Hirschsprung disease  is due to a failure of enteric precursor cells derived from neural crest (EPCs) to proliferate, migrate, survive or differentiate during Enteric Nervous System (ENS) formation. This is a complex process which requires a strict regulation that results in an ENS specific gene expression pattern. Alterations at this level lead to the onset of neurocristopathies such as HSCR. Gene expression is regulated by different mechanisms, such as DNA modifications (at the epigenetic level), transcriptional mechanisms , postranscriptional mechanisms (3′UTR and ncRNA) and regulation of translation. All these mechanisms are finally implicated in cell signaling to determine the migration, proliferation, differentiation and survival processes for correct ENS development. In this review, we have performed an overview on the role of epigenetic mechanisms at transcriptional and posttranscriptional levels on these cellular events in neural crest cells (NCCs), ENS development, as well as in HSCR. Hirschsprung disease  is a rare congenital disorder that occurs in approximately one per 5000 live births. It is characterized by the absence of enteric ganglia in the rectum and a variable continuous segment of the proximal intestine resulting in intestinal dysfunction. Based on the length of the aganglionic segment, the pathology is classified as short segment HSCR, long segment HSCR, and total colonic aganglionosis or total intestinal aganglionosis. Most often the disease appears as sporadic HSCR, although familial cases have also been reported. Although 70% of HSCR cases appear without any additional clinical manifestations (isolated HSCR), the remaining 30% of cases manifest with other disorders or congenital malformations (syndromic HSCR) [HSCR results from a failure to fully colonize the gut by enteric precursor cells (EPCs) derived from neural crest cells (NCCs). Such incomplete gut colonization is due to alterations in EPCs proliferation, survival, migration and differentiation during enteric nervous system (ENS) development . VariousEpigenetic events are defined as “the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity state” . Mostly,In addition, in this review, we also highlight the above-mentioned regulatory mechanisms that may finally result in the onset of HSCR.de novo methyltransferases [DNA methylation involves the insertion of a CH3 group at the C-5 position of the cytosine ring of DNA. Susceptibility regions of methylation (CpG islands) represent around 40% of all mammalian promoters. These regions are usually unmethylated when gene expression occurs . DNA metsferases ,14.DNMT3A and its paralog DNMT3B, some evidence has shown their crucial role for normal mammal development, as well as their involvement in diseases [Dnmt3A homozygous knockout mice die some weeks after birth, and rostral neural tube defects and growth impairment have been observed for Dntm3B homozygous knockout embryos that finally leads to death, suggesting that both enzymes are essential during embryonic development [Dnmt3A downregulation leads to a reduced expression of genes which is directly implicated in neural crest specification  [Dnmt3B is upregulated during chicken embryo neural crest formation [DNMT3B mutations have been found in the immunodeficiency-centromeric instability-facial anomalies syndrome (OMIM#242860) [DNMT3B knockdown leads to early neural crest differentiation as well as the upregulation of neural crest specifier genes [versus controls correlated with a decrease of global DNA methylation levels. In addition, the synergistic effect of mutations in both DNMT3B and other HSCR–related genes on the severity of the phenotype in HSCR patients has been reported [DNMT3B as a susceptibility gene for HSCR and demonstrates the crucial role of DNA methylation in ENS development and in the onset of HSCR.With respect to diseases ,16,17,18elopment . Variousnd Pax3) . On the ormation . Regardi#242860) ,22. In eer genes . The conreported  and an areported . TherefoRET proto-oncogene encodes a receptor tyrosine kinase that plays crucial roles in ENS development. It is the main gene associated with HSCR, contains a 5′-CG-3′ rich region within its promoter, and the methylation levels of this region have been demonstrated to be related to its expression level in peripheral white blood cells from HSCR patients [GFRA4 has been widely proposed as a susceptibility gene in the pathogenesis of HSCR [. It encodes for a RET co-receptor inducing neuronal survival and differentiation [GFRA4 in HSCR can be partly due to hypermethylation at its promoter region. Therefore, it has been proposed that DNA methylation contributes to the regulation of the neuroprotective role of GFRA4 on NCCs [EDNRB (endothelin receptor type B) is another susceptibility gene for HSCR because the Endothelin 3-Endothelin Receptor B Signalling Pathway is crucial for the correct formation of enteric ganglia [EDNRB might play a role in ENS development and in the onset of HSCR. Specifically, the upregulated expression level of EDNRB in HSCR tissue compared with controls correlated with a significantly lower ratio of its methylation level in these patients. [SHH) promoter are significantly increased in patients with congenital anorectal malformations (ARM), which is correlated with lower levels of its expression [SHH may be responsible for abnormal ENS development, which is related to the onset of ARM. De Pontual et al. have identified an aberrant CpG dinucleotide methylation within the PHOX2B promoter in neuroblastoma, an embryonic tumor originating from NCCs. This outcome suggests that aberrant methylation patterns within PHOX2B might be also implicated in this pathology [Aberrant DNA methylation patterns affecting genes related to ENS development and HSCR have been described. The patients . GFRA4 h of HSCR . It encontiation  through ntiation  since RE on NCCs . EDNRB ( ganglia . Tang etatients. . Additiopression . This epathology .CD47 and CUL3) in colon tissues from HSCR patients compared with controls. Moreover, such upregulation of CD47 and CUL3 reduced proliferation and migration of 293T  and SH-SY5Y (subline of the neuroblastoma cell line SK-N-SH) cell lines. These results suggest that the methylation status of the promoter of the miR-141 gene might be a key factor in the pathogenesis of HSCR [Furthermore, an important role of the methylation level of genes that encode for microRNA (miRNA) has also been described in HSCR. In this sense, miR-141, which belongs the tomiR-200 family that has been highly associated with different human pathologies , showed  of HSCR .Histones are the main binding proteins associated with chromatin, and their association with the compacted DNA strand results in nucleosomes. Each nucleosome consists of four duplicated units of histones , resulting in a structure formed by the combination of eight histones (nucleosome core) around which DNA rolls up with unstructured tails . There aSpecifically in eukaryotic cells, histone acetylation is established by two different enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs) ,42,43. HVarious histone acetylation and methylation mechanisms have been associated with NCC development, although their implication in HSCR is still unknown. All this evidence suggests a potential role in the onset of this pathology that should be investigated.hdac1 is required for eye development, the central nervous system and NCC populations [hdac1 function during the development of the neural crest in zebrafish and therefore in ENS formation [In most cases, the histone acetylation corresponds to cis-regulatory regions in the neural crest. The HDAC repression complex promotes trunk crest cell specification  and reguulations ,65,66,67ormation .JmjD2A knockdown resulted in a drastic loss of Snail2, FoxD3 and Sox10 expression. When H3K9m3 modification is present in the promoter regions of Sox10 and Snail2, their interaction with JMJD2A is unraveled by Chromatin immunoprecipitation (ChIP) assays [JmjD2A is necessary to correct neural crest establishment during embryo development. Moreover, PHF8, can demethylate the H4K20me1 and H3K9me1 marks close to the start of the transcription site that turns active. Interestingly, this transcriptional regulator was previously associated with the regulation of neural crest development in diverse vertebrate models [Regarding histone methylation, JMJD2A mediates neural crest gene expression by modulating the epigenetic modification H3K9m3, that finally establishes the neural crest in the embryonic stage. ) assays . Thus, Je models ,70,71,72MECP2 (Methyl-CpG binding Protein 2). Its association with HDACs and histone methyltransferases (HTMs) forms stable repressor complexes for gene expression [MECP2 in HSCR patients and, interestingly, the downregulation of this gene in SH-SY5Y caused a decline in cell proliferation. Nevertheless, in the methylation level of MECP2, there was no difference when analyzing both groups. Moreover, similar outcomes were found in miR-34b, which is a regulator of MECP2 expression. These results suggest that alteration in the expression level of MECP2 may be relevant in the etiology of HSCR through the regulation of histone modifications [The only histone modulator factor that has been related to HSCR thus far is pression . Zhou etications .This complex is formed by a series of proteins that prevent the transcription of their target genes by catalyzing H3K27me3 epigenetic complex ,76. TherAebp2 gene, which encodes for a component of PRC2 expressed in NCCs [Sox10. This result is similar to the reduced SOX10 dosage frequently observed in Waardenburg syndrome type 4 [Aebp2 misregulation might be responsible for HSCR and the Waardenburg syndrome due to an aberrant epigenetic regulation of neural crest genes. In the same way, EED (Embryonic Ectoderm Development), one of the two core catalytic subunits of PRC2 has been described as a regulator of neural crest gene expression during NCC determination and migration [EED in EPCs from HSCR patients with respect to controls [Heterozygous mutant mice, with respect to the  in NCCs , show si in NCCs ,82,83. Iigration . In the controls .This epigenetic mechanism is mediated by protein complexes, such as CHD (chromodomain helicase DNA-binding), ISWI (imitation switch) and SWI/SNF (mating-type switch/sucrose nonfermenting), that change the structure of chromatin by ATP-hydrolysis. They promote regions with a lack of nucleosomes to facilitate transcription factor binding and binding of other regulatory proteins in these regions ,86. SpecSeveral classes of ncRNA have emerged to play key roles in modulating many cellular processes, such as micro RNA (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). In particular, miRNAs are highly conserved RNAs (20–24 nucleotides) that inhibit gene expression by posttranscriptional mechanisms through complementary binding to the 3′-untranslated regions (3′-UTR) of target mRNA ,93. LncRDifferent research studies have related miRNAs, lncRNA and circRNA to HSCR ,120,121.RET variants and HSCR has been widely described. Fitze et al. characterized several RET polymorphisms in a group of HSCR patients and controls and found two variants located at the 3′UTR, c.3187+47T>C (rs2075912) and 3′UTR+124A>G) with a strong association with HSCR [RET haplotype characterized by the presence of an SNP, g.128496T>C (rs3026785) in the 3′UTR of RET [RET 3′UTR in the Chinese population and identified a combination of 7 SNPs that seems to act as protective haplotypes [RET polymorphisms still needs to be further elucidated.Finally, a relationship between 3′UTR ith HSCR . In contR of RET . MoreoveHSCR is a human congenital disorder due to an incorrect process in ENS formation attributed to an aberrant migration, proliferation, differentiation or survival of NCCs. Several epigenetic events have been related to ENS development and HSCR. Nevertheless, their potential role in the context of HSCR is just beginning to be defined. In this review, we have summarized the epigenetic mechanisms at transcriptional and posttranscriptional levels implicated in NCCs, ENS development and HSCR described so far . Nonethe


Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is regulated by many transcriptional factors (TFs) that control the expression of quorum sensing and protein secretion systems. Here, we report a genome-wide, network-based approach to dissect the crosstalk between 20 key virulence-related TFs. Using chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), as well as RNA-seq, we identify 1200 TF-bound genes and 4775 differentially expressed genes. We experimentally validate 347 of these genes as functional target genes, and describe the regulatory relationships of the 20 TFs with their targets in a network that we call ‘Pseudomonas aeruginosa genomic regulatory network’ (PAGnet). Analysis of the network led to the identification of novel functions for two TFs (ExsA and GacA) in quorum sensing and nitrogen metabolism. Furthermore, we present an online platform and R package based on PAGnet to facilitate updating and user-customised analyses.The virulence of  Pseudomonas aeruginosa is regulated by many transcriptional factors (TFs). Here, the authors study the crosstalk between 20 key virulence-related TFs, validate 347 functional target genes, and describe the regulatory relationships of the 20 TFs with their targets in a network that is available as an online platform.The virulence of  P. aeruginosa harbours multiple key determinants of virulence, including quorum sensing (QS), Type III (T3SS) and Type VI secretion systems (T6SS), flagella and type IV pili. Taken together, these determinants comprise a highly complex and well-ordered virulence system8.PP. aeruginosa is a typical Gram-negative bacterium equipped with population-dependent QS systems that synthesise and release small chemical signals to the environment. To date, at least four QS pathways  have been characterised in P. aeruginosa, and these were found to be strongly connected and co-regulated9. The las and rhl systems are based on acyl-homoserine lactone (AHL) signals11, whereas the pqs system is based on 2-alkyl-4-quinolone (AQ) signals12. The regulatory activities of LasR and RhlR are induced by binding to the LasI product N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and the RhlI product N-butanoyl-homoserine lactone (C4-HSL), respectively. The IQS signal molecule is synthesised by the abmBCDE operon13. The DNA binding affinity of IQS is induced through interactions with IqsR. Subsequently, IQS regulates the production of PQS, C4-HSL, rhamnolipids, elastase and pyocyanin13. These accumulated signals finely tune the transcription of hundreds of target genes, including those encoding virulence factors14. The QS system in P. aeruginosa controls a large group of genes involved in biofilm formation, motility, cytotoxicity and antibiotic resistance15. Several key regulators  were found to strictly regulate QS and responses to environmental changes. Our recent studies elucidated the regulatory mechanisms of five regulators associated with QS, including VqsR16, VqsM17, AlgR18, CdpR19 and RsaL20. The findings suggest that these regulators form a complex regulatory network.P. aeruginosa can also be controlled by the T3SS and T6SS. In this bacterium, the T3SS is self-equipped with a needle-shaped supramolecular apparatus used to transport T3SS effectors to host cells21. ExsA, an AraC family regulator of transcription22, controls the expression of most T3SS genes. The expression of ExsA is self-regulated via binding to an adenine-rich region upstream of the putative -35 RNA polymerase binding site23. The T3SS is positively regulated by PsrA24, HigB25, Vfr26 and DeaD27, but negatively regulated by MexT28, AlgZR, GacAS/LadS/RetS33 and MgtE34. The P. aeruginosa T6SS uses a bacteriophage-like structure with a sharp spike and an outer contractile sheath to translocate proteins into neighbouring prokaryotic or eukaryotic cells35. P. aeruginosa harbours 3 different T6SS loci: H1-T6SS, H2-T6SS and H3-T6SS38. Similar to the T3SS, the T6SS is tightly regulated by the GacAS/Rsm pathway. Once activated by GacAS, RsmY/Z positively regulates both H1-T6SS and H3-T6SS by inhibiting the binding activity of RsmA or RsmN to fha1 and tssA139. TseF, which is secreted by H3-T6SS, is incorporated into outer membrane vesicles (OMVs) with assistance from PQS. Subsequently, TseF facilitates the import of the PQS–Fe3+ complex into cells40.The virulence of 41. These TFs bind to gene promoters and closely coordinate the initiation of transcription in response to environmental conditions41. A recent study of 10 sigma factors  in P. aeruginosa revealed that these factors formed a network with an exquisite modular architecture42. Another study summarised the known targets of regulators related to P. aeruginosa virulence3. To date, several virulence-related TFs of P. aeruginosa, including VqsM17, AlgR18, AmrZ43, CdpR19, RsaL20, BfmR44, VqsR16, MvfR45 and LasR46, have been studied individually. However, little is known or verified about the crosstalk between strictly virulence-related TFs in this bacterial species.Transcription factors (TFs) are DNA-binding proteins that control downstream gene expression by promoting or blocking the recruitment of RNA polymerase to specific genesPseudomonas aeruginosa Genomic regulatory network, PAGnet, which encodes the regulatory relationships of these 20 TFs with their functional targets. Subsequently, crosstalk between the TFs in the PAGnet, defined as the co-regulation of genes by at least 2 TFs, was experimentally verified using electrophoretic mobility-shift assay (EMSA) and real-time quantitative PCR (RT-qPCR) analyses. This network not only revealed the master regulators of individual pathways, but also genes that participate in crosstalk and are thus involved in multiple virulence and metabolic pathways. Finally, we developed an online platform and a downloadable R package to enable network visualisation and analysis. We expect that these tools will significantly facilitate future studies of the global regulation of virulence in P. aeruginosa.In this study, therefore, we performed a genome-wide characterisation of the regulons of 20 virulence-related TFs involved in virulence regulatory processes such as QS, T3SS and T6SS, based on an integrative analysis of transcriptome profiling (RNA-Seq) and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) profiles. Using an integrative network approach, we mapped a P. aeruginosa, including AlgR, AmrZ, BfmR, CdpR, GacA, LasR, MexT, MvfR, QscR, RhlR, RsaL, SoxR, VqsM, VqsR, PhoB, GbdR, PchR, SphR and FleQ 50. Subsequently, the enriched loci for each TF were annotated using the R package ChIPpeakAnno51, and these data were used to globally characterise TF-binding peaks on the P. aeruginosa genome . Five TFs co-bound to the promoter of a group of genes that included algD , fimV (encoding the motility protein FimV), phdA, rhlR, rocA2 (involved in antibiotic resistance), acpP (encoding an acyl-carrier protein) and 6 hypothetical protein encoding genes . An additional 20 promoters were co-bound by 4 TFs. In other words, these TFs appear to be linked intricately. All genes bound by multiple TFs are listed in Supplementary Data Here, we mainly focused on 1200 annotated genes that contained binding peaks in their promoter regions  and were directly regulated by 1 or more TFs , a 16-bp PhoB-binding motif (ATGACNNNTNNATGAC) and a 12-bp PchR-binding motif (C/TGGC/TGCTG/TGCGG)  Fig. .Fig. 2Ge58, AmrZ59, BfmR44, VqsR60, ExsA61, GacA62, MexT57, RsaL63, SoxR56 and VqsM64) under different conditions and growth phases were previously characterised through RNA-seq or microarray analyses. In this study, we first standardised the experimental conditions for all transcriptomes as an optical density at 600 nm (OD600) of 0.6 and culture in Luria-Bertani (LB) medium. We repeated the RNA-seq experiment with 16 PAO1 deletion strains  under the same conditions . Detailed information about RNA-seq experiment is provided in the Methods section and Supplementary Table rhlQS in M8-glutamate minimal medium but not in LB medium65, we did not obtain additional RNA-seq data under our set conditions . Similarly, we did not subject SoxR to a repeat RNA-seq analysis because this TF mainly functions under oxidative stress56.As we observed profound crosstalk among the tested TFs, we next performed an integrated characterisation of their regulons. The transcriptomes of 10 of the 20 TFs  was co-regulated simultaneously by 16 regulators . The expression of lasB (encoding elastase) was co-regulated by 14 TFs . In addition, more than 10 operons  were jointly regulated by multiple TFs  in the .7% in thlasI, lasB, hcnA-C, cdpR, pqsA-E, rhlR, pslA and amrZ) together with 11 other TFs. Thirteen genes were co-regulated by AmrZ or PhoB with other TFs. Particularly, phzA1-G1 was directly co-regulated by 7 TFs, including CdpR, GbdR, PchR, PhoB, QscR, RhlR and RsaL. Three QS genes  were also co-regulated by 4 TFs  its upstream region was bound by TFs and (2) its expression level was significantly affected by the deletion of TFs. The virulence-related TF regulome revealed 347 functional target genes, including 42 genes as functional targets controlled by multiple TFs , pyocyanine biosynthesis (phzA1-G1 and phzA2-G2) T6SS (amrZ and hsiA2), metabolism , signal transduction (phnC), biofilm formation (pslA) and unknown functions   based on PseudoCAP66 and gene sets from Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases   xcpR , nosR (encoding nitrous-oxide reductase expression regulator), PA0123, PA4108, phoB (positive control), PA1736, narK1 and rhlR   and mucR (positive control) , mexG, PA3718, mqoB, oruR, PA4330 and mexS , flp, flgG, dctP and fleQ  Fig. . AlgR alol) Fig.  and S7f.ol) Fig. . LasR boP. aeruginosa.RT-qPCR for validation revealed that the PAGnet yielded a good performance     Fig.  and S8a.ol) Fig. .fabG (encoding 3-oxoacyl-[acyl-carrier-protein] reductase), which is involved in lipid synthesis , which plays a role bacterial colonisation  , dbpA (encoding RNA helicase DbpA), rho (encoding Rho-dependent termination protein) and wbpH (encoding glycosyltransferase) and wbpA (encoding UDP-N-acetyl-d-glucosamine 6-dehydrogenase). Taken together, these results strongly suggest that ExsA plays important regulatory roles in pathways other than T3SS.In addition, ExsA bound 4 promoters of non-T3SS genes. Specifically, it negatively regulated ion Fig. , and theion Fig. , which ipqs-QS system by binding directly to the pqsH-cdpR intergenic region and the pelB promoter to control biofilm formation  and nasA (encoding nitrate transporter) promoters to regulate adaptation to nitrogen stress , magD (encoding endopeptidase inhibitor), mifS (involved in α-Keto-glutamate utilisation) and napF (encoding ferredoxin protein)   Fig. . GacA atPA3637 and PA5531 were co-regulated by GacA and AlgR. PA4221 was co-regulated by GacA and PchR. In summary, GacA regulated multiple pathways and interacted with other TFs in the PAGnet.The PAGnet also revealed 6 genes that were directly co-regulated by GacA and other TFs  to obtain a subnetwork of interest. A brief summary of the subnetwork, with information about the TFs and their target genes, is also provided.A web application implementing PAGnet , observed hits (signature genes in the regulon of the TF) and a p-value calculated based on a hypergeometric test for each TF. This table will be sorted according to the statistical significance indicated by the p-values, allowing the prioritisation of the top significant TFs as master regulators.In addition to these basic functions, the PAGnet online platform also enables analysis of master regulators for the identification of key TFs that mediate a biological process or pathway . This package can be freely downloaded, locally installed and run by the user on his/her personal computer. The R package includes all functionalities available on the ‘PAGnet’ website and is powered by a local Shiny-based graphical user interface (GUI) within R. Moreover, the master regulator analysis can be performed using the function ‘pagnet.mra’ in the R console without running the GUI. More detailed instructions about installation and a step-by-step guide for use are provided in the GitHub-hosted vignette.To provide more flexibility, we also made the PAGnet available as an R package, ‘PAGnet’, on GitHub  Although the study by Schulz et al.42 focused on sigma factors in PA14, the present study focused strictly on TFs in PAO1. (2) Most importantly, we reported a large set of new data which was not included in the previous 2 studies. Specifically, we reported 16 RNA-seq datasets for AlgR, CdpR, ExsA, GacA, LasR, MvfR, QscR, RhlR, RsaL, VqsM, VqsR, PhoB, GbdR, PchR, SphR and FleQ, as well as 12 ChIP-seq datasets for ExsA, GacA, MexT, MvfR, QscR, RhlR, SoxR, PhoB, GbdR, PchR, SphR and FleQ. These data are expected to provide valuable resources for this field of research. (3) We verified our RNA-seq and ChIP-seq results through EMSA, qRT-PCR and statistical evaluations. (4) Finally, we also developed a freely accessible website (http://pagnetwork.org/) and R package based on PAGnet. We hope that these tools will serve as a useful database and platform for the community.Previously, a fine regulatory network of 10 sigma factors  revealed the crosstalk that orchestrates complex cellular processes in 600 = 0.6 in LB) for all mutants, we performed 10 RNA-seq analyses  for which the regulons had been published previously in other strains and under other conditions. Compared with the previous results, the present study significantly extended the regulons of AlgR, ExsA, GacA, RsaL, PhoB, GbdR and SphR , the nirN-S operon (involved in haeme biosynthesis), nosR-L operon (involved in denitrification pathway), narL and narK1-I (nitrogen metabolism). In the present study, we found that ExsA regulated 1,249 DEGs, a significantly greater number than the previous regulon61. Notably, ExsA regulated important non-T3SS pathways, including Pqs-QS (pqsA-E/phnAB), pprB (regulates biofilm formation and cell adhesion), pchA-G/fptA (pyochelin/siderophore pathway), the spuA-H operon (spermidine catabolic process), aotJ-argR (arginine biosynthetic and catabolic process) and siaD/ptrR/N (cyclic-di-GMP and biofilm). Strikingly, under our current growth conditions, PhoB regulated 1828 DEGs, including multiple virulence regulons such as PQS-QS (mvfR/pqsA-phnAB), Rhl-QS (rhlAB and rhlRI), T6SS  and glycine betaine catabolism (gbdR). A previous study elucidated the crosstalk between PhoB and TctD under phosphate-limited conditions73.To normalise the growth conditions . We believe that our study has provided a valuable online platform that will allow the efficient integration of new data and facilitate future studies of the complex regulatory mechanisms associated with P. aeruginosa virulence and metabolism.The present study, which focused on virulence-related TFs, defined 347 direct target genes in a regulome of 20 TFs. We quantified the relative contributions of these TFs to the overall transcriptome plasticity of 62, we newly discovered that GacA regulated multiple pathways by directly binding to the promoters of cdpR, pelB, glnD, nasA, mifS, napF, cbpD and magD. PAGnet also revealed that 5 genes were directly co-regulated by GacA and other TFs. Moreover, we newly identified a group of genes  that were directly regulated by ExsA. In this crosstalk, the AlgZR two-component AlgZR system also acted as a negative regulator of T3SS gene expression. Altogether, we revealed a complex relationship of interactions between these TFs and between the T3SS, T6SS and other virulence pathways.Although the known functions of these TFs were characterised previously, the present PAGnet identified some new functions. In addition to activating the induction of H1, H2 and H3-T6SSP. aeruginosa pathogenicity. The development of inhibitors against these newly identified master regulators may lead to the discovery of novel drugs targeting P. aeruginosa. The methodology and conclusions of this work may be applicable to other bacterial pathogens in the future.Collectively, this study mapped an integrated regulatory network, PAGnet, comprising the interactive regulons of 20 TFs. This network provided new insights regarding functional crosstalk and revealed new potential functions of several TFs, including PhoB, FleQ, AlgR, ExsA and GacA. Furthermore, the present study yielded a valuable online platform and R package that will enable the efficient integration of new data and user-customised analyses online or offline. In summary, this study revealed a highly modular structure of complex crosstalk among virulence-related TFs and clearly depicted the complicated network regulating P. aeruginosa PAO1 strain and its derivatives were grown at 37 °C in LB (Luria-Bertani) broth with shaking at 220 rpm or on LB agar plates. Antibiotics were used for Escherichia coli at the following concentrations: kanamycin at 50 μg/ml and ampicillin at 100 μg/ml.The bacterial strains, plasmids and primers are used in this study are listed in Supplementary Data 76. The pEX18AP plasmids was digested by using EcoRI and HindIII. The upstream(~1500-bp) and downstream (~1000-bp) of TF opening reading frame were amplified from PAO1 genome and digested with XbaI respectively (All primers are listed in Supplementary Data XbaI digested upstream(~1500-bp) and downstream fragments were ligated with T4 DNA ligase, the ligated DNA products were inserted into the EcoRI and HindIII digested pEX18AP plasmids using ClonExpress MultiS One Step Cloning Kit  generating the pEX18AP-TF plasmid. pEX18AP-TF was digested by XbaI and a 0.9 kb gentamicin resistance cassette cut from pPS858 with XbaI was then cloned into pEX18AP-TF, yielding pEX18AP-TF-Gm. The resultant plasmids were electroporated into PAO1 with selection for gentamicin resistance. Colonies were selected for gentamicin resistance and loss of sucrose (5 %) susceptibility on LB agar plates containing 50 μg/ml gentamicin and 5 % sucrose, which typically indicates a double-cross-over event and thus gene replacement. The ΔrsaL mutant was further confirmed by PCR.A SacB-based strategy was employed for the construction of gene knockout mutantspstream~100-bp andHindIII/BamHI for the overexpressed TFs through HindIII site by using ClonExpress MultiS One Step Cloning Kit . Wild-type P. aeruginosa containing empty pAK1900 or pAK1900-TF-VSV-G/FLAG was cultured in LB medium supplemented with ampicillin until mid-log phase (OD600 = 0.6), then treated with 1 % formaldehyde for 10 min at 37 °C. Cross-linking was stopped by the addition of 125 mM glycine. Bacterial pellets were washed twice with a Tris buffer (20 mM Tris-HCl [pH 7.5] and 150 mM NaCl), re-suspended in 500 μl IP buffer , and then subjected to sonication to produce 100-300 bp DNA fragments. Insoluble cellular debris was removed by centrifugation at 4 °C and the supernatant was used as the input sample in IP experiments. Both control and IP samples were washed with protein A beads  and then incubated with 50 μl agarose- conjugated anti-VSV antibodies (Sigma) in IP buffer. Washing, crosslink reversal, and purification of the ChIP DNA were conducted77. DNA fragments (150-250 bp) were selected for library construction and sequencing libraries were prepared using the NEXTflex™ ChIP-Seq Kit (Bioo Scientific). The libraries were sequenced using the HiSeq 2000 system (Illumina). All the experiment has two repeats. The two repeats were merged together for the following analyses. ChIP-seq reads were mapped to the P. aeruginosa genomes (NC_002516) using Bowtie (Version 1.2.2). Only the uniquely mapped reads were kept for the subsequent analyses. Binding peaks (P < 1 × 10−5) were identified using MACS software (version 2.1.0). Consensus Motifs were identified using MEME with all significant peaks as input. TF target genes were annotated by peaks locating in gene promoters (upstream of gene start site or overlapping with gene start site).For the VSVG-tagged and FLAG-tagged plasmids, the ORF was amplified by PCR from PAO1 genome and cloned into pAK1900 plasmid by 5 times to generate an empirical background distribution of co-occurrence of the TFs on the promoter of interest. (iii) Based on the empirical background distribution, we calculated the number of co-occurrence (or co-binding) events by chance (termed N) on the promoter of interest. iv) P-values were derived from N/105. (v) The raw p-values were finally adjusted for multiple hypothesis testing by the Benjamini–Hochberg procedure.A permutation test was performed to evaluate the statistical significance of the co-occurrence (co-binding) of multiple TFs on the same promoter of a gene using the following steps: (i) For each of the TFs co-binding a particular promoter of interest, we counted the real number of binding sites for the TF on all promoters in the entire genome and randomly distributed the same number of binding sites to all promoters. (ii) Step (i) was iterated 10The ChIP-seq data files have been deposited into Gene Expression Omnibus (GEO) and can be accessed through GEO Series accession number GSE121243 and GSE128430.600 = 0.6) bacterial cultures (PAO1 and TF mutant strains) were collected by centrifugation . A RNeasy mini kit (Qiagen) was used for subsequent RNA purification with DNaseI (NEB) treatment. After removing rRNA by using the MICROBExpress kit (Ambion), mRNA was used to generate the cDNA library according to the NEBNext® UltraTM II RNA Library Prep Kit protocol (Illumina), which was then sequenced using the HiSeq 2000 system (Illumina). Each sample in RNA-seq assay was repeated twice. RNA-seq reads were mapped to the P. aeruginosa genomes (NC_002516) using STAR, only the uniquely mapped reads were kept for the subsequent analyses. Differentially expressed genes were identified using DESeq2 (BH-adjusted P < 0.05 and |log2 Fold Change| > 1)78. The statistical significance of co-occurrence of differential gene expression observed in multiple mutant TFs was evaluated by permutation tests similarly to what was performed in the ChIP-seq data analysis. All the experiment has two repeats. The RNA-seq datasets have been deposited in National Center for Biotechnology Information (NCBI) with an accession number GSE121243 and GSE128430.To examine the effect of TFs (taking an example of RpoN) on the transcriptome, 2 ml of mid-log-phase . RNA concentration was measured by Nanodrop 2000 spectrophotometer (ThermoFisher). The cDNA synthesis was performed by using a FastKing RT Kit (Tiangen Biotech). RT-qPCR was performed by SuperReal Premix Plus (SYBR Green) Kit (Tiangen Biotech) and prepared by following the manufacturer’s instruction. Each reaction was performed in triplicate in 25 μl reaction volumes with 800 ng cDNA and 16S rRNA as an internal control. For each reaction, 200 nM primers (Supplementary Data -(ΔΔCt). All the reactions were conducted with three repeats.For real-time quantitative PCR (RT-qPCR), all strains were cultured at 37 °C, 220 rpm overnight in LB until ODP. aeruginosa genome DNA amplified by polymerase chain reaction (PCR) to obtain the region encoding the protein . The PCR products were inserted into pMCSG19 vector79 and transformed into E. coli BL21 (DE3) strain carrying pRK1037 plasmid80. The pET28a vector was transformed into E. coli BL21 (DE3) strain. Briefly, a single colony on the plate was inoculated into 10 ml sterilized LB broth containing 100 μg/ml ampicillin and 50 μg/ml kanamycin for 12 h. Then, we transferred the culture into 1 L the same medium as above and the cells were grown at 37 °C, 220 rpm to OD600 = 0.6. 0.5 mM IPTG  was added into the culture to induce protein expression at 16 °C for 16 h. The culture was centrifuged at 4 °C, 7000 rpm, for 5 min to harvest the bacteria. The whole processes were performed at 4 °C. The pellet was suspended in 20 ml buffer A ). The cells were lysed with sonication at six seconds interval and centrifuged at 4 °C . The supernatant was filtered with a 0.45 μm filter and the filtrate was added into a Ni-NTA column (Bio-Rad) which had been equilibrated with buffer A before using. After the Ni-NTA column was washed three times with buffer A, the column was eluted with 30 ml gradient of 60-500 mM imidazole prepared in buffer A respectively. Fractions from 300 mM to 500 mM were pooled and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to verify the molecular weight of target protein.Oligonucleotides, restriction enzymes and vectors used for cloning of His tagged proteins analysed in this study are listed in Supplementary Data 2, 10% glycerol). After incubation at room temperature for 20 min, the samples were analysed by 6% polyacrylamide gel electrophoresis (90 V for 60 min for sample separation). The gels were subjected to DNA dye for 5 min and photographed by using a gel imaging system (Bio-Rad). The assay was repeated at least three times with similar results.DNA probes were PCR-amplified using primers listed in Supplementary Data P < 0.05). R package RedeR was used to visualize the network81.Virulence regulatory network was mapped by integrating regulatory relationships experimentally validated in the literature or newly identified in our integrative analysis of ChIP-seq and RNA-seq data. For transcription factors with ChIP-seq and mutant RNA-seq data generated from our lab or known differentially expressed genes from previous studies, the transcriptional targets that are differentially expressed were identified as functional targets (Regulons). Experimentally validated regulons  were integrated to virulence regulatory network directly. The statistical significance of a gene functionally co-regulated by multiple TFs was evaluated by permutation tests similarly to what was performed in the ChIP-seq data analysis. Master regulator analyses for QS, T3SS and T6SS were performed identified using Hypergeometric test (BH-adjusted t-tests were performed using Microsoft Office Excel 2010. *P < 0.05, **P < 0.01 and ***P < 0.001 and results represent means ± SD. All experiments were repeated at least three times.Two-tailed Student’s Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting SummarySource Data


Arabidopsis. Over the past decade, genomic DNA (gDNA) methylation and transcriptome studies have revealed signatures of chromatin regulation during active growth and winter dormancy. To date, only a few chromatin modification genes, as candidate regulators of these developmental stages, have been functionally characterized in trees. In this work, we summarize the major findings of the chromatin-remodeling role during growth-dormancy cycles and we explore the transcriptional profiling of vegetative apical bud and stem tissues during dormancy. Finally, we discuss genetic strategies designed to improve the growth and quality of forest trees.In temperate and boreal regions, perennial trees arrest cell division in their meristematic tissues during winter dormancy until environmental conditions become appropriate for their renewed growth. Release from the dormant state requires exposure to a period of chilling temperatures similar to the vernalization required for flowering in  Populus sp.), photoperiod plays a major role in cell division arrest, and bud formation , suggesting comparable DNA methylation dynamics during vernalization and chilling requirements (Genomic DNA methylation refers to the addition of a methyl group to the carbon atom at the fifth position of a cytosine (5 mC). DNA methylation plays a major role in gene expression, genome protection and stability through transposon silencing, DNA recombination, and other biological processes . Variatiirements . CollectPopulus tremula × alba INRA clone 717 1B4), grown under natural conditions in Pozuelo de Alarcón, Madrid, over the period of January 13th to April 14th 2015 for apical buds, and from November 7th 2014 to April 9th 2015 for stems, coinciding with bud break. Weekly time points were grouped according to the Pearson correlation for samples within the groups and considered as a biological replicate. A detailed list of sample names, dates and groups is shown in the 1, under the expression tab for each gene.To investigate in poplar how the DNA methylation machinery could create this winter hypermethylation-hypomethylation wave in vegetative SAM and stem tissues, we performed a RNA-seq mediated gene expression profiling on weekly collected vegetative apical buds and stems of hybrid poplar , are highly and constantly expressed from autumn to spring including winter dormancy in apical bud and stem tissues (DML10). DML10 showed a steady expression decline during early dormancy followed by a progressive increase in mRNA levels from mid-winter, with maximum expression observed at bud break in apical bud and stem tissues (methyltransferase 1 (MET1) and chromomethylase 3 (CMT3), that operate in CG and CHG contexts, were found induced just before the onset of bud break in apical bud and stem tissues  and SAHH17 (Potri.017G059400). Interestingly, both genes have been genetically associated with vegetative bud phenology during dormancy by DNA methyltransferases use S-adenosyl methionine (SAMe) as a methyl group donor . The end+ . During  of SAHH . In appl of SAHH . Transcr release . We suggdormancy associated MADS-box (DAM) genes, the master regulators of vegetative and reproductive bud dormancy in Rosaceae and other perennial species, resemble chromatin dynamics of known regulators of vernalization, and seed dormancy and germination in Arabidopsis  in a wider region of the gene  gene encodes a putative APETALA2/Ethylene responsive transcription factor that reactivates vegetative meristem growth and bud-break after dormancy release in poplar  was sharply up-regulated under short-photoperiod treatments correlating with growth cessation and dormancy induction in poplar (FIE suppression by RNAi prevented the establishment of dormancy in transgenic hybrid aspen (Populus × spp.), even though growth cessation and bud formation were not affected , containing components conserved in animals and plants . In peacn poplar . Moreoveaffected . In peacaffected , suggestbidopsis .PICKLE (PKL), and putative modifiers involved in histone deacetylation (HDA14 and HDA08), histone lysine methylation (SUVR3), and histone ubiquitination (HUB2) are also up-regulated during the transition to dormancy in Populus (PKL expression restores photoperiod-induced dormancy in abi1-1 hybrid aspen mutants with a defective abscisic acid (ABA) response, suggesting that ABA promotes dormancy by repressing PKL  in hybrid poplar resulted in transgenic lines in which apical bud formation during dormancy establishment was accelerated showing no other visible alterations  and SWINGER (SWN) could provide additional evidences on PCR2 participation in dormancy mechanisms and also serve as candidates for phenological manipulation. However, the putative targets of PCR2 activity may constitute more suitable and specific objectives for biotechnological approaches.The functional role of histone modifications during tree annual growth-dormancy cycles needs further clarification. The PRC2 complex seems to play a key role in dormancy regulation through the H3K27me3 modification on regulatory genes, although only the function of the PRC2 component FIE has been initially explored in poplar . Tree orDAM genes located in tandem causes a non-dormant phenotype in peach, highlighting the functional relevance of these genes in regulating dormancy   . Thus, pAdditional research is needed on the study of detailed characterization of the seasonal growth cycles involving frequently sampled intervals along the growth-dormancy-growth cycle. Detailed, subtle changes in chromatin modifications may be occurring outside the published data sets. In addition, new analytical techniques are emerging that may allow high-throughput characterization of DNA methylation and histone acetylation  should be applied to such periodically sampled tissues.Other genes mentioned in this study involved in DNA methylation, RNA interference, chromatin remodeling, histone modification and transcriptional regulation of meristem growth, and dormancy are interesting candidates for biotechnological applications in tree phenology. The CRISPR/Cas9 system emerges as a promising technique due to its simplicity, design flexibility and high degree of efficiency. However, so far, few studies involving the use of this technique in tree species have been conducted, and no study has examined the impacts of epigenetics mark modifications . Future DC, MP, AL, MB, PG-M, GR, and IA participated in the discussions described here. DC, MP, AS, GT, and IA implicated in the RNA-seq analysis. DC, MP, GT, GR, and IA wrote 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.


Elimination of GBS1 and 4 attenuates transcriptional activation by GLI1. Elimination of GBS1, 2, and 4 attenuates transcriptional activation by GLI2. Eliminating all sites essentially eliminates reporter gene activation. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 can regulate GLI1 expression through protein-protein interactions involving complexes of transcription factors, histone variants, and reader proteins in the regulatory intron of the GLI1 gene. GLI1 acting in trans on the GLI1 intron provides a mechanism for GLI1 positive feedback and auto-regulation. Understanding the combinatorial protein landscape in this locus will be important to interrupting the GLI positive feedback loop and providing new therapeutic approaches to cancers associated with GLI1 overexpression.GLI1 is one of three transcription factors  that mediate the Hedgehog signal transduction pathway and play important roles in normal development. GLI1 and GLI2 form a positive-feedback loop and function as human oncogenes. The mouse and human GLI1 genes have untranslated 5′ exons and large introns 5′ of the translational start. Here we show that Sonic Hedgehog (SHH) stimulates occupancy in the introns by H3K27ac, H3K4me3 and the histone reader protein BRD4. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region. We identified six GLI binding sites (GBS) in the first intron of the human GLI1 gene that are in regions of high sequence conservation among mammals. GLI1 and GLI2 bind all of the GBS  It isA significant disease burden is associated with mis-regulation of this signal transduction pathway . ConstitGLI1 is transcriptionally activated by GLI2, and non-canonical signaling, independent of SHH, PTCH and SMO, has been observed through several pathways  and with transcription factors, including c-MYC and EWSR1-FLI1, that directly activate transcription of GLI1 -18. ImpoThe interplay of chromatin landscape and transcription factors is key to regulation of gene expression. It is well established that epigenetic modification of histones correlates with transcriptional activity. In particular H3K27ac and H3K4me3 are present at sites of active transcription . AlteredGiven the widespread association of GLI1 with many human cancers including glioblastoma, basal cell carcinoma, lung cancer, GI cancers, prostate cancer ,29,30 an2.2.1.The first exon of the human GLI1 gene is non-coding and the ATG translational start is at the 5′ end of the second exon . The fir2.2.In order to interrogate the occupancy of the region by activating epigenetic histone marks and their response to SHH we utilized mouse LIGHT2 (LT2)  cells beLT2  cells deThe highest occupancy by BRD4, H3K27ac and H3K4me3 occur around the TSS and the 5′ end of the mouse first and second intron , 3. In oBecause inhibition of BRD4 binding by I-BET151 in mouse cells reduced GLI1 expression and is a candidate cancer therapy  we wishe2.3.In addition to the public data our sequencing previously identified six highly conserved GLI putative binding sites GBS,  with 8/92.4.We determined that both human GLI1 and GLI2 protein bind all six of the conserved GBS by gel shift analysis . Purifie2.5.We then determined whether the first intron of the human GLI1 locus is competent to activate transcriptional activity through GLI1 or GLI2 binding to the GBS by cloning the full sequence of the intron into luciferase activity reporter vectors  utilizin2.6.A GLI1-GFP construct was produced and activates transcription (data not shown) following transfection into HeLa cells. Proteins were isolated with GFP trap beads and separated on SDS PAGE gels and mass spectrometric analysis performed. In two independent isolations histone variant H2A.Z demonstrated high LFQ (label-free quantification) and high LFQ ratio between the GLI1-GFP and control plasmid pull down . The anaThe interaction between GLI1 and histone H2A.Z was confirmed with proximity ligation assays PLA;  utilizinPublic ChIP seq data showed multiple peaks of H2A.Z reads in the human GLI1 first intron in trophoblast, hES cells, mesenchymal stem cells derived from hES, neural progenitor cells derived from hES cells, HeLa, A549, HepG2 cells and a variety of others . The H2A3.Here we show that the first intron of the human GLI1 gene is a regulatory region. Further, the histones present in the mouse and human GLI1 introns carry the active marks of H3K27ac and H3K4me3. Both GLI2 occupancy of the regulatory region and active epigenetic histone marks are SHH dependent. This SHH effect requires GLI2 and/ or GLI3 as it is absent in GLI2/GLI3 null cells. The human first intron contained six conserved GBS in regions of high sequence conservation that are capable of binding GLI1 and GLI2 in vitro. Deletion analysis demonstrates that binding to some but not all of the GBS significantly affects gene expression and that overall the elimination of all sites greatly reduces gene reporter expression. We show that SHH drives occupancy of GLI2, H3K27ac and H3K4me3 in this region and that the human intron can regulate GLI1 expression. The intron contains elements capable of activation of GLI1 expression explaining in part GLI1 auto-regulation.While the epigenetic landscape is consistent with a nucleosome free region our work and the public data demonstrate the presence of histones within the intron and indeed our results show binding of GLI1 to H2A.Z, a developmentally relevant histone. The region is primed for remodeling and the histone reader protein BRD4 is present. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region with the small inhibitory molecule I-BET151 despite the fact that I-BET151 reduces GLI1 expression in both mouse  and humaConcepts of transcriptional regulation through promoters and enhancers are rapidly evolving and the notion of a highly conserved cluster of GBS within the GLI genes themselves offers a potential mechanism for self-regulation by a positive feedback (feed forward) loop of the principal activating transcription factors in the pathway, GLI1 and GLI2. This region then is an ideal location to further study both function and regulation of GLI1. Interruption of the feedback loop could be exploited in novel cancer therapy. Additionally, the regulatory region has been shown in the public data to bind dozens of other transcription factors. Interestingly GBS6 can be seen to line up in ChIP seq with the largest number of transcription factors . Our datAccumulating evidence suggests that transcriptional regulation occurs in a landscape of protein complexes designed to not only create a platform for transcriptional machinery but to facilitate the formation of elongating RNA. This has led to the concept of transcriptional factories . The rolThe presence of direct GLI1 and histone H2A.Z interaction is surprising. H2A is one of the four core histone proteins and is known to have several important variants. The H2A.Z variant is required for embryonic development and its knock out is embryonic lethal. Incorporation of H2A.Z into nucleosomes weakens the DNA winding and positioning of such nucleosomes around transcriptional start sites affects expression of the associated genes ,44. H2A.In humans, a significant cancer burden is associated with mis-regulation of the Hedgehog/Patched/GLI (HH/PTCH/GLI) pathway ,53. GLI1Transcription factors have proven to be difficult therapeutic targets because of specificity, selectivity and differential sensitivity . OvercomMechanisms of control of GLI1 expression are important because GLI gene transcription is auto-regulated. Mouse GLI3 directly binds to the GLI1 promoter and induces GLI1 transcription in response to SHH. Other ex4.4.1.10 million cells were cross-linked in 1% formaldehyde (Thermo scientific) for 10–15 min in room temperature and then blocked by 0.125 mM glycine (Ameresco) for 5 min in room temperature. Cell pellets were harvested and washed with pre-chilled PBS twice. Then 350 μl of lysis buffer was added to lyse cells. Cell lysates were then sonicated (Bioruptor™ UCD-200) to yield DNA fragments that have sizes between 300 and 800 base pairs. Thereafter, cell lysate were cleared after 10 min at 15,000 xg centrifugation. Supernatants were collected and diluted with ChIP dilution buffer by 10 fold. Diluted lysates were then precleared by protein A/G beads (Invitrogen) that had been blocked by BSA (Sigma) and salmon sperm DNA (Trevigen). Pre-cleared lysates were then aliquoted and incubated with 4 μg ChIP antibodies for 1 h. Then pre-blocked A/G beads were added for overnight incubation. On the second day, the beads were spun down and washed with low salt wash buffer, high salt wash buffer and lithium chloride buffer sequentially. Elution buffer was then added to elute protein-DNA complexes from the beads. Then the eluates were subjected to reverse crosslinking, RNA digestion (RNAse A: Sigma) and protein digestion  sequentially. A DNA purification kit (QIAGEN) was employed to purify DNA from previous steps. Purified DNAs were then utilized for q-PCR to quantify the abundance of associated proteins in specific regions of the gene locus.4.2.BRD4 , Rabbit IgG , H3K27ac  and H3K4me3 , GLI2 , Myc .4.3.Gli2−/−Gli3−/− MEFs [LIGHT2 (LT2) cells , Gli2−/−−/− MEFs , BL1648 −/− MEFs  were use4.4.3).ChIP lysis buffer , ChIP dilution buffer , low salt wash buffer , high salt wash buffer , Lithium chloride buffer , elution buffer  was cloned from HeLa cell lysate and ligated into pGL3 luciferase expression vectors using Gibson Assembly (NEB). Potential GLI binding sites (8/9 consensus bases) were identified using MacVector and deleted individually or in groups via inverse PCR. For luciferase assays, plasmids were cotransfected into HeLa cell culture using HilyMax (Dojindo) reagent. 30 h post transfection, cells were lysed and relative luciferase and renilla signal was detected using the Dual-Luciferase Reporter Assay system (Promega) with a Lumat luminometer (Berthold). After subtracting baseline noise and normalizing signal against renilla activity, statistical significance was determined by ANOVA (R).Deletion primers:TargethGLI1 intron1Reverse Primer: CGCCTGTAATTCCAACGCTTForward Primer: GCCTGGGGTGAGACATTAGAD1Reverse Primer: AGGAGGGGCCTCGATCCCCForward Primer: TCTGAATCCTCTTTCAGGTD2Reverse Primer: ACTGGCTGCAGACGGCTCCForward Primer: AAGGCGGGACCGGGAGTAGD3Reverse Primer: AGGCTAGTAAAAGGAAAATGForward Primer: TGTAGCCCCATTTCCTTGGD4Reverse Primer: GACCTTGGAACTAATGTTGForward Primer: TCCGCACCTCGGTTGGAAAAGD5Reverse Primer: TGGGATTTAGGGTGAGGGCForward Primer: CCGGCCCGCTCCCGGTGGD6Reverse Primer: TGTTTTGGACTAATTGTGCForward Primer: ACCCAGGCAAAGCTCCCACD7Reverse Primer: AGGAGGGGCCTCGATCCCCForward Primer: TGTAGCCCCATTTCCTTGGD8Reverse Primer: GACCTTGGAACTAATGTTGForward Primer: ACCCAGGCAAAGCTCCCACD9Reverse Primer: ACTGGCTGCAGACGGCTCCForward Primer: CCGGCCCGCTCCCGGTGGD10Reverse Primer: GACCTTGGAACTAATGTTGForward Primer: CCGGCCCGCTCCCGGTGGD11Reverse Primer: AGGAGGGGCCTCGATCCCCForward Primer: ACCCAGGCAAAGCTCCCAC4.7.32P labeled probe and the GLI1 protein preparation was described previously [Escherichia coli using the pETBlue-2 blunt cloning kit . The GLI2 aa 84–355 cDNA was prepared by PCR (sense primer:5′- GGAGCAGCTGGCTGACCTCAAG GAA-3′ and antisense 5′- CATCTCCACGCCACTGTCATTGTTG-3′) and cloned into the EcoRV site of the pETBlue-2 plasmid DNA (Novagen). For protein production, pETBlue-2 GLI2 construct was introduced into the Tuner (DE3)pLacI competent cells (Novagen) and GLI2 protein was induced with 1–2 mM isopropyl-1-thio-1-D-galactopyranoside (IPTG) for 3–4 h at 37°C. Bacteria were then harvested, sonicated, and cleared by centrifugation. The 6xHisTag GLI2 aa 84–355 in the clear lysate was purified using His-Spin Protein Miniprep kit with  with modifications. EMSA was performed using DIG gel shift kit. 1 μl of GLI2 or control protein was used and the rest of the method is the same with GLI1 procedure.EMSA was performed using DIG gel shift kit  or with eviously . Prepara2O, and 0 or 1 μl (20 pmol) of unlabeled competitor oligonucleotides. The mixture was incubated at 4° C for 10 min. 1 μl (155 fmol) of double stranded digoxigenin-labeled probe was added and the mixture was incubated at 4°C for 20 min. Probes were designed using MacVector software . Probe sequences are shown, listing the sense sequence (5′→3′) followed by the antisense sequence (5′→3′) used to produce the double-stranded probe. We used the following probes (GBS sequences are in the 5 μl of GLI1 protein (aa 211-1106) or control protein (pinpoint protein) was mixed with 2 μl of 5X binding buffer (Roche), HGBS #2; sense 5′- GGCGGCGACTTGGGTGGGCCGAGGAGGCA -3′ and antisense 5′- TGCCTCCTCGGCCCACCCAAGTCGCCGCC -3′GBS #3; sense 5′- AGGGGAGATATGGGTGGGCTGTGGAACGC -3′ and antisense 5′- GCGTTCCACAGCCCACCCATATCTCCCCT -3′GBS #4; sense 5′- AGCATCCCGGGATCACCCACCGCGCCGGC -3′ and antisense 5′- GCCGGCGCGGTGGGTGATCCCGGGATGCT -3′GBS #5; sense 5′- AAGGTCGAGTTGGGAGGTCTTGGATGCGG -3′ and antisense 5′- CCGCATCCAAGACCTCCCAACTCGACCTT -3′GBS #6; sense 5′- TCTACACACAGACCACACAGGCAAAGCTC -3′ and antisense 5′- GAGCTTTGCCTGTGTGGTCTGTGTGTAGA -3′non-specific competitor; sense 5′- TCTACACACAGACCACACAGGCAAAGCTC -3′ and antisense 5′- CGCAGACACACACCTGGGGTTACCTC -3′. The GLI1- DNA complexes were separated by 5% TBE gel electrophoresis, transferred onto Zeta-Probe GT membranes (Bio-Rad), and the shifted bands were visualized by anti-digoxigenin antibody and chemiluminescence reagent .4.8.4.8.1.7 cells and harvested via trypsinization. Cell pellets were then lysed with modified RIPA buffer  and cleared by centrifugation. Lysates were then incubated with pre-cleared, equilibrated GFP-Trap beads (Chromotek) overnight at 4 °C. Next, beads were collected by centrifugation and washed with modified RIPA buffer . Beads were then heated to 70 °C for 5 min in SDS loading buffer to elute proteins from beads. The supernatant was then collected by centrifugation and size-separated by SDS-PAGE. After gel fixation, lanes were divided by size and subjected to mass spec.Adherent HeLa cells were transiently transfected with a plasmid encoding either GFP-GLI1 fusion protein or GFP alone under constitutive control of the CMV promoter. Expression of recombinant protein was confirmed visually via fluorescent microscopy and by western blotting. The fusion protein was shown to be functional with a luciferase GBS reporter (data not shown). Cultures were expanded to approximately 3.5 × 104.8.2.www.uniprot.org on 2014-01-23). Mass tolerance for searches was set to maximum 4.5 ppm for peptide masses and 20 ppm for HCD fragment ion masses. Data was searched with carba-midomethylation as a fixed modification. A maximum of three mis-cleavages was allowed while requiring strict trypsin specificity [All mass spectrometric experiments were performed on a nanoscale UHPLC system connected to an Orbitrap Q-Exactive HF equipped with a nanoelectrospray source . Each peptide fraction was auto-sampled and separated on a 15 cm analytical column (75 μm inner diameter) in-house packed with 1.9-μm C18 beads  using a 1 h gradient ranging from 5% to 40% acetonitrile in 0.5% formic acid at a flow rate of 250 nl/min. The effluent from the UHPLC was directly electrosprayed into the mass spectrometer. The Q Exactive HF mass spectrometer was operated in data-dependent acquisition mode and all samples were analyzed using the previously described ‘fast’ acquisition method . All rawcificity , and onlcificity . Protein4.9.PLA was performed as described by the manufacturer . Rh30 cells , and HeLa cells  were interrogated with anti-GLI1 antibody  and/or anti-H2A.Z antibody  for PLA.PLA labeled cells were imaged on a confocal microscope . Z-stacks were acquired to allow for three-dimensional rendering in Volocity software . DAPI fluorescence images were processed in the EBImage package or the R statistical programming environment to better define the nuclear boundary. Differential Interference Contrast data from brightfield images were processed in Photoshop  using high pass filtering and median filtering to allow for rough visualization of cell boundaries. No processing was done to PLA fluorescence. All images were handled equivalently in Volocity, they were rendered in “3D Opacity” mode and channel opacity, density and black levels were all set to the same between images. Each square in the grid is approximately 22 μm on a side.4.10.7 Rh30 cells were resuspended and incubated in 1 ml of cell lysis buffer  for 30 min with gentle rocking. The lysate was then homogenized by passing through a 26 G needle for several times and cleared by centrifugation for 10 min. at 10,000 rpm. The clear lysate was incubated with 20 μl of Anti-GLI1 antibody (Rockland) or control antibody  for 30 min and then was incubated with 20 μl of protein A/G-magnetic beads (Thermo Scientific) for 30 min with gentle rocking. GLI1-H2A.Z Immune complex was collected by using a magnetic rack for 1 min, washed with lysis buffer, and then eluted with low pH IgG elution buffer (Thermo Scientific). The eluate was then neutralized with an equal volume of 1 M Tris.Cl (pH 7.6). All the following procedures were carried out at room temperature. The GLI1-H2A.Z eluate was separated on a SDS-PAGE gel, transferred onto a nitrocellulose membrane and incubated in PBST buffer with 5% milk for 30 min. The membrane was washed with PBST buffer, incubated with polyclonal rabbit H2A.Z antibody (Active Motif) (1:4000 dilution) in PBST buffer with 5% milk for 1 h. The membranes were washed with PBST buffer  and incubated with secondary antibody conjugated with HRP  for 1 h (1:8000 dilution in PBST with 5% milk). The membrane was then washed 3 times with PBST buffer. The H2A.Z protein was visualized using SuperSignal West Pico chemiluminescence kit (Thermo Scientific).The following procedures were carried out at 4°C. Approximately 2 × 10 010203040506


Oikopleura dioica, a member of the closest living relatives to vertebrates.Development is largely driven by transitions between transcriptional programs. The initiation of transcription at appropriate sites in the genome is a key component of this and yet few rules governing selection are known. Here, we used cap analysis of gene expression (CAGE) to generate bp-resolution maps of transcription start sites (TSSs) across the genome of % of male-specific transcription, including the use of cryptic internal promoters within operons. In many cases this led to the exclusion of trans-splice sites, revealing a novel mechanism for regulating which mRNAs receive the spliced leader. Binding of the cell cycle regulator, E2F1, is enriched at the TSS of maternal genes in endocycling nurse nuclei. In addition, maternal promoters lack the TATA-like element found in zebrafish and have broad, rather than sharp, architectures with ordered nucleosomes. Promoters of ribosomal protein genes lack the highly conserved TCT initiator. We also report an association between DNA methylation on transcribed gene bodies and the TATA-box.Our TSS maps revealed promoter features in common with vertebrates, as well as striking differences, and uncovered key roles for core promoter elements in the regulation of development. During spermatogenesis there is a genome-wide shift in mode of transcription initiation characterized by a novel core promoter element. This element was associated with > 70Our results reveal that distinct functional promoter classes and overlapping promoter codes are present in protochordates like in vertebrates, but show extraordinary lineage-specific innovations. Furthermore, we uncover a genome-wide, developmental stage-specific shift in the mode of TSS selection. Our results provide a rich resource for the study of promoter structure and evolution in Metazoa.The online version of this article (10.1186/s12864-018-4504-5) contains supplementary material, which is available to authorized users. Sites for the initiation of transcription are frequently marked in the genome by specific sequence elements, which are recognized and subsequently bound by basal transcription factors , 2. The Caenorhabditis elegans and Drosophila) [Ciona intestinalis [In the promoters of vertebrates 5-methylcytosine (5mC) DNA methylation is associated with transcriptional repression. When DNA is methylated within gene bodies, however, it is instead associated with active transcription and splicing , a featusophila) , 8. The stinalis , 10 is gstinalis , 10. HowC. intestinalis they are sharp with a TCT initiator, but lack a TATA-box [The identification of core promoter elements, and mapping of transcription start sites (TSSs), at single-nucleotide resolution, has been facilitated by Cap Analysis of Gene Expression (CAGE) . This haTATA-box . RecentlTATA-box . MaternaOikopleura dioica is a marine, larvacean, chordate in the sister group to vertebrates and is well positioned to examine the evolution of TSS features and the dynamics of TSS selection. The O. dioica genome is the most compact of any animal genome sequenced so far and 27.8% of its genes are organised into operons [trans-splicing [Trans-splicing in O. dioica [trans-spliced [trans-splicing a portion of the original 5’ sequence upstream of the trans-splicing acceptor site is removed. Here, we mapped TSSs at single-nucleotide resolution, using CAGE, in six key stages of O. dioica development, covering the entire 6-day life cycle. In order to maximise the mapping of original TSSs (rather than trans-splice sites) we sequenced only mRNAs without the SL sequence. We used our TSS maps, together with previously generated genome-wide maps of trans-splice sites [O. dioica has some promoter features in common with vertebrates, including evidence of nucleosome positioning at broad promoters and tissue-specific expression of TATA-dependent promoters, but it differs markedly in its mode of maternal transcription initiation, which is characterized by the ordering of nucleosomes and the binding of the cell cycle regulator E2F1. O. dioica also employs a remarkable genome-wide shift in mode of TSS-selection during spermatogenesis, associated with a distinct, tissue-specific, TCTAGA core promoter motif, that has not been previously identified. operons . Each opsplicing , 18 of a. dioica  also occ-spliced . During ce sites , E2F1 bice sites , to deriO. dioica at six stages of development across the 6-day life cycle of the animal  of all monocistronic (non-operon) trans-spliced genes allowing us to analyse promoter features of trans-spliced genes. Since trans-splicing is thought to occur co-transcriptionally, and some monocistronic genes can be both trans-spliced and non-trans-spliced depending on the developmental stage, we did not expect to capture a larger proportion of the promoters of all potentially trans-spliced monocistronic genes.We extracted RNA for CAGE from mal Fig. a. Illumi. dioica  using poWe defined 13,771 consensus promoter regions in the genome by clustering TCs, with > 5 tpm, across stages , 23. Exp1trans-spliced genes, were found predominantly within broad TCs  with a TCTAGA within 100 bp upstream of the annotated start site compared to 2088 (12.5%) that had a consensus TATA-box motif. This suggests a larvacean, lineage-specific evolution of this mode of TSS-selection for the activation of the spermatogenesis transcriptional program.In order to determine whether or not this mode of regulation is found in mammals we re-analyzed existing CAGE data , 24 fromA single gene may have several alternative TSSs selected at different developmental stages. We identified all promoter regions with a shift in TSS usage , 23 betw1O. dioica genome contains 1765 operons that comprise multiple genes that are transcribed from a single promoter into polycistronic mRNA. Cryptic internal promoters within operons, which drive tissue-specific expression of downstream genes, have been described in C. elegans [O. dioica genome is unknown. We identified 693 internal promoters within operons in O. dioica: male-specific promoters (208) were over-represented and the TCTAGA element was found more frequently  than expected . This suggests that during spermatogenesis the TCTAGA element plays a role in selecting TSSs at internal promoters within operons that are otherwise transcribed from a single upstream promoter in other stages. We therefore analyzed patterns of enrichment of the H3K4me3 promoter mark from ChIP-chip data [The  elegans  but the hip data  in the otrans-splicing are determined by the presence of an unpaired AG acceptor site, which is usually followed by an adenine [trans-splice site . Transcription downstream of these TCTAGA elements during spermatogenesis therefore results in mRNAs that lack a trans-splice acceptor site and are therefore not trans-spliced with the SL sequence. Transcription driven by alternative upstream promoters during other stages of development leads to mRNAs with the trans-splice site intact and are therefore trans-spliced with the SL. This finding reveals a novel mechanism for the developmental regulation of trans-splicing.Sites for  adenine . Remarkap < 0.05; Fig. 22trans-splicing.Male-specific TCTAGA promoters had significantly higher expression levels compared to other promoter types in males  maternal promoters in O. dioica.Maternal promoters in zebrafish tend to be sharp, or multiple sharp, with a degenerate TATA-like motif (W-box) determining TSSs . In contoad Fig. c and lacO. dioica.We also found that the nucleosome-free region at the TSS of broad promoters in the day 6 female corresponds to an enrichment of the activating transcription factor E2F1 Fig. a, a key O. dioica were located on the X-chromosome more frequently than expected, compared to zygotic promoters , revealing a female-bias of X-linked genes [O. dioica.Maternal promoters in ed genes  in O. diO. dioica , in line with other trans-spliced gene promoters in this animal . This indicates that these promoters have lost the specific transcriptional regulation conferred by the TCT initiator in other species and provides further evidence that the trans-spliced SL replaces the role of the TOP motif [Whereas a typical Initiator (Inr) CA dinucleotide was present in 53% of consensus promoter regions in  operons ). UnlikeOP motif , which sO. dioica development. A TATAW element was present in 10.7% of consensus promoter regions, in line with the percentages of TATA-dependent promoters in mammals [4O. dioica and vertebrates. As in other species, the preferred location of the TATA box was 28–31 bp upstream.We next searched all our CAGE-defined promoters for the ancient TATA-box promoter element in order to assess the usage of this promoter motif in  mammals . A lowerp = 2.04×10−8), promoter width  and downstream CpG content  the presence of the most common core TATAA motif was a significant, independent, positive predictor  of downstream DNA-methylation levels in ovary, testis and tailbud, . H3K4me3, which inhibits the interaction of DNA methyltransferases with histone proteins [4O. dioica. We found a similar association in zebrafish, although the increase in DNA methylation compared to TATA-independent promoters was at the TSS rather than the gene body  data  from ova4proteins , was depO. dioica  with a TATA-like upstream element whereas zygotic promoters are broad with transcription initiation guided by ordered nucleosome positioning . In compg canals , 32.trans-spliced in O. dioica [trans-splicing removes the 5’ end of a pre-mRNA (the “outron”) it follows that this sequence has little, if any, role in the post-transcriptional regulation of its mRNA. Indeed, one hypothesis for the function of trans-splicing in monocistrons is that it removes deleterious sequences at the 5’ end of an mRNA (e.g. premature start codons). There is mounting evidence that the SL sequence itself plays an important role in translational control, particularly for TOP mRNAs, which are trans-spliced in O. dioica [O. dioica ribosomal protein TSSs. We hypothesize that there is no requirement for a strict site of transcription initiation for trans-spliced genes since the spliced leader provides any necessary 5’ post-transcriptional regulatory motifs. Promoters of trans-spliced genes are then permitted to adopt a broad architecture governed by chromatin state rather than sequence motifs.Most maternal transcripts are . dioica  and this. dioica . We havetrans-splicing during spermatogenesis: many mRNAs that are trans-spliced in other stages (often in operons) are transcribed during spermatogenesis from an alternative TSS, driven by a TCTAGA promoter located downstream of the trans-splice acceptor site. This trans-splice acceptor site is thereby excluded from resulting mRNAs, which are no longer trans-spliced with the SL sequence. This may lead to a switch in the translational control of these transcripts to one that is independent of nutrient levels [O. dioica [Our data also revealed a remarkable genome-wide shift in mode of TSS-selection during spermatogenesis to one associated with a position-specific core promoter motif (TCTAGA). This shift is likely regulated by a basal transcription factor that is specifically expressed in the male. With a distance (44 bp) from the TSS similar to that of the TATA-box (36 bp) it is tempting to speculate that a TFIID complex containing a male-specific variant of the TATA-binding protein (TBP) is the factor binding TCTAGA and driving expression of male-specific genes. Indeed, variants of the basal transcription factor (TF) machinery are known to play roles in development and gametogenesis across metazoans . One cont levels . We hypo. dioica . The TCTO. dioica. This relationship is present during early development as well as in both the male and female germ lines, despite these differing substantially in their chromatin landscapes [O. dioica genes is driven by core promoter features, specifically the TATA-box. A study in C. intestinalis found that gene bodies in near identical sets of genes are methylated in different cellular contexts [O. dioica. This study also showed, however, that features within two ubiquitously expressed promoters are not the primary determinant of gene body DNA methylation. Analysis of additional C. intestinalis promoters may nevertheless reveal a relationship with the TATA-box similar to what we observe in O. dioica. Further exploration of sequence context in both species may also reveal a role for additional factors.A recent study found that genes with transcription-associated gene body methylation encode more highly conserved proteins with typical “housekeeping” functions . We discndscapes . Promotecontexts , which iGiven that DNA methylation in gene bodies suppresses transcription from alternative downstream promoters , 8 it istrans-splicing and the reorganization of promoter architectures for a conserved set of core metabolic genes, probably arising at least in part, because of regulatory sequences encoded in the SL. We also show shifts in TSS selection associated with a previously unidentified core promoter motif during the spermatogenic program. Further work on a range of additional models would provide a better framework in understanding the evolution of core promoter architectures, particularly with respect to innovations within major lineages.Our results support previous findings of overlapping promoter codes , while rTM 5’-Phosphate-Dependent Exonuclease to deplete excess small RNAs. A modified CAGEscan protocol [trans-spliced from non-trans-spliced transcripts by first using a custom designed 5’ linker, specific to the 5’ spliced leader sequence, before using standard linkers for non-trans-spliced mRNAs. Sequenced libraries for each stage therefore included only non-trans-spliced transcripts.Total RNA from each stage of development was isolated using RNAqueous Micro (Ambion) and treated by Terminatorprotocol  was carrO. dioica reference genome [Illumina sequencing generated a total of 39,124,333 reads, 37 nt in length. We mapped these to the e genome  using Boe genome  with defq0.1−q0.9) of promoter widths (a measure of how broad/dispersed or peaked/focused a promoter’s TSS usage is that is more robust to expression level than using the full promoter width). We used this to group promoters into four classes using the mean and upper and lower quartiles as thresholds. We defined the upper and lower quartiles as “broad” and “sharp” respectively. We categorized promoters by CpG frequency in a 200 bp window centered on the dominant CTSS. Promoters with a CpG frequency in the upper quartile of CpG frequencies were classed as high CpG (HCG) and promoters with a CpG frequency in the lower quartile classed as low CpG (LCG). Using CAGEr we grouped all tag clusters with > 5 tpm across all stages into consensus promoter regions, using the interquantile range (q0.1−q0.9) of tag cluster widths and a distance of 100 bp to merge clusters into one region. We used SOM clustering both at the level of individual CTSSes and consensus promoter regions to generate 25 expression profiles in each case.We used the R package “CAGEr”  to clustp-value of Kolmogorov-Smirnov test for all consensus promoters for all pairwise comparisons. We used a score > 0.6 and FDR < 0.01 to define a promoter shift – identifying promoters that have at least 60% of transcription initiation in the sample with lower expression occurring either upstream or downstream of transcription initiation in the compared sample.We calculated a shifting score and www.genoscope.fr) to classify genes into operons and non-operons. A CTSS was associated with a gene model if it overlapped a gene body or its 500 bp upstream region. Using previously published CAGE data for trans-spliced transcripts [trans-spliced if there was a SL CTSS within the gene body or within a 500 bp upstream region, if it was supported by > 1 tag count and if it had an ‘AG’ acceptor site motif immediately upstream.We used Genoscope gene model predictions and annotations of polycistrons  by scanning core promoter regions for the occurrence of all possible k-mers (for k=1–6). We used TOMTOM to match position-weight matrices of motifs identified by MEME to known transcription factor binding sites . We plotO. dioica ovaries and testes [2(Cy5/Cy3). We used the NimbleGen normalization method, which adjusts for systematic dye and labeling biases by subtracting from individual log2 ratios the Tukey’s biweight mean, computed across each sample’s log2 ratios. To reduce noise in the data we smoothened the normalized log2 ratios using a running median across a 150 bp window (the approximate size of a single nucleosome) with a minimum threshold of 3 non-zero probes. For each group of promoters we plotted the mean log2 ratio at each probe position for all probes in a 1000 bp window centred on the dominant CTSSes of promoter regions of interest. We excluded promoters with flanking regions that overlap. We defined regions of ChIP-enrichment genome-wide as previously described [We analysed previously published meDIP-chip data and ChIP-chip data for E2F1, H3 and histone modifications H3K4me1, H3K4me2, H3K4me3 and H3K27me3 from mature d testes . We usedd testes  for pre-escribed .O. dioica testes and ovaries [We used tiling array data generated from  ovaries  to categRACE was performed using SMARTER RACE kit from Clontech according to manual.http://promshift.genereg.net/CAGEr/InputData/consisting of TSSs from 8 stages of mouse testis development. Briefly, we used the CAGEr [Analysis of TSS data followed that found in  using dahe CAGEr  package he CAGEr . We plotC. intestinalis genes for “TCTAGA” motif and the consensus TATA-box motif “TATAWAR” using the “Biostrings” R package.We searched the 100 bp region upstream of all annotated Ensembl 87 KH Additional file 1Table S1, Table S2, Figures S1 - S12 and Supplemental References. (PDF 13 464 kb)Supplementary Material. Supplemental Results, Supplemental Methods,


CATSPER1 gene encodes a pore-forming and pH-sensing subunit of the CatSper Ca2+- permeable channel, a protein in the flagellum essential for sperm hyperactivation. Previous studies have shown that the murine Catsper1 gene promoter is regulated by different Sox proteins. Likewise, it is acknowledged that the human CATSPER1 gene promoter sequence is enriched in potential interaction sites for the sex-determining region Y gene (SRY), which suggest a novel regulatory transcriptional mechanism for CatSper1 channel expression. Therefore, in this work, we sought to determine whether the human CATSPER1 gene expression is regulated by the SRY transcription factor. To this end, a series of deletions and mutations were introduced in the wild- type CATSPER1 gene promoter to eliminate the SRY sites, and the different constructs were tested for their ability to activate transcription in human embryonic kidney and murine spermatogonial germ cell lines  using luciferase assays. In addition, by using a strategy that combines electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) we investigated whether the CATSPER1 gene expression is regulated by the SRY transcription factor both in vitro and in vivo. Our results show that the transcriptional factor SRY specifically binds to different sites in the promoter sequence and has the ability to control CATSPER1 gene transcription. Inactivation of the CatSper genes produces alterations in sperm hyperactivation and subsequent lower fertilization capability or infertility [CatSper is a voltage-gated Calagellum ,2. It islagellum –5, also lagellum –8. In coertility –9.CATSPER1 gene share significant homology with its murine orthologue, is located in chromosome 11 and encodes a protein of 780 amino acids with a histidine-rich domain located in the amino-terminal region [Catsper1 mRNA expression has been detected in early stages of spermatogenesis [Catsper3, which has been detected in the brain, the expression of the CatSper genes has been observed only in meiotic and post-meiotic sperm cells [Catsper2 is expressed before Catsper1, 3 and 4, and this differential expression may be associated with different groups of genes expressed specifically in testis [The human l region . This furm cells –14. It hCATSPER1 mRNA expression is significantly lower in subfertile patients [CATSPER1 expression are presently unknown, mainly because there is limited information regarding the transcriptional regulation of its promoter and the factors that repress or activate its gene expression [CATSPER1 genes are driven by TATA-less promoter sequences located adjacent to the first exon. Also,we reported that the murine Catsper1 promoter is responsive to testis transcriptional factors including Sox9 and Sox5 [in silico analysis revealed multiple sites for the sex-determining region Y gene (SRY) in the human CATSPER1 promoter sequence. The SRY gene encodes a protein with a highly conserved DNA-binding site (79–80 amino acids), known as the HMG box that is expressed during fetal development, as well as in the adult gonadal tissue [Likewise, it has been reported that patients ; howeverpression ,16. We hand Sox5 ,18. Likel tissue ,20. SRY l tissue ,22.ER71 and SOX9 are upregulated by SRY during gonadal differentiation, and both factors regulate their expression by a transcriptional loop in the adult testis [Th gene promoter in the brain, and a role as a regulator of the Renin-Angiotensinogen system in rat and humans has also been suggested [CATSPER1 gene expression by SRY. Our results show that SRY may regulate either negatively or positively the CATSPER1 gene transcription via multiple SRY binding sites located in the promoter sequence.Similar to the murine Sry, the actions of human SRY have been widely documented during testis development. However, less is known regarding its functional relevance in the adult testis. Hence, t testis . Sry reguggested . Here, wCATSPER1 gene were identified by MatInspector (http://www.genomatix.de/cgi-bin/matinspector_prof/mat_fam.pl) and the ConSite web server (http://consite.genereg.net/cgi-bin/consite). pCAT1, pCATΔbasal, pCATΔ3’ and pCAT739 constructs of the human CATSPER1 promoter and Renilla luciferase gene were described previously [CATSPER1 promoter as well as 20- to 27-bp of the desired deletions. Three deleted promoter plasmids were generated  which were verified by restriction enzyme analysis and automatic sequencing. Likewise, point mutations were made for each site to prevent SRY binding to the CATSPER1 promoter. The pCATΔ3’ was used as the template, and specific oligonucleotides , the following primers were designed:The cDNA expression vector containing the sequence encoding the human SRY was initially obtained from PCR amplification of the intronless SRYF-5’-CACGGATCCATGCAATCATATGCTTCTGC-3’, SRYR-5’-TTTGAATTCC TACAGCTTTGTCCAGTGG-3’. After an initial denaturation step at 94°C for 5 min, PCR was carried out for 35 cycles at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min, followed by one step to 72°C for 10 min. A 616 bp PCR fragment was purified and subcloned into BamHI/EcoRI-digested pIREShr-GFP1a vector, and then transformed into competent E.coli DH5α cells. Positive clones were sequenced to verify the correct inserts and to obtain the pIRES-SRY vectors.5 cells/well in 24-well plates for 24 h and then transfected using TurboFect transfection reagent (Dharmacon), with 1 μg of each promoter construct with deletions or 5 ng of pRL-CMV control plasmid. Fifty ng of pGL-CMV plasmid expressing Photinus luciferase was used as a control for transfection efficiency. Subsequent co-transfections were performed in HEK-293 cells since the mouse Catsper1 promoter is inactive in the GC1-spg [The human embryonic kidney (HEK-293) cell line and the murine spermatogonial germ cell line (GC1-spg) were purchased from American Type Culture Collection (ATCC). Cells were kept in culture in DMEM medium containing 10% heat-inactivated fetal bovine serum (FBS) and incubated at 37°C in a humidified incubator with 5% CO2 and 95% air. Cells were seeded at 2.5 × 10 GC1-spg . Co-tranCell lysates were obtained from untransfected and pIRES-SRY HEK-293 transfected cells, harvested and washed with PBS and lysed in RIPA buffer. Lysates were incubated for 2 h on ice and centrifuged for 20 min at 10,000 rpm. Total protein was quantified by the Bradford method, and 15 μg were used in SDS-PAGE. Gels were transferred to PVDF membranes (Sigma-Aldrich), which were blocked and incubated with a monoclonal SRY antibody  at 1:1,000 dilution; a RNAPol II antibody  at 1:500 dilution; a GAPDH antibody (Millipore) at 1:500 dilution, and secondary mouse or rabbit antibody (Sigma-Aldrich) at 1:15,000 dilution. Signals were revealed using a Infrared Imaging System (LI-COR Biosciences).32P)-ATP  using T4 polynucleotide kinase and purified in a centrisep column. Binding reactions were carried out with 100 fmol of the end-labeled probe and 10 μgof nuclear protein SRY in binding buffer 10X (Promega), 1 μg/ml poly-dIdC, 0.05% Nonidet P-40, and 300 μg/ml BSA. Reactions were incubated at room temperature (RT) for 20 min, loaded onto 5% polyacrylamide gels and electrophoresed. For competition analysis, 2 pmol of the cold-competitor probe was included in the reaction. All experiments were performed in triplicate.Nuclear proteins from untransfected or SRY transfected HEK-293 cells were prepared using nuclear and cytoplasmic extraction reagents (Fermentas). Protein concentration was determined using the Bradford method. Nuclear proteins were collected and stored at −80°C. The mutated and wild-type SRY site oligonucleotides used are listed The wild-type SRY site oligonucleotides  were 3´-7 untransfected and SRY transfected HEK-293 cells, as well as immature spermatogenic cells from human semen, were used. Cells were washed in PBS and treated with 1% formaldehyde in PBS for 10 min, and 125 mM glycine for 5 min at RT. Cells were washed in PBS with a protease inhibitor cocktail (Roche) and prepared for nuclei isolation with SDS lysis buffer. Nuclear extracts were sonicated to yield DNA fragments of 200 to 900 bp. Sheared chromatin was precleared with 30 μl of protein G agarose beads overnight at 4°C. Cleared supernatant was used as input in the ChIP assay. For immunoprecipitation, crosslinked chromatin samples were added with 1 μg of each antibody for SRY (Santa Cruz Biotechnologies), RNA polymerase II (Santa Cruz Biotechnologies), a nonrelated antibody , and mouse IgG as negative control. An aliquot of these samples was taken as input for Western blotting. Immunocomplexes were absorbed with 30 μl of protein G agarose beads for 24 h at 4°C. Samples were also taken as supernatant for Western blotting [3, pH 8) from beads twice, and NaCl2 was added overnight at 64°C to eluate input samples. After reverse cross-linking, RNAse and proteinase K was added for 1–2 h. DNA was purified by phenol/chloroform extraction followed by ethanol precipitation and resuspended in 30 μl. PCR experiments were carried out with Go Taq (Promega) using SRY0ChIP, SRY1ChIP, SRY2ChIP and SRY3ChIP oligonucleotides , SRY2 (-822/-802) and SRY3 (-1405/-1386), as well as a single SRY site in a downstream region called SRY0 , while deletions of 1718 and 1398 bp, respectively, in the pCATΔSRY1 and ΔSRY2 constructs also showed a significant increase in activity (~10-fold) in relation to pCATΔ3’, the construct that contained all the SRY binding sites . This coCATSPER1 is not detectable in HEK-293 cells even after SRY overexpression. In addition, expression of CatsSper1 was not detected in the GC1spg cell line though these cells are derived from spermatogonia. Despite the fact that the promoter is specifically activated in sperm under physiological conditions, our data suggest that transcriptional activity in transfected cells is twice as high in GC1spg as in HEK-293 cells, and in both cases, this activity is higher than in the mock condition , and their basal and induced transcriptional activities determined by luciferase assays after 48 h of transfection . These results suggest a regulatory role for the SRY transcription factor on the CATSPER1 promoter activity mediated by the three predicted binding sites.Given that these plasmids represent large regions in the promoter sequence, our results suggested a potential regulation through SRY but does not indicate necessarily whether the transcription factor occupies the conserved binding sites found in the onstruct . DeletioCATSPER1 promoter, we next introduced mutations in the four identified binding sites. As mentioned earlier, three of these sites are located upstream of the TSS (SRY1-3), and one site (SRY0) is located downstream of the TSS and the start codon. Bioinformatic analysis was performed to verify that the mutant sequences did not introduce possible new SRY sites or any other site for additional transcription factors. Mutations were made using the pCATΔ3’ construct as a template by PCR site-directed mutagenesis and verified by automatic sequencing.In order to corroborate the functional role of the SRY binding sites in the CATSPER1 promoter (pCATΔ3’). We used pCAT1 basal activity to normalize data. Our results show small changes (~20%) in the relative activity of pCATmutSRY3 and pCATmutSRY1 mutant construct with respect to the WT pCAT1Δ3’ construct  onstruct . Only pConstruct . Likewis32P-labeled oligonucleotide probes for the WT SRY sites. Initially, nuclear proteins from SRY-transfected HEK-293 cells were tested using 32P-labeled and unlabeled duplex WT oligonucleotides under binding conditions. Each of the three upstream SRY sites (SRY1-3) showed the formation of a covalent DNA-protein complex after incubation of the probes with the nuclear extract obtained from pIRES-SRY transfected cells. Such DNA-protein complexes diminished when an excess of non-specific duplex oligonucleotides was added to the reaction, suggesting that other nuclear proteins are bound to these oligonucleotides in an unspecific way. However, the complex formation was abolished in the presence of a specific unlabeled competitor oligonucleotide . SRY sites were represented in duplex oligonucleotides as a WT and mutated versions. The overexpression of SRY and its nuclear location was corroborated by Western blot of SRY in nuclear and cytoplasmic extracts from pIRES-SRY transfected HEK-293 cells . EMSA asin vitro. Though the other mutant oligonucleotides eliminated two of the SRY1 and SRY3 sites, DNA-protein complexes were still observed given that other weak binding sites might still be recognized by SRY or by other Sox factors  cells as well as human ejaculates using SRY antibodies. Immunoprecipitation of SRY during ChIP was verified by Western blot analysis from chromatin samples of HEK-293 (SRY) and sperm cells . Interestingly, Sox proteins and SRY belong to the same family of HMG-box proteins, and their consensus binding sites contain the same core (AACAA) with minimal differences of one or two bases at the end of the sequence. However, it is possible that different Sox factors may recognize the same binding site in the promoter, and then the differential expression of Sox factors according to the cell type becomes relevant as well as the recruitment of other factors to the promoter.The murine  factors . HoweverCATSPER1 promoter contains three overlapping SRY sites upstream the TSS (SRY1-3) and one single SRY site downstream the TSS (SRY0). Deletion analysis of the promoter shows a loss of transcriptional activity upon removal of the region containing the SRY3 site, which is recovered when the region containing the SRY2 site is removed. Since both regions are extensive, these findings could be the result of a loss of sites for other transcription factors. However, a mutation in the SRY1 site introduced into the pCATΔSRY2 construct virtually eliminates basal transcriptional activity. On the other hand, SRY overexpression increases transcriptional activity in all constructs, since they contain at least one of the SRY sites. Indeed, we found that mutation of SRY2 had a decreasing effect on basal transcriptional activity when the other SRY sites remained intact in the promoter sequence, even with the mutated SRY1 site in the double mutant pCATmutSRY1-2. This decrease in activity was prevented after SRY overexpression. On the other hand, the SRY mutants did not reach the same level of transactivation by SRY overexpression with exception of SRY0 mutant. Last, EMSA, supershift and ChIP analysis revealed a specific interaction between SRY and the SRY2 site. These findings imply that SRY2 is an important site for the regulation of the CATSPER1 gene promoter activity.The https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/; August 2017) [CATSPER1 promoter sequence may determine a differential expression pattern among individuals. In this context, it is worth noting that low expression of CATSPER1 is related to impaired fertility because of reduced sperm mobility [CATSPER1 expression may occur in populations of sperm with normal and altered motility [CATSPER1 expression also present poor sperm motility. Likewise, the presence of overlapping SRY sites in the CATSPER1 promoter might help to prevent the negative effects of the mutations or SNPs on CATSPER1 expression, ensuring gene expression and preventing alterations in sperm motility.Interestingly, three SNPs have been detected in the SRY2 site, called rs7129846, rs201387881, and rs200309988. These SNPs are present in the core of the SRY binding site and correspond to a C↔A change, an AC/A deletion and a C/CA insertion, respectively. The rs7129846 is observed with an allele frequency of C = 0.08 A = 0.91 in the Mexican ancestry from Los Angeles, CA, USA, (st 2017) . Thus, imobility ,13,27. AIt is worth noting here that ENCODE and roadmap epigenomics data suggest multiple transcription factors binding (DNase and ChIP-Seq), histone modification (H3K27Ac) of an active state in nonexpressing cells near exon 2 of the gene, and a general open chromatin state for the entire gene. Yet there is no expression in these cells. These data suggest the presence of much more complex patterns of promoter regulation.CATSPER1 during spermatogenesis. Sry expression has been documented in different tissues such as kidney, adrenal gland, and brain, as well as in the adult testis [SRY gene promoter contains multiple sites for the transcription factor Sp1. Likewise, the Sry promoter lacks the TATA box sequence, and experimental data suggest a differential SRY transcription that depends on tissue- and species-specific factors. In vitro studies during gonadal differentiation have shown that transcription factors WT1, NR5A1, Sox9, Gata4, and Sp1 may bind to the promoter and activate Sry gene transcription. Similarly, it has been shown that during Sry expression, the levels of a histone demethylase (H3K9me2) are reduced favoring transcription initiation. Last, it has been reported that an isoform of the WT1 protein (Wt1+ KTS) contributes to the posttranscriptional regulation of the Sry messenger RNA [In addition, SRY may serve as an important transcriptional factor to regulate the tissue-specific expression of t testis ,29. Likenger RNA .CATSPER1 gene transcription via multiple SRY binding sites located in the promoter sequence. This is likely to assist in defining the key factors responsible for physiological and pathophysiological CatSper expression. Given the proposed importance of this protein in fertilization, identification of the mechanisms controlling its expression could also be of clinical use. Furthermore, since variations in the sequence of the SRY2 site have recently been shown to give rise to individual variability of CATSPER1 expression, knowledge of the promoter sequence and regulation might eventually help to ensure gene expression and prevent alterations in sperm motility.In summary, here we show that SRY may regulate S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.