–15 min after each round of spawning and washed with system water three times. Collected unfertilized oocytes and embryos were washed three times with system water to remove potential follicle cells or other contaminants and then were transferred to 90 mm glass petri dishes with approximately 10 ml system water before proceeding to next step. Embryos were grown at 28 °C and staged according to standard morphological criteria , Three mTubes with pellets were filled with cold PBS buffer to 1 ml, 37% formaldehyde solution (sigma) was added to a final concentration of 1%, and then tubes were rotated for 10 min at room temperature. Cross linking was halted by adding fresh glycine solution to a final concentration of 125 mM, and tubes were rotated for another 5 min. The cross-linked cells were centrifuged for 10 min at 10,000 g and washed two times with cold PBS buffer. The supernatant was removed, and cell pellets were stored at − 70 °C or directly used in chromatin immunoprecipitation steps.The bead-antibody complexes were prepared before cell lysis using 2 μg of H3K4me3 (Abcam ab8580) antibody or 2.5 μg of H3K27me3 (Millipore 07–449) antibody per 10 μl of protein A Dynabeads in low binding PCR tubes. Beads were washed two times with 100 μl ChIP buffer , then the total volume was brought up to 100 μl per assay. The bead-antibody complexes were incubated on a rotator at 4 °C for 2 h. The ChIP buffer was removed before adding the sonicated chromatin. Cell were lysed with 100 μl of lysis buffer  on ice for 30 min. Lysed cells were sonicated in Bioruptor for 25 cycles (30s on and 30 s off per cycle) to achieve fragments that were 300–500 bp in length. The tubes were centrifuged for 10 min at 4 °C , and 100 μl of supernatant was transferred to the bead-antibody complex tubes. The tubes were rotated slowly at 4 °C overnight (8–10 h). The next day, the tubes were placed on a magnetic rack for one minute, and the supernatant was removed. The beads were washed two times with low salt buffer  and high salt buffer ; one time with LiCl buffer . The supernatant was removed, and TE buffer was added  before the beads were transferred to another new tube. The supernatant was discarded, and 100 μl of elution buffer was added . The tubes were placed on a thermal shaker operated at 68 °C and 1000 rpm for 6–8 h. DNA fragments were purified with AMPure XP beads (Beckman) directly before library preparation following Illumina’s instructions. For each sampled stage, two ChIP replicates were performed, and libraries were prepared individually for each replicate except for H3K27me3 ChIP with oocytes. For the H3K27me3 ChIP assay performed in oocytes, precipitated DNA fragments were pooled from two replicates to provide enough DNA for library preparation.http://broadinstitute.github.io/picard). The replicates of each stage were merged with equivalent read pairs and peaks were called using MACS2 [ChIP-Seq data generated in this study or public ChIP-Seq data (dome and 48 hpf stages) used in this study were processed as following. Low quality and adapter-containing reads were trimmed using Trimmomatic . High-qung MACS2  with defng MACS2  against ng MACS2 . ChIP-seng MACS2  package Gene ontology and INTERPRO domain enrichment analyses were carried out using the DAVID web service , 46 withThe Wilcox test was used in this study to determine the significant differences between groups unless otherwise specified in the manuscript. The significance level was uniformly set to 0.01.Additional file 1:Figure S1. Data quality assessments. a Pearson correlation of genome wide coverage between replicates. For H3K27me3 ChIP assay in oocyte, we pooled all ChIP DNA from two replicates as DNA from single replicate is not adequate for library construction. b Overlaps of marked genes between our data and previous ChIP-chip results. c Comparison of proportion of H3K27me3 marked genes in 1 K stage that are covalent with H3K4me3 between our data and previous ChIP-chip results. Note:‘n.s.’indicates there is no significant difference in proportion of genes according to Chi-Square test. Figure S2. Differences of peak width in distal and promoter peak for H3K4me3 and H3K27me3. a Width of promoter peaks is significantly larger than it in distal peaks for H3K4me3. b Width of promoter peaks is significantly larger than it in distal peaks for H3K27me3. ‘***’ denotes that there is significant difference by Wilcox test (p < 0.01). Figure S3. Differencesof CpG dinucleotidedensity in distal and promoter peak for H3K4me3 and H3K27me3. a CpG dinucleotidedensity of promoter peaks is significantly larger than it in distal peaks for H3K4me3. b CpG dinucleotidedensity of promoter peaks is significantly larger than it in distal peaks for H3K27me3. ‘***’ denotes that there is significant difference by Wilcox test(p < 0.01); ‘n.s.’ means there is no significant difference by Wilcox test (p > 0.01). Figure S4. Examples of genes showing histone modification reprogramming patterns during ZGA. Figure S5. Histone modification landscapes of hoxgene cluster in zebrafish. Figure S6. Histone modifications dynamics in mouse. a Histone modification dynamics for genes in mouse. b Hierarchy clustering of sampled developing stages. Figure S7. Correlation between RNA abundanceand histonemodification enrichment. a RNA-Seq FPKM is positively correlated with H3K4me3 FPKMand it increased as embryo developing. b RNA-Seq FPKM is negatively correlated with H3K27me3 FPKM. Figure S8. Relationship between DNA methylation and histone modification enrichment. a DNA methylation level and histone modification enrichment are negatively associated in promoter regions (TSS +/− 3 kb) for both H3K4me3 and H3K27me3. b Histone modification and DNA methylation landscapes of dnmtgene family. Figure S9.. DNA methylation levels in distal and promoter peak regions are significantly different in mouse. Note, ‘***’ denotes there is significant difference between group with Wilcox test (p < 0.01). Figure S10. Functional conservation in bivalent and active gene groups between zebrafish and mouse. a Conserved INTERPRO domains between zebrafish and mouse for bivalent gene group. b Conserved GO terms between zebrafish and mouse for bivalent gene group. c Conserved INTERPRO domains between zebrafish and mouse for active gene group. d Conserved GO terms between zebrafish and mouse for active gene group. (PDF 2365 kb)Additional file 2:Table S1. Pairwise dissimilarities for each stage in zebrafish (XLSX 9 kb)Additional file 3:Table S2. Pairwise dissimilarities for each stage in mouse (XLSX 9 kb)Additional file 4:Table S3. Enriched GO and INTERPRO domains for co-occupied gene group in zebrafish. (XLSX 11 kb)Additional file 5:Table S4. Enriched GO and INTERPRO domains for active gene group in zebrafish. (XLSX 15 kb)Additional file 6:Table S5. Enriched GO and INTERPRO domains for co-occupied gene group in mouse. (XLSX 11 kb)Additional file 7:Table S6. Enriched GO and INTERPRO domains for active gene group in mouse. (XLSX 15 kb)Additional file 8Table S7 Gene age preference for each modification status in each stage of zebrafish. (XLSX 12 kb)Additional file 9:Table S8. Gene age preference for each modification status in each stage of mouse. (XLSX 11 kb)


Vegf gene expression in granulosa cells (GCs) undergoing luteinization during ovulation. To understand the factors involved in this increase, we examined the roles of two transcription factors and epigenetic mechanisms in rat GCs. GCs were obtained from rats treated with eCG before, 4 h, 8 h, 12 h and 24 h after hCG injection. Vegf mRNA levels gradually increased after hCG injection and reached a peak at 12 h. To investigate the mechanism by which Vegf is up-regulated after hCG injection, we focused on C/EBPβ and HIF1α. Their protein expression levels were increased at 12 h. The binding activity of C/EBPβ to the Vegf promoter region increased after hCG injection whereas that of HIF1α did not at this time point. The C/EBPβ binding site had transcriptional activities whereas the HIF1α binding sites did not have transcriptional activities under cAMP stimulation. The levels of H3K9me3 and H3K27me3, which are transcriptional repression markers, decreased in the C/EBPβ binding region after hCG injection. The chromatin structure of this region becomes looser after hCG injection. These results show that C/EBPβ regulates Vegf gene expression with changes in histone modifications and chromatin structure of the promoter region in GCs undergoing luteinization during ovulation.The ovulatory LH-surge increases  After the surge of luteinizing hormone (LH), endothelial cells in the theca cell layer proliferate and start to invade the granulosa cell layer during ovulation. This is the first process to form the CL3. Endothelial cells rapidly form blood vessels and vascular network in the CL, which makes CL highly vascularized organs. This event is necessary for CL continuing to produce progesterone for establishing and maintaining pregnancy. In previous studies, angiogenic factors that are responsible for luteal development have been identified4. Vascular endothelial growth factor (VEGF) is one of them and is highly expressed in rat and human CL5. VEGF in the ovary is involved in physiologic development, maintenance and maturation of blood vessels and contributes to rapid vascularization during CL formation. Therefore, VEGF is one of important genes, which leads to CL development7. The LH surge changes a number of gene expressions in granulosa cells (GCs) during ovulation8. Although VEGF gene expression is increased during ovulation, the detailed molecular mechanism of this regulation has been unclear. It is well known that hypoxia inducible factor 1-alpha (HIF1α) induced by hypoxic conditions increases VEGF expression in many types of cells11. However, it is unclear whether HIF1α also regulates VEGF expression in GCs undergoing luteinization.Angiogenesis is required for the corpus luteum (CL) formation13. In C/EBPα and β knockout mice, the expressions of angiogenesis-related genes are suppressed and vascular development in CL is defective12. These findings led us to hypothesize that Vegf expression is regulated by C/EBPβ in GCs during ovulation.CCAAT/enhancer-binding protein beta (C/EBPβ) is a transcription factor involved in follicle rupture and following CL formation16. Histone modification is one of them and affects the chromatin structure, which regulates the accessibility of transcription factors into gene promoter or enhancer region19. In previous studies of GCs in rats undergoing luteinization21, we reported that changes of histone modifications and chromatin remodeling are involved in the regulation of three luteinization-related genes: steroidogenic acute regulatory protein (StAR), aromatase (Cyp19a1) and cytochrome P450 cholesterol side-chain cleavage enzyme (Cyp11a1). These findings raised the possibility that Vegf expression is regulated not only by transcription factors but also by an epigenetic mechanism.Not only transcription factors, but also epigenetic mechanisms regulate gene expressionVegf expression in rat GCs undergoing luteinization.In this study, we showed that C/EBPβ, but not HIF1α, regulates Vegf mRNA levels in the GCs gradually increased and reached a peak at 12 h after hCG injection  was injected to rats to induce luteinization. GCs were isolated from ovarian follicles. We examined by Western blot analyses whether C/EBPβ and HIF1α protein expression levels were increased in rat GCs after hCG injection. C/EBPβ protein Fig.  and HIF1Vegf promoter, including C/EBPβ (−1115 bp to −1106 bp) and HIF1α   primers surrounding these sites, −1148 bp to −1024 bp (C/EBPβ binding region), −976 bp to −857 bp (HIF1α binding region-1), −724 bp to −645 bp (HIF1α binding region-2) and −470 bp to −369 bp (HIF1α binding region-3), respectively  Fig. . Becauseely Fig. . The binion Fig. . The binion Fig. , 0 h. Hoion Fig. , 12 h. TVegf mRNA expression, we examined the effect of C/EBPβ knockdown on Vegf mRNA expression. Because the efficiency of knockdown in primary GCs is quite low, we used KGN cells (human granulosa tumor cells) that are widely used for experiments on luteinization22. Because KGN cells do not respond to hCG stimulation, cAMP, a second messenger of hCG, was used to induce luteinization in KGN cells as reported previously23. First, we confirmed that VEGF mRNA was increased by cAMP stimulation in KGN cells . These findings show that HIF1α binding sites in the rat Vegf promoter region have the transcriptional activities under CoCl2 stimulation, but not under cAMP stimulation.The transcriptional activity of the C/EBPβ binding site in the rat cts Fig. . When ce16, we examined in rat GCs whether hCG stimulation changes histone modification levels of the C/EBPβ binding region in the Vegf promoter. The levels of H3K9me3 and H3K27me3, which are correlated with the suppression of transcription, significantly decreased at 12 h after hCG injection. The level of H3K4me3, which is correlated with the activation of transcription, was not affected by hCG injection -qPCR. The relative FAIRE enrichment ratio in this region increased by hCG injection Fig. , indicat26, to the C/EBPβ binding region. The EZH2 binding activity to the C/EBPβ binding region, as measured by ChIP assay, significantly decreased after hCG injection  in rat Vegf promoter region are not involved in the regulation of Vegf expression by cAMP. In this study, we did not show the involvement of HIF1α in the regulation of Vegf in our experimental condition including the use of KGN cells. However, we do not exclude the role of HIF1α in Vegf induction during luteinization, because HIF1α may regulate Vegf expression at different time points of luteinization process, or bind to an unknown promoter or enhancer region of Vegf gene undergoing luteinization in GCs. On the other hand, Rico et al.35 showed that HRE deficient mice responded to LH with an increase in Vegf expression in granulosa cells in vivo, suggesting that Vegf expression by LH is not regulated by HIF1α in mouse granulosa cells, which supports our findings. Kim et al.10 used Echinomycin, which inhibits the binding ability of HIF1α to HREs, and found that Echinomycin inhibits hCG-increased Vegf mRNA expression in mice granulosa cells. Although they concluded that hCG up-regulates Vegf expression through HIF1α, it should be noted that Echinomycin directly affects many gene expressions. Taken together, we concluded that C/EBPβ is an important factor to regulate Vegf gene expression in rat GCs undergoing luteinization after the LH surge.HIF1α has been reported to regulate ngs Fig. . Becauseion Fig. , 0 h, buion Fig. , 12 h. TVegf mRNA expression in GCs, most of them are based on the data from in vitro experiments done under the conditions of hypoxia or CoCl2 stimulation. Martinez-Chequer et al.36 reported that CoCl2 increased VEGF expression in monkey GCs. Alam et al.37 reported that CoCl2 induced HIF1α protein expression in rat GCs. Kim et al.10 showed that HIF1α is involved in Vegf expression induced by CoCl2 in mouse GCs. Yalu et al.24 showed the involvement of HIF1α in the CoCl2-induced VEGF expression by knockingdown HIF1α in human GCs. Our results also showed that HIF1α contributes to Vegf expression under CoCl2 stimulation, but not under cAMP stimulation . From these results, it is likely that HIF1α is involved in the regulation of Vegf expression in GCs under CoCl2 stimulation in vitro.Although there are several reports showing that HIF1α is associated with Vegf promoter region in luteinizing rat GCs. These changes included decreases of H3K9me3 and H3K27me3 levels along with a decrease of EZH2 binding to the C/EBPβ binding region in the Vegf promoter all of which are associated with the activation of transcription by loosening the chromatin38. This result is consistent with our previous findings that stimulating rats with hCG caused decreases in H3K9me3, H3K27me3 and EZH2 binding of the Cyp11a1 promoter region in GCs and increases in their mRNA expressions20. Our results showed that hCG loosened the chromatin structure of the Vegf promoter region, allowing C/EBPβ to access its response element of the Vegf promoter region. These findings suggest that these epigenetic changes are closely associated with the up-regulation of Vegf expression in GCs undergoing luteinization. H3K4me3 is a histone modification related to active transcription40. In our study, the levels of H3K4me3 were not changed by hCG stimulation. This is not surprising, because the changes of H3K4me3 levels are not always correlated with those of other histone modifications18, and because gene expression is regulated by not only one histone modification, but also the combination of various histone modifications13.Our data also showed that epigenetic changes such as histone modifications and chromatin remodeling occurred in the Vegf is up-regulated in rat GCs undergoing luteinization after the LH surge. We found that C/EBPβ, but not HIF1α, regulates Vegf gene expression by binding to the novel binding site in the rat Vegf promoter region. In addition to transcription factors, histone modifications and chromatin structure of the Vegf promoter region are involved in the regulation of Vegf expression. Because Vegf plays a key role in angiogenesis, our results should help to better understand the regulation of angiogenesis in GCs undergoing luteinization during ovulation.In summary, the present study shows a molecular mechanism by which This study was reviewed and approved by The Committee for the Ethics on Animal Experiment in Yamaguchi University Graduate School of Medicine. All experiments were performed in accordance with relevant guidelines and regulations.Female Sprague Dawley rats (21- to 24-day old) were purchased from Japan SLC . They were injected subcutaneously with 15 IU of equine chorionic gonadotropin (eCG)  to promote follicular growth followed by 15 IU of hCG (Sigma) injection to induce ovulation and luteinization. The ovaries were obtained before hCG (0), and 4, 8, 12, and 24 hours (h) after hCG injection. The follicles were punctured to isolate GCs. Cells were centrifuged and washed in PBS and provided for the experiments.KGN cells , which were derived from a human granulosa cell tumor, were cultured in DMEM/Ham’s F-12 medium.41. Quantitative real time PCR reaction was carried out as described previously43. Primer sequences are listed in Table RNA was extracted with RNeasy Mini  and was converted to cDNA with reverse transcriptase  as reported previously44. The membranes were incubated for overnight with the following antibodies: C/EBPβ (Santa Cruz Biotechnology), HIF1α  and Histone H3 . Amersham ECL Western Blotting Detection Reagent and hyperfilm-ECL  were used to detect immunoblot bands.Total protein were extracted and subjected to SDS-PAGE and then transferred to membrane as described previously45 with some modifications. The following antibodies were used in this assay: H3K4me3 , H3K9me3 , H3K27me3 (generous gift from Dr. Kimura), EZH2 , C/EBPβ, HIF1α and non-specific normal IgG . Real-time qPCR was performed with primers listed in Table The histone modification levels and recruitments of transcription factors were examined by ChIP assays as described previously46. After 24 h of transfection, KGN cells were incubated with or without dibutyryl-cAMP  (Sigma) for 24 h.Transfection of siRNAs was performed as reported previouslyVegf gene; 1) the construct including both C/EBPβ binding site and HIF1α binding sites (−1171 bp to +115 bp), 2) the construct lacking the C/EBPβ binding site (−976 bp to +115 bp) and 3) the construct that four HIF1α binding sites are mutated (−1171 bp to +115 bp), were amplified using the primer sets in Table 20 and then cells were incubated with or without cAMP (0.5 mM) or CoCl2 (0.5 mM) for 24 h. Luciferase activities were examined as described20. Luciferase assays combined with C/EBPβ knockdown were performed as reported previously23. The reporter construct (the construct of −1171 bp to 115 bp) and siRNA (C/EBPβ siRNA or non-targeting siRNA) were transfected simultaneously. Then, KGN cells were incubated with or without cAMP and luciferase activities were examined. Luciferase assays were done in triplicate and repeated three times.Three 5′-flanking regions of the rat Vegf promoter, FAIRE-qPCR was performed as described48 with some modifications. Real-time qPCR was performed to determine and calculate the FAIRE enrichment with same primers used in ChIP assay (Table To examine the chromatin accessibility of C/EBPβ binding region in the ay Table .Unpaired student t-test was used to compare the mean values in two groups. To analyze differences between groups, one-way ANOVA followed by Tukey-Kramer test was used. P < 0.05 was considered significant.Supplementary information file


Cc2d1b and Purβ, which have no described roles in myelinating glial cells. We demonstrate that silencing of either Cc2d1b or Purβ limits the formation of myelin segments. These data provide a deeper insight into the myelin gene transcriptional network and the role of YAP/TAZ in myelinating glial cells.Schwann cells (SCs) are exquisitely sensitive to the elasticity of their environment and their differentiation and capacity to myelinate depend on the transduction of mechanical stimuli by YAP and TAZ. YAP/TAZ, in concert with other transcription factors, regulate several pathways including lipid and sterol biosynthesis as well as extracellular matrix receptor expressions such as integrins and G-proteins. Yet, the characterization of the signaling downstream YAP/TAZ in SCs is incomplete. Myelin sheath production by SC coincides with rapid up-regulation of numerous transcription factors. Here, we show that ablation of YAP/TAZ alters the expression of transcription regulators known to regulate SC myelin gene transcription and differentiation. Furthermore, we link YAP/TAZ to two DNA binding proteins,  YAP and TAZ regulate positive and negative myelin regulators.in vitro.Cc2d1b and Purβ are necessary for Schwann cell myelination EGR2, YY1, ZEB2, Topilko et al., The function of the nervous system relies on the ability of peripheral nerve fibers to transmit information to and from the target tissues. The speed of propagation of action potentials in these fibers is regulated by myelin, a multilamellar structure produced by Schwann cells  sciatic nerves, SCs lacking YAP/TAZ are unable to myelinate and experience a global dysregulation of transcription  harbor TEAD elements and are downregulated in Yap cHet; Taz cKO sciatic nerves ,  and Purβ ,  and control (non-targeting) were bought from Dharmacon SMARTvector library. SCs were infected with five virions per cells, incubated for 72 h and collected for qRT-PCR analysis. Coculture experiments were done with shCc2d1b #1 and shPurβ #2.shRNA virions were produced as Poitelon et al. . shRNA tActb was used as endogenous gene of reference and 18S was used as to validate the stable expression of Actb. The primers and probe used are the following: 18S , mouse Actb , mouse Cc2d1b ; mouse Purβ ; rat Cc2d1b ; rat Purβ .Sciatic nerves were dissected, stripped of epineurium, frozen in liquid nitrogen, pulverized and processed as described . Myelin segments number and length were quantified using ImageJ softwareImmunohistochemistry, immunocytochemistry and immunoblotting were performed as described  and Two-way ANOVA were used for statistical analysis of the differences between multiple groups according to the number of sample groups. Values of P ≤ 0.05 were considered to represent a significant difference.Experiments were not randomized, but data collection and analysis were performed blind to the conditions of the experiments. Data excluded are reported in the legend of the figures. Data are presented as mean ± standard error of the mean (SEM) or SD. No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those generally employed in the field. Two-tailed Student’s Yap cHet; Taz cKO sciatic nerves at 3 days of age (NCBI GEO: GSE79115). We identified 2,071 misregulated transcripts (Yap/Taz dysregulated 64 genes (Tbx2) and immature SCs (Oct6/Pou3f1/Scip), as well as genes inhibiting differentiation (Id4) and myelin formation (Sox2) were highly expressed  and P20  protein family, which includes of PURα, PURβ and PURγ. There is substantial evidence for PUR role in DNA binding (Rumora et al., Cc2d1b and Purβ expression appear to be downstream YAP/TAZ/TEADs, the signals contributing to the regulation of their expression are essentially unknown. Elucidation of the upstream pathways and signals that induce CC2D1B and PURβ will be important both for understanding the molecular control of the myelination program, but also potentially for identifying strategies to promote remyelination in demyelinating disease. Indeed, numerous transcription factor involved in developmental myelination is also involved in remyelination following injury. Thus, it will be critical to characterize the role of CC2D1B and PURB in peripheral nerve repair.Although the Finally, our study extends regulatory mechanisms directing SC myelinogenesis (Hung et al., All datasets generated for this study are included in the manuscript and/or the supplementary files.SB and YPo designed research and interpreted data. SB and YPo performed experiments with JH assistance. YPa contributed to analytical tools. YPo wrote the manuscript with SB assistance. SB and YPo analyzed the data. YPa, JV, and LF critically reviewed the 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.


During T cell differentiation and activation, specific stimuli, and a network of transcription factors (TFs) are involved in orchestrating chromatin accessibility, establishing enhancer-promoter interactions, and regulating gene expression. Over the past few years, there have been new insights into how chromatin interactions coordinate differentiation during T cell development and how regulatory elements are programmed to allow T cells to differentially respond to distinct stimuli. In this review, we discuss recent advances related to the roles of TFs in establishing the regulatory chromatin landscapes that orchestrate T cell development and differentiation. In particular, we focus on the role of TFs  in mediating chromatin accessibility and interactions and in regulating gene expression in T cells, including gene expression that is dependent on IL-2 and IL-21. Furthermore, we discuss the state of knowledge on enhancer-promoter interactions and how autoimmune disease risk variants can be linked to molecular functions of putative target genes. Transcriptomic profiles determine the phenotype and function of cells, and this process is tightly controlled by various transcription factors (TFs), epigenetics, and chromatin interactions to define transcriptional patterns in response to cellular signals. More specifically, control of gene expression depends not only on the binding of sequence-specific TFs to target DNA sequences, but also on chromatin accessibility, which is controlled by the proper packaging of DNA/nucleosomes (chromatin) within the nucleus, leading to the arrangement of the genome into distinct spatial structures. Differences in chromatin composition can determine gene expression profiles in cells by providing relative accessibility (open or closed) of key regions to TFs that bind to DNA. Within the immune system, upon cellular stimulation by extracellular signals  or cytokines), chromatin composition is modified through the concerted actions of signal-specific TFs and chromatin modifiers via a dynamic process. Naturally occurring genetic mutations in binding sites for TFs that alter the chromatin landscape can potentially disrupt or establish chromatin interactions, thereby resulting in altered gene expression profiles, and predisposing to cancer, autoimmune disease, allergy, immunodeficiency, or other immune disorders. In this review, we focus on the cooperative actions of TFs that play critical roles in shaping the chromatin landscape and accessibility in early and mature T cell development, and how these dynamic changes can alter gene expression profiles.Lineage specific transcription factors (LSTFs) or master regulators are expressed at critical times during lymphoid development or differentiation, and they contribute to cell type determination. During the development of T cells in the thymus, master regulators such as TCF-1, BCL11B, GATA3, PU.1, and RUNX family TFs are critical for T-lineage commitment Figure , has rev−CD8− or double-negative (DN) thymocytes, which can then progress through four stages of maturation . T cell commitment occurs at the DN2a to DN2b developmental transitional stages  cells, which express both CD4 and CD8  was shown to function as a specialized nucleosome-binding transcription factor during the DN1-DN2 transition  family of cytokines  through cytokine receptors IL-2R and IL-21R Figure , 33. Cytytokines , 41. Durh2 cells , 43 in th2 cells  and for h2 cells  and IL-4h2 cells –48, withl4 locus . IL-2 vil4 locus , 50, potl4 locus , 51, thel4 locus . Moreovel4 locus  and limil4 locus , 54 wherl4 locus , 55, 56.+ T  cell differentiation and act as pioneer factors for the differentiation of type 1 Treg (Tr1) cells  mainly in T cells and dendritic cells –59 and t+ T  cells  cells . BACH2, mily TFs , 66.Irf4−/− CD4+ T cells compared to WT cells  motifs. IL-2 potently activates STAT5, whereas IL-21 primarily activates STAT3. This differential STAT activation leads to differential gene expression by these cytokines. It is established that STAT proteins are critical components of cytokine-activated enhancers, but recently their roles related to super-enhancers , revealing that super-enhancers are critical for both the positive and negative regulation of IL-2 signaling  may result in altered target gene expression that leads to disease development Figure . Anotherll types . StrikinIn summary, studies of the transcriptional and epigenetic regulation of T cells have identified several mechanisms of cross-regulation between TFs, chromatin modifiers, and the pre-existing chromatin landscape. The interactions between chromatin and TFs are influenced by a range of stimuli, including TCR and cytokine signals. Transcription factors are important for cell function, and they collaborate combinatorically with other factors to influence gene regulation. Their binding to DNA depends on epigenetic landscapes, and their function may depend on chromatin interactions to juxtapose distal regulatory elements with gene promoters. The ability of cytokine-activated proteins to modify nucleosome packing and influence histone modifications allows them to control developmental processes. The gene regulatory networks that determine T cell development are broad and involve chromatin accessibility, epigenetic status, and distant chromatin interactions in both time- and context-dependent manners. Our evolving understanding of gene regulatory networks will help to comprehensively link genetic variants to putative gene targets, furthering our understanding of molecular mechanisms for a range of immune diseases. Achieving a deeper understanding of the mechanisms involved has now been greatly facilitated by genetic manipulations including CRISPR/Cas9 gene editing but still awaits other advances, such as the ability to comprehensively study single cells in real time.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with the authors.


In the current report, we describe biochemical and genetic data demonstrating that thiS motif RNAs function as sensors of the thiamin precursor HMP-PP, which is fused with HET-P ultimately to form the final active coenzyme thiamin pyrophosphate (TPP). HMP-PP riboswitches exhibit a distinctive architecture wherein an unusually small ligand-sensing aptamer is almost entirely embedded within an otherwise classic intrinsic transcription terminator stem. This arrangement yields remarkably compact genetic switches that bacteria use to tune the levels of thiamin precursors during the biosynthesis of this universally distributed coenzyme.We recently implemented a bioinformatics pipeline that can uncover novel, but rare, riboswitch candidates as well as other noncoding RNA structures in bacteria. A prominent candidate revealed by our initial search efforts was called the ‘ Many bacteria use small genetic devices called riboswitches to sense molecules that are essential for life and regulate the genes necessary to make, break or move these molecules. Riboswitches are made of molecules of RNA and appear to have ancient origins that predate the evolution of bacteria and other lifeforms made of cells. Inside modern bacteria, chunks of DNA in the genome provide the instructions to make riboswitches and around 40 different types of riboswitch have been identified so far. However, it has been proposed that the instructions for thousands more riboswitches may remain hidden in the DNA of bacteria.All of the currently known riboswitches contain a region called an aptamer that binds to a target molecule. This binding causes another structure in the riboswitch RNA to switch a specific gene on or off. For example, the aptamer binding might cause a hairpin-like structure called a terminator to form, which stops a gene being used to make new RNA molecules.thiS motif. This potential new riboswitch appeared to be associated with a gene that encodes a protein required to make a vitamin called thiamin .In 2019 a team of researchers reported using a computational approach to identify new riboswitches in bacteria. This approach identified many different chunks of DNA that might code for a riboswitch, including one known as the thiS motif. The experiments revealed that the motif binds to a molecule called HMP-PP, which bacteria use to make thiamin. Unexpectedly, the aptamer of the riboswitch was nested within a terminator, rather than being a separate entity.To test the new computational approach, Atilho et al. including several of the researchers involved in the earlier work used genetic and biochemical techniques to study the The findings of Atilho et al. reveal that riboswitches can be even more compact than previously thought. Furthermore, these findings reveal new insights into how bacteria use riboswitches to manage their vitamin levels. In the future it may be possible to develop drugs that target such riboswitches to starve bacteria of these essential molecules. Approximately 40 distinct riboswitch classes that regulate gene expression in various bacterial species have been experimentally validated to date . Based oi) percent guanosine and cytidine (GC) nucleotide content and (ii) length in nucleotides. For many bacteria, structured ncRNAs are GC-rich compared to other regions of the genome , and then uses comparative sequence and structural analyses to identify novel candidate RNA motifs . Specifie genome , and thee genome , includie genome .thiS motif’ −4-methylthiazole phosphate (HET-P) (thiS motif RNAs appear to code for proteins that participate in the production of HET-P or its fusion to 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate (HMP-PP), to ultimately produce the bioactive coenzyme thiamin pyrophosphate (TPP)  . Other gte (TPP) .thiS motif RNAs was derived from the fact that this novel RNA structure occasionally resides in tandem with TPP riboswitches . This same reporter-fusion construct yields no reporter gene expression in host B. subtilis cells that naturally carry a thiS gene. These findings were consistent with our hypothesis that thiS motif RNAs function as riboswitches, but the precise ligand sensed by the unusual architecture of this RNA remained unknown.Previously , we creathiS motif RNAs is the TPP biosynthetic intermediate HMP-PP. Furthermore, our findings demonstrate that this unusually small riboswitch employs a distinct architecture to regulate RNA transcription termination. This regulatory RNA provides cells with an efficient mechanism to balance the production of two key biosynthetic intermediates, HMP-PP and HET-P, which are then fused to make the essential coenzyme TPP.In the current report, we describe a series of bioinformatic, genetic and biochemical analyses that provide conclusive evidence that the ligand for thiS motif exhibits an unusual arrangement wherein the predominant secondary structure, derived by thermodynamic modeling, is an extended hairpin structure that maximizes conventional Watson/Crick base pairing , and that each tandem TPP-thiS motif system would function as a two-input Boolean logic gate  more of the desired metabolite. Although thiS motif representatives associate with genes for the production of HET-P (thiE gene (coding for thiamin-phosphate synthase) is occasionally associated with thiS motif representatives, suggesting that the RNA motif determines if cellular conditions are suitable to couple the two key precursors of thiamin monophosphate (TMP) to eventually yield TPP. Taken together, all these bioinformatic observations are consistent with the hypothesis that thiS motif RNAs function as ON riboswitches for the TPP precursor HMP-PP.Furthermore, if the two secondary structure states called the ‘terminator configuration’ and the ‘aptamer configuration’  represenof HET-P , it doesthiS motif RNAs, we employed a chromosomally-integrated reporter construct  riboswitch to a lacZ reporter gene, which was integrated into the amyE locus, exhibited no β-galactosidase activity in response to HMP added to rich (LB) liquid culture media  or M2 (C35G)  , which a2 (C35G) , are not2 (C35G) . ImportaB. subtilis cells grown in LB medium are expected to suppress the genes needed to produce both the pyrimidine moiety HMP-PP and the thiazole moiety HET-P. Thus, as expected, expression of the reporter gene fused to the putative HMP-PP riboswitch is off, regardless of the genetic background tested. Likewise, WT cells grown in minimal (GMM) medium also do not express the reporter gene, presumably because HMP-PP does not accumulate due to its rapid and efficient conversion to the final product, TPP. By contrast, ΔthiS and ΔthiE cells  exhibit high levels of reporter gene expression  required to phosphorylate HMP-P to make HMP-PP. Reporter gene expression is not observed in ΔthiD cells , and three progressively truncated variants  represent only the RNA regions that would be exposed when RNA polymerase is stalled at various locations within the run of U nucleotides  for this interaction. However, if we assume that all HMP was enzymatically converted into HMP-PP, this product was fully recovered after removal of protein by filtration, and that there was no degradation over the time frame of the assays, the KD value cannot be greater than 500 μM for this variant aptamer construct.We suspect that the ligand concentration is insufficient to saturate all RNAs in the sample. Regardless, by quantifying the band intensities at the three sites of structural modulation and by assuming that these values could become zero (or background) upon ligand binding, we generated a partial HMP-PP binding curve for 52 thiS M3 , which ithiS M3 construct exhibits evidence of both binding to HMP-PP and of structural switching to favor the aptamer configuration (‘ON’ state) (thiS motif sequences by having the U nucleotides at positions 46–48 remain sequestered in the RNA-exit channel (A series of shortened constructs carrying the M3 mutations  recapitu’ state) . This fi’ state) , which s channel  of an RNthiS motif RNAs function as riboswitches that directly bind to HMP-PP. As a result, we favor renaming thiS motif RNAs as HMP-PP riboswitches. The pursuit of more precise biochemical and biophysical characteristics, and further evidence for the mechanism proposed above, will require both the judicious use of RNA constructs that form the aptamer configuration, and either the preparation of pure samples of HMP-PP with consideration for its relative instability or the use of more stable analogs that can trigger riboswitch function.Overall, our biochemical data are strongly consistent with the hypothesis that As noted above, approximately 30% of the representatives of HMP-PP riboswitches reside in tandem with TPP riboswitches. In these tandem systems, the TPP riboswitch always occurs first, and the TPP aptamer is always associated with its own expression platform, which is routinely a readily recognizable intrinsic terminator stem. The HMP-PP riboswitch follows each complete TPP riboswitch, and by its unique architecture, carries its own intrinsic terminator expression platform.Clostridium lundense  to state 4 . As predicted by the truth table, ligand states 2 and 4 do not yield expression of the reporter gene . We alsothiS motif RNAs represent an unusual class of riboswitches that sense and respond to the thiamin pyrophosphate biosynthetic intermediate HMP-PP. Its architecture is unusual because the majority of the nucleotides and structures required for ligand recognition appear to be encompassed by an intrinsic transcription terminator, which forms in a mutually-exclusive manner to regulate gene expression. All other riboswitch classes reported to date  or exclusively (54 thiS) adopt the terminator configuration  methanol, which was purchased from Enamine Ltd. The radiolabeled molecules , and lysed with a microfluidizer. The resulting lysate was clarified via centrifugation, applied to NiNTA resin, and washed with 10 column volumes of Buffer A. Tagged protein was eluted from the column with three column volumes of Buffer B  and applied to a size-exclusion column equilibrated in Buffer C . 10% Glycerol  was added to the protein sample before storage at −80°C.An N-terminal 6xHis-tagged Salmonella typhimurium HMP-P kinase  to a 90 μL reaction preparation to yield a final concentration of 5 mM HMP, 20 mM ATP, 50 mM Tris-HCl buffer (pH 7.5 at 23°C), 2 mM TCEP, and 5 mM MgCl2. This 100 μL reaction mixture was allowed to incubate at 23°C overnight, at which time the protein was removed by ultrafiltration using an Amicon Ultra-0.5 centrifugal filter unit with a 3 kDa cutoff membrane. The HMP-PP generated in this manner was used without further purification, typically on the same day but no later than 2 weeks after production. HMP-PP stock solutions were stored at −20°C. Control assays for transcription termination and in-line probing conducted without the addition of enzymatically prepared HMP-PP contained an equivalent amount of the enzymatic reaction preparation wherein the ThiD protein was excluded. These control assays therefore contain HMP, whereas test reactions contain HMP-PP.Pyrophosphorylation of HMP was carried out enzymatically as described previously . BrieflyEnzymatically prepared HMP-PP samples were sent to the MS and Proteomics Resource at Yale University for analysis. The presence of HMP-PP was confirmed by data  generatelysC gene from B. subtilis, the riboswitch aptamer, and the expression platform of the thiS gene from C. sp Maddignly to 33 nucleotides following the terminator stem. Additional non-native nucleotides were added to the 5′ region upstream of the HMP-PP aptamer to increase the amount of [α-32P]-ATP incorporation.The protocol used for single-round in vitro transcription assays was adapted from that described previously . DNA con2, 100 µM EDTA, 10 µg mL−1 bovine serum albumin, 130 µM ApA dinucleotide, 1% glycerol, 0.04 U µL−1E. coli RNA polymerase holoenzyme, 2.5 µM GTP, 2.5 µM UTP, and 1 µM ATP]. Approximately 8 µCi [α-32P]-ATP was added to the 90 µL transcription reaction and transcription was allowed to proceed at 37°C for 30 min, leading to formation of a stalled polymerase complex before the first cytidine of each transcript. The reaction mixture was then distributed in 8 µL aliquots into separate microfuge tubes, which contained 1 µL of an HMP or HMP-PP solution plus 1 µL of 10x elongation buffer . For transcription termination assays, the maximum concentration of HMP-PP is 1.5 mM, assuming 100% enzymatic conversion of HMP and no loss to instability. However, HMP-PP concentration is likely substantially lower due to incomplete conversion of HMP to HMP-PP , 20 mM NaCl, 14 mM MgClo HMP-PP . Transcr32P]-ATP incorporation were considered negligible.The transcription products were separated by denaturing (8 M urea) 10% polyacrylamide gel electrophoresis (PAGE) then imaged and quantified using a Typhoon Phosphorimager and ImageQuaNT software. The fraction of full length (FL) and terminated (T) RNA transcripts was calculated by measuring band intensity values and using the equation Fraction FL = (FL intensity)/(FL intensity +T intensity). The differences in specific activities between the FL and T products due to [α-32P-labeled, and repurified as previously described .Thank you for submitting your article "A bacterial riboswitch class for the thiamin precursor HMP-PP employs a terminator-embedded aptamer" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:thiS motif is a functional riboswitch that responds to an important precursor of thiamine pyrophosphate, the active form of vitamin B1. The size of this riboswitch is remarkably small, and yet not only can this riboswitch recognizes the ligand very specifically, it can also function in conjunction with another riboswitch to precisely balance the expression of relevant genes. Undoubtedly, this work will attract attention of many researchers in different fields and will force researchers to re-think composition and role of one of the most widely-spread molecular elements – transcription terminator. The results will be of great interest to broad readership of eLife. Collectively, the reviewers have a few major points for clarification.The manuscript by Atilho et al. presents a new twist in the riboswitch field: an unexpected finding of a ligand-sensing domain embedded in the expression platform, a transcriptional terminator. It begs the question: how many of these mini-systems are hidden out there? The authors did excellent work proving that Essential revisions:thiS-thiM regulated bacterial physiology would certainly further improve the impact of the work.). Figure 1B shows several genes associated with this motif and it would be helpful to identify and discuss these genes. Is each of these genes preceded by its own copy of the thiS motif? Are these genes controlled by a single thiS motif and not by a tandem this-thiM motif? The third paragraph of the subsection “The unusual architecture and genetic distribution of the thiS riboswitch candidate”, says that 30% of the known representatives reside near TPP riboswitches. Is there a genetic bias for the tandem riboswitch arrangement? Why the consensus motif  is presented differently in the terminator and aptamer configurations? In the aptamer configuration, the loop in P1 is shown as variable while no extra nts are indicated for this region in the terminator configuration. The variable loop size contradicts the statement: "This structure exhibits all the features characteristic of bacterial intrinsic terminator stems, including an uninterrupted and strong base-paired stem". Actually the entire 5' end is different in the two configurations. Answers to these questions will help to convince this reviewer that the thiS motif is indeed a HMP-PP binder and will also help microbiologists to better understanding regulatory networks associated with vitamin production in bacteria.1) There is a lack of sufficient information on the distribution and genetic arrangements of 2) The novel computational pipeline the authors describe is from unpublished work. Though not the subject of this manuscript, it is hard to see how this new approach differs from approaches used by this lab in the past. What makes this new approach more sensitive to rare riboswitch elements? Does this sensitivity arise from looking at smaller groups of bacteria (5 species) instead of across numerous species? The novelty of the approach is touted throughout, but not well explained. 1) There is a lack of sufficient information on the distribution and genetic arrangements of thiS riboswitches. The authors cited an unpublished paper by Stav et al., 2019 but did not provide this manuscript and did not reveal details relevant to the current work. It is unclear when the Stav et al. work will be published and it is recommended to give a better summary of the findings and perhaps a supplementary figure with genetic arrangements, which would nicely complement a supplementary figure with riboswitch sequences.What are "uncommon" genes that reside downstream of thiS motif? Are all of them mentioned in the third paragraph of the Introduction? Can the assigned specificity of this motif explain regulation of these uncommon genes? If no, could it be possible for the riboswitch to recognize more than one ligand, especially given the fact that HMP-PP binding may be weak? Can this motif be a family of distinct riboswitches? Were there any attempts made to separate the motif on submotifs based on the genetic association? What are transporter genes controlled by the motif? ThiW appears to be an essential thiazole transporter and although the presence of the thiS motif controlling this protein suggests the regulation of coupling of the two TPP moieties, one can imagine direct regulation of a subtype of this motif by precursors of HET-P . Figure 1B shows several genes associated with this motif and it would be helpful to identify and discuss these genes. Is each of these genes preceded by its own copy of the thiS motif? Are these genes controlled by a single thiS motif and not by a tandem this-thiM motif? The third paragraph of the subsection “The unusual architecture and genetic distribution of the thiS riboswitch candidate”, says that 30% of the known representatives reside near TPP riboswitches. Is there a genetic bias for the tandem riboswitch arrangement? Why the consensus motif  is presented differently in the terminator and aptamer configurations? In the aptamer configuration, the loop in P1 is shown as variable while no extra nts are indicated for this region in the terminator configuration. The variable loop size contradicts the statement: "This structure exhibits all the features characteristic of bacterial intrinsic terminator stems, including an uninterrupted and strong base-paired stem". Actually the entire 5' end is different in the two configurations. Answers to these questions will help to convince this reviewer that the thiS motif is indeed a HMP-PP binder and will also help microbiologists to better understanding regulatory networks associated with vitamin production in bacteria.thiS motif that is otherwise typically supplied for novel riboswitches.Point 1A: The reviewers note a lack of bioinformatics information on the thiS riboswitch candidate (and numerous other RNA motifs). Unfortunately, this manuscript had experienced a remarkably long delay in the initial review process (~9 months). On March 7, 2019 we received notice that this original discovery manuscript has been accepted for publication by BMC Microbiology. Regardless, we have also decided to include an updated and detailed bioinformatics dataset as a Supplementary file 1 to the current revised manuscript for eLife. The original two supplementary files have been renamed accordingly. See also our response to Major Point 2 below.As the reviewers correctly speculate, this comprehensive information set was included for publication in an earlier manuscript  reporting the bioinformatics discovery of the thiS motif and to discuss the possibility that the riboswitch aptamer might recognize different ligands to regulate these uncommon genes.Point 1B: The reviewers encourage us to comment on uncommon gene associations for the ykkC orphan riboswitch” RNAs that have adapted to respond to at least five distinct ligands. However, nearly all the genes associated with thiS motif RNAs appear to code for proteins that participate in the production of HET-P or its coupling to HMP-PP. In some instances, associated genes from organisms whose genomes have not been annotated appear as ‘proteins of unknown function’. However, we have examined these genes and nearly all of these can easily be classified as thiS. Therefore, we do not have any indication that thiS motif RNAs have changed their specificity to regulate genes unrelated to thiamin pyrophosphate biosynthesis in general, or HET-P biosynthesis in particular. Also, the sequence alignments are quite uniform (matching the consensus), and so we do not believe that the RNAs have specialized based on any particular gene association. To clarify these consistent characteristics, we have edited the following text to the paragraph noted by the reviewers:This question from the reviewers is an important one, particularly given the recent and striking examples of variant “thiS motif RNAs appear to code for proteins that participate in the production of HET-P or its fusion to 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate (HMP-PP), to ultimately produce the bioactive coenzyme thiamin pyrophosphate (TPP) .”“Most other genes associated with thiS motif association with a putative thiazole transporter.Point 1C: The reviewers ask for specific clarification on the thiS motif RNAs would make most sense if they import molecules related to HET-P production. Specifically, we believe that the thiW gene association is a logical association for an HMP-PP “ON” switch because excess HMP-PP would trigger the import of the thiazole derivative HET, which can be used to biosynthesize more HET-P. In other words, we see no reason to doubt the current annotation of this transporter gene, and thus we have no evidence of a ligand specificity switch based on the current transporter gene annotations associated with thiS motif representatives.We have found that transporter annotations can be uncertain (and are frequently incorrect) and so we must speculate with considerable caution. However, the transporter genes associated with thiS motif RNAs and to describe the arrangements of their associated riboswitches.Point 1D: The reviewers encourage us to discuss each of the genes associated with thiS gene is the gene most proximal to the RNA motif, whereas the other genes appear to be part of an operon regulated by HMP-PP riboswitches. Based on these gene names, and these operon arrangements, we believe the gene products catalyze the reaction steps for HET-P or TPP biosynthesis as depicted in Figure 1B. This list of gene functions has now been included in the new Supplementary file 1 that reports the various thiS representatives and their associated genes. Unfortunately we do not have a basis for discussing these predicted functions beyond that implied by the figure.Generally, the thiS) riboswitch systems.Point 1E: The reviewers ask if there is any special bias in the distribution of tandem TPP and HMP-PP  caused slight differences in the consensus sequence model near the 5’ terminus . To avoid depicting these differences, we have revised Figure 1A so that we depict the two structural states using only the consensus nucleotide sequence model for the aptamer configuration alignment. This updated figure is included in the revised manuscript file.Point 1G: The reviewers state that our claim that the motif has the classic characteristics of an intrinsic terminator stem is incorrect.left side of Figure 1A (terminator configuration) has all the key characteristics expected for a classic intrinsic terminator. Specifically, this part of the figure depicts a long hairpin with strong and continuous base-pairing, followed by a run of U nucleotides. Even as originally drafted, we do not believe that readers will be unduly confused by the right side of Figure 1A (aptamer configuration), as we carefully label these two structural states. Thus, given the clarity of the text and the figure annotations, we feel that the original text and the revised graphics should serve the readers well, and so no additional edits beyond those noted in Point 1F have been made.We disagree. As we indicate in the original manuscript, the structural model as drawn on the 2) The novel computational pipeline the authors describe is from unpublished work. Though not the subject of this manuscript, it is hard to see how this new approach differs from approaches used by this lab in the past. What makes this new approach more sensitive to rare riboswitch elements? Does this sensitivity arise from looking at smaller groups of bacteria (5 species) instead of across numerous species? The novelty of the approach is touted throughout, but not well explained.thiS RNAs compared to our previously used methods. Specifically, how is the current pipeline able to uncover rare riboswitch candidates?The reviewers ask us to clarify the differences between the current discovery pipeline used to identify These questions on the bioinformatics pipeline are fully addressed in the preceding manuscript  that is mentioned in the response to Major Point 1A above. Briefly, our approach is a substantially updated version of a search strategy we first published in 2009. Specifically, this approach enriches for stretches of bacterial genomes that are likely to carry novel noncoding RNA motifs like riboswitches by collecting long, GC-rich intergenic regions (IGRs). This massively reduces the number of false positive candidates, and allows us to focus on motifs that are more likely to have the biochemical functions we seek. Although the delay in the initial review of the Stav et al. manuscript has been unusually long, as noted in the response to Point 1A, we have recently received word that our revised manuscript has been accepted for publication.


In eukaryotes, three RNA polymerases  are responsible for the transcription of distinct subsets of genes. Gene-external type 3 Pol III promoters use defined transcription start and termination sites, and they are, therefore, widely used for small RNA expression, including short hairpin RNAs in RNAi applications and guide RNAs in CRISPR-Cas systems. We report that all three commonly used human Pol III promoters  mediate luciferase reporter gene expression, which indicates Pol II activity, but to a different extent (H1 ≫ U6 > 7SK). We demonstrate that these promoters can recruit Pol II for transcribing extended messenger transcripts. Intriguingly, selective inhibition of Pol II stimulates the Pol III activity and vice versa, suggesting that two polymerase complexes compete for promoter usage. Pol II initiates transcription at the regular Pol III start site on the 7SK and U6 promoters, but Pol II transcription on the most active H1 promoter starts 8 nt upstream of the Pol III start site. This study provides functional evidence for the close relationship of Pol II and Pol III transcription. These mechanistic insights are important for optimal use of Pol III promoters, and they offer additional flexibility for biotechnology applications of these genetic elements. Transcription in eukaryotes is conducted by three RNA polymerase (Pol) enzymes: Pol I, II, and III. These polymerases contain respectively 14, 12, and 17 subunits and share a 10-subunit catalytic core,Type 3 promoters are unique among Pol III elements in that they solely utilize upstream regulatory elements, which resemble the architecture of canonical mRNA-type Pol II promoters.8cis-acting elements that are similar to elements of Pol II promoters, including the proximal and distal sequence elements (PSE and DSE) of snRNA-type Pol II promoters and the TATA box of mRNA-type Pol II promoters.7G) cap at the 5′ end and a 3′ polyadenylated tail. It thus is doubtful whether Pol III-expressed transcripts can act as an efficient template for protein synthesis.20Most knowledge of type 3 Pol III promoters is based on comprehensive studies of the U6 promoter that encodes the U6 snRNA.We now report that all three commonly used human type 3 Pol III promoters  can drive Luc translation. By systematic molecular analysis, we demonstrate that these Pol III promoters synthesize extended Luc mRNA transcripts by the recruitment of Pol II. Thus, these Pol III promoters have dual-polymerase activity. These three promoters exhibit similar Pol III activity, but they differ profoundly in Pol II strength (H1 ≫ U6 > 7SK). We demonstrate that the Pol III activity can be boosted by selective inhibition of the Pol II activity and vice versa, indicating Pol II-III competition for binding to the same promoter sequences.To directly examine the Pol III activity of these human promoters, we constructed a set of P-N44 vectors in the same plasmid backbone in which the respective promoters transcribe an artificial 44-nt sequence (N44) followed by the efficient Pol III termination signal TTTTTT (T6) . The Luc expression trends were very similar in all cell types C. The 7STo determine if Luc expression is due to Pol III readthrough transcription, we first inspected the Luc coding sequence for the presence of Pol III termination signals. This survey revealed 7 × T4, 1 × T5, and 1 × T6 signals A. We recTo confirm this idea, we inserted an efficient T6 Pol III termination signal upstream of the Luc gene in all four constructs  signal (pA) upstream of the Luc gene  to generate ∼200-nt transcripts. Two major signals were observed: the ∼44-nt Pol III and the ∼200-nt Pol II signals C. The laWe next attempted to apply the P-T6-pA-Luc design to all three Pol III promoters for the simultaneous visualization of Pol II and III transcripts A. The SVWe next tested the Luc mRNA transcription of the P-T6-Luc constructs with or without α-amanitin treatment. As in The TATA box of Pol III promoters acts as a major determinant of Pol III specificity.Finally, we set out to determine where Pol II transcription initiates on these Pol III promoters. We used the 5′-rapid amplification of cDNA ends (RACE) method to map the 5′ end of the Luc mRNA, which reflects the transcriptional start site in the promoter DNA. An equal amount of total cellular RNA from P-Luc-transfected HEK293T cells was used as a template. To specifically amplify Pol II transcripts, 5′ cap-removing reagent (Cap-Clip) and the Oligo(dT) primer were used for cDNA synthesis A. As shoType 3 Pol III promoters have been widely used for synthesis of small functional RNAs, such as the shRNA in RNAi applications and gRNA in CRISPR-Cas genome-editing platforms. In this study, we report that three commonly used human Pol III promoters  also have Pol II activity, with significant variation in strength (H1 ≫ U6 > 7SK). Pol II activity of the H1 promoter reaches approximately 30% of the Pol III activity, and this Pol II activity surpasses that of the regular early SV40 Pol II promoter. These Pol III promoters can produce extended mRNAs that are translation competent. The selective inhibition of either polymerase (II with α-amanitin or III by TATA-box mutation) stimulates promoter usage by the other polymerase, indicating that they compete for binding to the promoter.Extensive transcription studies of the human Pol II snRNA and Pol III U6 snRNA promoters revealed close similarity in their structure.The tested Pol III promoters show significant variation in Pol II strength (H1 ≫ U6 > 7SK). Pol II on the 7SK and U6 promoters initiates from the regular Pol III +1 site, whereas Pol II on H1 starts 8 nt upstream of the regular Pol III +1 position. These striking differences are likely due to a variation in promoter structure. The 7SK and U6 promoters have a very similar architecture with the DSE/PSE motifs at a distance of ∼150 bp,There is accumulating evidence for an important regulatory role of chromatin structure on Pol III promoter activity.The dual-polymerase activity of these type 3 Pol III promoters has important implications for small RNA expression. A few potential adverse effects of unwanted Pol II transcripts can be predicted. For therapeutic H1-shRNA vectors, the extended Pol II transcripts will have the perfect complementary target sequence and may be attacked by the Pol III-generated shRNA, and this may comprise the therapeutic silencing efficiency. The strong Pol II activity of H1 promoter may also interfere with the activity of downstream Pol II transcription units.The dual-polymerase activity of H1 and U6 promoters may also be exploited for specific purposes. First, one could try to identify promoter mutants that have selectively lost the Pol II activity, thus constituting a pure Pol III promoter. Second, these promoters can be used as pure and strong Pol II promoter for protein synthesis by deleting Pol III-specific elements. As a proof of principle, we achieved this goal by mutation of the TATA box that kills Pol III transcription and, consequently, boosts Pol II-mediated Luc expression. Third, we envisage that the dual II-III activity could be used for the simultaneous expression of a small RNA and a protein. For example, the popular CRISPR-Cas9 genome-editing system requires a small gRNA plus the Cas9 protein, and both components could in theory be expressed from a single Pol III promoter. This would minimize the size and the complexity of transgene cassettes and benefit the construction of viral vectors with limited packaging capacity, such as adeno-associated virus and lentiviral vectors.The vectors psiRNA-h7SK hygro G1 (InvivoGen), pSilencer 2.0-U6 (Ambion), and pSUPER (Oligoengine) were used as sources of human Pol III promoter sequences. The U6 variant used was U6-1, which is one of the strongest U6 promoters.2. C33A is a human cervical cancer cell line and HCT116 is a human colon cancer cell line. Cells were trypsinized and seeded 1 day prior to transfection. At 1 day prior to transfection, 3 × 105 and 1.5 × 106 cells were seeded per well in 12-well plates and 25 cm2 flasks, respectively.HEK293T cells, C33A cells, and HCT116 cells were grown as monolayer in DMEM (Life Technologies) supplemented with 10% fetal calf serum (FCS), minimal essential medium nonessential amino acids, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C and 5% CO6 PBMCs were transfected with 30 μg Luc constructs using the P3 Primary Cell 4D-Nucleofector X Kit (Lonza) with the EO-115 program. Luc expression was measured 24 hr post-transfection.At 3 days before transfection, PBMCs were thawed and cultured in the presence of phytohemagglutinin . Culture medium was supplemented with interleukin-2 (IL-2) to 1 μg/mL 1 day prior to transfection. 2 × 10Equimolar amounts  of P-Luc constructs and 2 ng Renilla luciferase plasmid (pRL) were co-transfected into HEK293T cells in 12-well plates using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. At 36 hr post-transfection, luciferase activity was measured with the dual-luciferase reporter assay system , according to the manufacturer’s protocol. The ratio of Firefly to Renilla was calculated to represent the relative Luc activity. Three independent transfections were performed. The luciferase data were corrected for between-session variation as described previously.In the initial test, different concentrations  of α-amanitin (Sigma) were realized at different time points after transfection. 2 μg/mL α-amanitin was used in the dual-luciferase reporter and RNA extraction assays.32P]-labeled decade RNA marker (Life Technologies) was used for size estimation. To check for equal sample loading, the gel was stained in 2 μg/mL 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 (LNA) oligonucleotides (Pol47: 5′-ATTACTACTGCCCCTTCAC-3′) were 5′ end labeled with the kinaseMax kit (Ambion) in the presence of 1 μL γ[32P]-ATP . Sephadex G-25 spin columns (Amersham Biosciences) were used to remove the unincorporated nucleotides. The membrane was incubated overnight with labeled LNA oligonucleotides in 10 mL ULTRAhyb hybridization buffer at 42°C. The membrane was washed with low  and high (0.1 × SSC and 0.1% SDS) stringency buffers. The signals were captured by Typhoon FLA 9500  and quantitated using ImageJ software.Northern blot for small RNAs detection was performed as previously described.For detection of extended RNA transcripts, agarose-formaldehyde gel electrophoresis and northern blotting were performed. 15 μg total cellular RNA was mixed with denaturing loading dye and heated for 10 min at 70°C, and then it was subjected to electrophoresis in 1% denaturing formaldehyde gels in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer at 120 V for 4 hr. The RiboRuler High Range RNA Ladder (Thermo Fisher Scientific) was run alongside for size estimation. The RNA was transferred onto a positively charged nylon membrane using 20 × SSC (3.0 M NaCl and 0.3 M Na-citrate [pH 7.0]) overnight by capillary force. The RNA was cross-linked to the membrane by a UV crosslinker. The membrane was incubated for 1 hr in ULTRAhyb hybridization buffer at 55°C. The probe, consisting of the NcoI- and XbaI-digested luciferase fragment of pGL3-control, was denatured for 10 min at 96°C and labeled with [α-32P] dATP by using the DecaLabel DNA Labeling Kit (Thermo Fisher Scientific). The probe was purified on a Sephadex G-25 spin column and added to the prehybridized membrane and hybridized overnight at 55°C. The membrane was washed three times for 15 min at room temperature in low-stringency buffer and at 50°C in high-stringency buffer. Signals were obtained and analyzed as described above.260/A280 ratio ≥ 1.8) and concentration were measured using NanoDrop 2000. DNA-free RNA was obtained by RURBO DNase (Ambion) treatment to remove residual genomic DNA, according to the manufacturer’s instructions. Briefly, 5 μg total cellular RNA was resolved in 50 μL TURBO DNase Buffer and incubated with 1 μL TURBO DNase (2 U) at 37°C for 30 min. RNA was extracted with phenol/chloroform and dissolved in 9 μL nuclease-free water. The DNA-free RNA sample was reverse transcribed in a 20-μL reaction by the Oligo(dT) primer using the First-Strand cDNA Synthesis Kit (ThermoScript RT-PCR system). The qPCR reactions were prepared with SensiFAST SYBR No-ROX Kit (Bioline), according to the manufacturer’s instructions. Reactions were run on the LightCycler 480 (Roche) according to the amplification protocol: 5 min 95°C followed by 35 cycles of 10 s 95°C, 30 s 60°C, and 30 s 72°C. Primers for Luc and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were designed to amplify a fragment of about 150 bp in length and the primer sequences were as follows: Luc: 5′-GAGGCGAACTGTGTGTGAGA-3′, 5′-GTGTTCGTCTTCGTCCCAGT-3′; and GAPDH: 5′-ACAGTCAGCCGCATCTTCTT-3′, 5′-GAGTCAACGGATTTGGTCGT-3′. The Luc primers were designed to amplify a Luc fragment between positions 1,159 and 1,293. The raw data were exported and analyzed using LinRegPCR program, and the relative expression of Luc RNA was normalized to GAPDH.Total cellular RNA was harvested 36 hr post-transfection using the mirVana miRNA isolation kit (Ambion). RNA purity  as described above. The DNA-free RNA was treated by Cap-Clip acid pyrophosphatase (CellScripts) to remove the 5′ cap from the mRNA molecules. Phenol-chloroform precipitation was used to remove the Cap-Clip enzyme. The resulting RNA was ligated to a 5′ RNA-adaptor (CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA). The Oligo(dT) primer was used for reverse transcription using the First-Strand cDNA Synthesis Kit (ThermoScript RT-PCR system). PCR amplification was carried out using a 5′ adaptor forward primer (5′-AGGACACTGACATGGACTGAA-3′) and a gene-specific reverse primer (5′-CGGACATTTCGAAGTACTCA-3′). PCR products of the expected size were gel extracted and cloned into the pCR2.1 TOPO vector by TA cloning (Invitrogen). Colony PCR was performed with T7 and M13R primers. The amplified inserts were subjected to sequencing using the BigDye Terminator v1.1 Cycle Sequencing Kit (ABI). The Pol II transcription start site was determined by aligning the sequencing reads with the sequence in the corresponding P-Luc constructs.Z.G., E.H.C., and B.B. designed the experiments. Z.G. and B.B. drafted the manuscript. Z.G. conducted the experiments, and all authors analyzed the data.The authors declare no conflicts of interest.